Accepted Manuscript Title: RNA binding proteins implicated in Xist-mediated chromosome silencing Author: Benoit Moindrot Neil Brockdorff PII: DOI: Reference:
S1084-9521(16)30029-5 http://dx.doi.org/doi:10.1016/j.semcdb.2016.01.029 YSCDB 1940
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Seminars in Cell & Developmental Biology
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19-10-2015 20-1-2016 21-1-2016
Please cite this article as: Moindrot Benoit, Brockdorff Neil.RNA binding proteins implicated in Xist-mediated chromosome silencing.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.01.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
RNA binding proteins implicated in Xist-mediated chromosome silencing Benoit Moindrot and Neil Brockdorff Dept of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK email; [email protected]
1
Abstract Chromosome silencing by Xist RNA occurs in two steps; localisation in cis within the nuclear matrix to form a domain that corresponds to the territory of the inactive X chromosome elect, and transduction of silencing signals from Xist RNA to the underlying chromatin. Key factors that mediate these processes have been identified in a series of recent studies that harnessed comprehensive proteomic or genetic screening strategies. In this review we discuss these findings in light of prior knowledge both of Xist-mediated silencing and known functions/properties of the novel factors.
Key words X chromosome inactivation Xist Chromatin non-coding RNA Epigenetic
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Introduction The X inactive specific transcript (Xist) gene was first identified as a candidate for the master switch locus required in cis for the process of X chromosome inactivation (XCI) [1-3]. Xist was shown to be a 15-17kb long non-coding (lnc) RNA that is retained in the nucleus, coating the inactive X chromosome (Xi) territory [4, 5]. Gene knockout and transgene studies demonstrated that Xist is both necessary and sufficient to establish chromosome silencing [6-9].
Key questions arising from these findings are what are the mechanisms for
developmental regulation of Xist expression that underpins establishment of appropriate XCI patterns in male and female embryos, and what is the basis for chromosome silencing by Xist RNA? Analysis of Xist RNA deletions indicates that the latter question can be divided into two parts, mechanisms that confer Xist RNA localisation in cis within the Xi territory, and mechanisms driving Xist RNA mediated chromatin modification and gene silencing [10]. Whilst our understanding of Xist gene regulation has advanced significantly (see review by ... in this issue), mechanisms governing Xist RNA localisation and establishment of chromosome silencing remain poorly characterised.
An early study estimated that there are around 1000 Xist RNA molecules/cell [11], and it was also noted that levels vary considerably between different mouse strains [2]. More recent analysis using 3D-SIM super-resolution microscopy determined that Xist RNA localises to around 200 resolvable foci, likely corresponding to individual molecules, within the Xi (Xi) territory [12]. Similar estimates were obtained in a subsequent study using PALM/STORM– super resolution microscopy [13]. These observations indicate a ratio of one Xist RNA molecule for every 1Mb of DNA on the X chromosome, corresponding on average to around 5 genes.
3
Xist RNA is tightly bound up within the insoluble nuclear matrix [14], and this has hampered efforts to apply biochemical approaches to identify the critical RNA binding proteins (RBPs) that interact directly with Xist RNA. A second factor that has limited progress towards identifying key factors in Xist-mediated silencing is that Xist function is normally limited to a window of opportunity during early development [15]. It follows that efforts to identify and validate candidate Xist interactors need ideally to be performed in Xist responsive cell types, for example embryonic stem (ES) cells, rather than in somatic cell lines. A further consideration is that multiple redundant pathways maintain XCI in somatic cells, and ongoing Xist expression is not required for maintenance of silencing [16-18].
It should be
noted that Xist RNA localisation/domain formation occurs normally when Xist is induced in differentiated cell types [15, 19], indicating that, unlike silencing, this process is not restricted to early developmental stages.
A recent series of studies that utilised either proteomic [20-22] or genetic [23, 24] screening strategies have led to a significant advance in the identification of key RBPs that interact with Xist RNA. Several, but not all, of the factors were identified independently in more than one study, lending added confidence to their candidacy as critical factors acting in the XCI pathway.
The proteomic screens used Xist antisense oligonucleotides to capture Xist RNA/protein complexes from soluble cell extracts. Chu et al [20] performed their analysis following formaldehyde cross-linking, which covalently links nucleic acid -protein interactions and protein-protein interactions, and therefore identifies factors that bind Xist RNA either directly or indirectly. Mass spectrometry data were filtered by comparing results obtained for Xist RNA and other nuclear non-coding (nc) RNAs, and further by comparing data from different cell types, including ES cells, providing a list of 81 candidate interactors. A further 4
comparison of full length Xist RNA with Xist RNA deleted for the repeat A element, a region required for chromosome silencing (see below), highlighted three key factors (SPEN, WTAP and RNF20). The study by McHugh et al [21], in contrast, used UV cross-linking in ES cells, and thereby identified only candidate direct interactors/RBPs.
Again mass spectrometry
data were filtered by comparing results obtained for other nuclear ncRNAs, resulting in the identification of ten candidate Xist interactors, all except one of which had protein domains commonly found in classical RBPs. Minajigi et al [22], also used UV cross-linking but their experiments were performed in a somatic cell line. They identified more than 200 proteins as direct Xist interactors, the majority of which did not have domains commonly found in RBPs.
Validation experiments focused on the role of factors identified in maintenance of
XCI, specifically on the role of cohesin complexes in topological organisation of the Xi in somatic cells, which is discussed in detail in another review in this series (see review by....in this issue).
In the genetic screens, Moindrot et al [23] set out to identify the primary factors involved in Xist localisation/silencing by using a reporter ES cell line with a GFP ORF knocked-in in cis with an inducible Xist transgene. The reporter line was screened with a pooled shRNA library targeting each of 5,000 nuclear factors with at least 9 independent hairpins. A ranking list of around 250 genes was determined based on enrichment in GFP-high cells and number of independent hairpins for which there was significant enrichment. Within the top ranked hits they identified multiple subunits of known complexes, including those linked to RNA and chromatin silencing. Monfort et al [24] used a different strategy, namely insertional mutagenesis in haploid ES cells in which inducible Xist RNA was engineered into the single X chromosome to screen for Xist silencing/localisation factors. Silencing of the single X chromosome in the reporter cell line results in cell lethality and it was therefore possible to screen for mutagenesis events that allowed cells to survive following Xist induction. The 5
frequency of mutagenesis events at individual loci was determined for induced and noninduced cells in order to identify candidate silencing factors.
Proteomic and genetic screens each have different benefits and limitations. Thus, for example, proteomic screens can identify direct interactors, but may miss factors with low abundance or for which there is a paucity of peptides suitable for detection in mass spectrometry. Additionally, the filtering process discards factors that bind equally well to Xist and control ncRNAs, some of which could have important functions in Xist-mediated silencing.
Genetic screens on the other hand have the advantage of potentially defining
multiple factors within an RBP complex, reinforcing the verity of the observation. Genetics can also identify factors that function more indirectly in the pathway of interest. Limitations on the other hand include not detecting factors because of functional redundancy or because loss of function is detrimental to cell survival, independent of the role of the factor in XCI. With these considerations in mind, in this review we compare and contrast the findings of the proteomic and genetic screening studies. Additionally, we examine how prior knowledge of the function of the key candidate RBPs identified in these studies provides clues as to their possible role in Xist localisation and/or chromosome silencing.
State of the Art Before discussing the recently discovered candidate silencing factors we will provide an overview of prior knowledge relevant to understanding establishment of XCI by Xist RNA. Xist is a large molecule, 15-17kb, and therefore has the capacity to interact with many factors simultaneously.
As illustrated in Figure 1, there are several tandemly arranged simple
repeats, labelled A-F, within the Xist sequence. Some of these repeats, and also some unique sequence regions, show a relatively high degree of sequence conservation [4, 5, 25, 26]. Key functional elements within Xist RNA have been defined in genetic experiments 6
(Figure 1).
The repeat A element at the 5’ end of the transcript, is uniquely required for
Xist mediated silencing, as determined by analysis of cell lethality in XY ES cells expressing Xist RNA transgenes with deletions of overlapping regions [10]. This same study further concluded that several elements, contribute in a redundant fashion to Xist RNA localisation, referring to the ability of the RNA to form a coherent cloud or domain in interphase nuclei following transgene induction. There is evidence pointing to a greater complexity. A small insertion downstream of the repeat A was found to disrupt Xist-mediated silencing, at least in part [27], and a distinct element, also in Xist exon 1, has been found to be critical for recruitment of the Polycomb Repressive complex 2 (PRC2), one of the known silencing factors that is recruited in an Xist dependent manner [28]. Additional Xist localisation elements, for example the repeat C, have been defined in studies that disrupt Xist localisation using short targeted LNA or PNA probes [29, 30], and using gene knockout analysis [31].
Developmental context is a further important consideration. The window of opportunity for Xist-mediated silencing (but not localisation) points to the involvement of factors that are themselves developmentally regulated. However, the reality may again be more complicated as some somatic cell lines, and adult cell types retain Xist responsiveness [32, 33]. Moreover, factors/chromatin modifications that are hallmarks of the Xi, for example recruitment of the PRC2 complex and associated histone H3 lysine 27 methylation (H3K27me3) [34, 35], occur in the absence of silencing, i.e. in response to expression of an Xist transgene lacking the repeat A element [19, 28]. Linked to this, XCI is faithfully maintained in somatic cells in which Xist is conditionally deleted, despite the fact that several Xist dependent factors/chromatin modifications, including PRC2 and H3K27me3, are lost [17, 35].
7
The nuclear matrix proteins SATB1/2 were found to be important for conferring Xist responsiveness in tumour cells and in adult somatic cell types [36], although apparently not in early embryos [37]. Nevertheless, the link between SATB1/2 and Xist could be important, especially in light of the evidence that Xist RNA domains are retained in insoluble nuclear matrix preparations. More recent studies have reinforced the link between Xist RNA function and the nuclear matrix. Notably, 3D-SIM super-resolution microscopy analysis demonstrated that Xist RNA is confined within perichromatin spaces, rather than on chromatin [12, 38], colocalising with the nuclear matrix protein hnRNP U (also referred to as SAF-A) [12]. hnRNP U was previously shown to have a role in Xist RNA localisation [39], and until recently represented the only XCI associated factor with a bona fide RNA recognition domain. Indeed UV cross-linking qRT-PCR analysis suggested that hnRNP U interacts directly with Xist RNA [39].
To date, the best studied factors with a role in Xist-mediated silencing are the Polycomb repressive complexes PRC2 [34, 35, 40], canonical PRC1 [41] and variant PRC1 [42, 43]. These complexes catalyse the histone modifications H3K27me3 and mono-ubiquitylation of histone H2A lysine 119 (H2AK119u1) respectively, both of which are hallmarks of the Xi. Polycomb complexes are recruited rapidly in response to Xist RNA expression and their association with the Xi is strictly dependent on ongoing Xist expression [19, 35, 42-44], suggesting that they might be primary silencing factors recruited by Xist RNA. In the case of PRC2 there is evidence for a direct interaction between the PRC2 subunit Ezh2 and the repeat A region of Xist RNA [45]. Ezh2 does not have a known RNA binding domain, and this conclusion has been challenged in other studies [28, 38]. Regardless of the mechanism of Polycomb recruitment, it needs to be borne in mind that Xist RNA, in which the repeat A region is deleted, does recruit Polycomb complexes [19], arguing that Polycomb is probably not sufficient for the establishment of chromosome silencing. 8
Studies aimed at
understanding Polycomb recruitment and function in X inactivation have been extensively covered in recent reviews [46-48], and we will instead focus on the possible roles of newly identified factors in XCI. Several other factors have been implicated in Xist-mediated silencing. Examples include the variant histone macroH2A [49], the chromosomal protein Smchd1 [50], the de novo DNA methyltransferase Dnmt3b [51], hnRNP U [39], the trithorax protein Ash2l [52], as well as HP1 [53] and Cdyl [54] proteins that are thought to bind directly to histone modifications enriched on Xi. There is evidence to suggest that most if not all of these factors function in maintenance rather than establishment of XCI. This conclusion is based in part on observations indicating a significant lag in the recruitment of these factors following the onset of Xist expression in an in vitro model system, differentiating XX ES cells [51, 52, 55-57]. Additionally, as mentioned above, none of these factors has a characterised RNA binding domain, with the exception of hnRNP U. Together, these considerations indicate that the critical RBPs required to initiate Xist-mediated silencing remain to be identified. Indeed, factors mediating well characterised Xi chromatin features, such as global histone hypoacetylation [58], depletion of histone H3K4 methylation [56] and of RNA polymerase II [59], are not known. These Xi features are established rapidly in response to the onset of Xist RNA expression [56, 57, 59], and as such may play a key role in initiating chromosome silencing.
hnRNP U hnRNP U is an approximately 800 aa protein with several conserved domains: an Nterminally located SAP domain, implicated in DNA binding, centrally located SPRY and ATP binding domains of unknown function, and an N-terminal RGG box linked to RNA binding (Figure 2). hnRNP U was identified in the proteomic screens [20-22], consistent with a previous study in which UV cross-linking analysis indicated a direct interaction between 9
hnRNP U and Xist RNA [39]. hnRNP U therefore served as a positive control that validated the proteomic screening strategies. Chu et al [20] used UV cross-linking RBP immunoprecipitation followed by qRT-PCR to determine binding sites for hnRNP U on Xist RNA. Binding was seen to occur broadly over much of Xist RNA. This is similar to prior findings from Hasegawa et al [39], although in this latter case there was evidence for preferred binding towards the 5’ end of the transcript. Genome wide analysis of hnRNP U binding has been determined using UV-cross-linking immunoprecipitation (CLiP) [60], although in cells that do not express Xist RNA. Further studies are therefore required to better define sites of interaction of hnRNP U with Xist RNA.
The genetic screen studies did not report hnRNP U amongst the top ranked hits (Figure 3). The reason for this is currently unclear. hnRNP U shRNAs were included in the library used by Moindrot et al [23], but none were enriched, indicating that knockdown may have been inefficient or alternatively, that knockdown had a strong effect on cell viability. The latter explanation seems unlikely given that other studies were successful in assaying Xist localisation following hnRNP U knockdown in ESCs. HnRNP U was not detected in Monfort et al either [24], maybe because the probability of having a mutational insertion in a small gene like hnRNP U (9kb) is relatively low. This lack of saturation of the screen may also explain why some other factors validated in the parallel studies, were not seen.
Knockdown of hnRNP U by both Chu et al [20] and McHugh et al [21], confirmed its important role in Xist RNA localisation. Specifically, Xist RNA showed a diffuse localisation in the nucleus, contrasting with the focal localisation observed in the parental cell lines. Perhaps surprisingly, validation of other factors found in the genetic and proteomic screens did not reveal detectable deficiencies in formation of Xist RNA domains, indicating that hnRNP U is likely to be the principal determinant for localising Xist RNA within the nuclear matrix 10
compartment. However, it cannot be ruled out that factors in the screens that were not validated contribute to this pathway. In this regard, it is interesting to note that Matrin3, another nuclear matrix protein which also binds RNA [61], was identified by Chu et al [20] and Moindrot et al [23]. The fact that McHugh et al [21] failed to find this protein using the high stringency UV cross-linking approach may indicate that it is an indirect interactor, perhaps functioning in cooperation with hnRNP U in defining the nuclear matrix environment in which Xist RNA is entrapped.
An unresolved issue regarding the role of hnRNP U in XCI is the paradoxical observation that enrichment of the protein within Xi domains is seen only late in the XCI cascade, as determined by analysis of differentiating XX ESCs [53]. It is not obvious how to reconcile this with the requirement for hnRNP U for Xist localisation at the onset of XCI in ES cell models [20, 21]. One possibility is that low levels of hnRNP U, below that required for detection by immunolocalisation but sufficient for Xist RNA localisation, interact with Xist RNA at the onset of XCI and that hnRNP U then accumulates in the Xi domain progressively thereafter.
Alternatively, it may be that a more general role of hnRNP U in nuclear
organisation is important for correct Xist RNA localisation and it is the disruption of this function in ES cells, rather than direct binding, that affects Xist domain formation.
hnRNP K hnRNP K is an approximately 450 aa protein that has three KH domains that mediate interaction with RNA (Figure 2) [62]. hnRNP K was identified by Chu et al [20] as one of two highly abundant proteins in the formaldehyde cross-linked Xist interactome, the other being hnRNP U. Like hnRNP U, UV cross-linking RBP immunoprecipitation followed by qRT-PCR suggested hnRNP K binds at similar levels across the Xist transcript. However, McHugh et al [21] did not identify hnRNP K using UV cross-linking mass spectrometry (Figure 3). 11
Further analysis, for example using CLiP, is therefore required both to confirm that hnRNP K interacts directly with Xist RNA, and to identify putative preferred binding sites. At present there are no published CLiP analyses of hnRNP K, although the protein is known to show a strong preference for cytosine rich RNA sequences, similar to the related proteins PCBP1/2 (reviewed in [63]).
Chu et al [20] tested the role of hnRNP K in Xist-mediated silencing in ESCs by assaying silencing of genes located in cis to an inducible Xist transgene. hnRNP K siRNA abrogated Xist-mediated silencing to a similar degree to that seen with hnRNP U knockdown. Moreover, it was found that Xist induced domains for the PRC1 and PRC2 mediated histone modifications H2AK119u1 and H3K27me3, were also reduced, suggesting a role for hnRNP K in chromatin modification on the Xi.
hnRNP K is found as a major component of nuclear matrix preparations [64], consistent with it having a role in Xist-mediated silencing. The protein has been suggested to act as a docking platform or hub for several complexes that mediate nucleic acid linked processes, notably gene activation and gene silencing, translation and RNA processing (reviewed in [65]). More recently hnRNP K has been implicated in coordinating transcriptional silencing by the histone modifying complex SETDB1 [66].
Whilst the findings of Chu et al [20] support hnRNP K direct role in Xist-mediated silencing, there are reasons to consider this with caution. As mentioned above, McHugh et al [21] did not identify hnRNP K as a direct Xist interactor, and neither of the genetic screening studies identified hnRNP K as a candidate silencing factor (Figure 3). The latter may indicate limitations in identifying silencing factors by genetic screening, but nevertheless needs to be taken into consideration. It should also be noted that hnRNP K is a common contaminant 12
in mass spectrometry experiments due to non-specific binding to agarose and magnetic beads used to purify protein complexes from extracts [67].
What then of the proposed role of hnRNP K in Xist-mediated chromatin modifications, notably recruitment of Polycomb repressors? As discussed above, Polycomb repressor complexes are implicated in maintenance rather that establishment of gene silencing in XCI, and as such hnRNP K may play a role in their recruitment regardless of its role in gene silencing in cis. Indeed, there is some evidence that hnRNP K can interact with the PRC2 protein EED [68]. However, it is important to consider the converse argument, that hnRNP K affects gene silencing, either directly or indirectly, and that this in turn reduces establishment of Polycomb mediated chromatin modifications, due to antagonism by chromatin modifications associated with gene activation (reviewed in [69]).
A similar
argument can be applied to Rbm15 and Spen, two other candidate factors identified in the genetic and proteomic screens, that were also found to have a role in Polycomb recruitment (see below). Mapping interaction sites for these factors in relation to elements implicated in silencing versus Polycomb recruitment should help to resolve this issue.
In relation to Polycomb recruitment by Xist RNA it should be noted that none of the proteomic studies identified core proteins uniquely found in PRC2 complexes, arguing against the prevailing model that PRC2 interacts directly with Xist RNA. Minajigi et al [22], did report the RbAp46/48 PRC2 subunit as a direct interactor, but this protein is also found in several other chromatin modification complexes, and in itself cannot be considered as representative of PRC2. PRC1 proteins on the other hand were identified by Chu et al [20] (and Minijigi et al [22]), but not by McHugh et al [21], indicating that they are recruited to Xist RNA either directly or via an RBP co-factor. Moindrot et al [23] also identified PRC1 proteins in their genetic screen, albeit with a relatively low ranking, whilst Montfort et al [24] identified 13
the PRC2 protein Eed. The fact that Polycomb is not sufficient for establishment of silencing by Xist RNA (see above), indicates that these findings are most likely due to indirect genetic interactions. Thus, in summary, whilst hnRNP K is an important candidate protein, further experiments are required to confirm its function both in Xist-mediated silencing and in chromatin modification by Polycomb repressor proteins.
SHARP/SPEN/MINT SPEN is an exceptionally large protein, approximately 3500 aa, and is the founding member of the Split-End family of proteins, first discovered in drosophila [70]. Members of this family are characterized by the presence of 3/4 RNA Recognition Motifs (RRMs) at the N-terminal and a SPEN paralog and ortholog conserved (SPOC) domain at the C-terminus (Figure 2). SPEN is also known as SMRT/HDAC1 Associated Repressor Protein (SHARP) in human, and Msx2-Interacting Nuclear Target (MINT) in mouse.
SPEN was identified and validated as a high confidence hit in the proteomic [20-22] and genetic [23, 24] screens (Figure 3). In the work of Chu et al, SPEN was one of only three factors found to be enriched only in the presence of the repeat A, suggesting that it may bind to this region [20]. McHugh et al, using the UV crosslinking approach, identified SPEN as the factor most highly enriched for Xist compared to control RNAs, further indicating that SPEN directly interacts with Xist RNA [21]. Moreover, Moindrot et al [23], using superresolution 3D-SIM, demonstrated that SPEN co-localizes with Xist RNA in the interchromatin compartment, consisting of a network of channels relatively devoid of DNA [71]. All four studies validated SPEN extensively, demonstrating that loss of function interferes with Xistmediated silencing but has no effect on Xist RNA localisation/domain formation (Figure 3). Given the large size of SPEN, it is possible to envisage that the protein bridges Xist
14
RNA/nuclear matrix and the underlying chromatin by binding via its N-terminal RRMs and C-terminal SPOC domain respectively.
SPEN, a well characterised RBP, is known to bind to the SRA transcript (steroid receptor RNA activator), and this interaction has been explored in detail [72]. The RNA recognition region of SPEN is organized into two modules. The first comprises the RRM3 and RRM4 domains, which both engage with the H12-H13 fragment of the SRA transcript. The second module comprises the RRM2 domain that is not essential for interacting with the SRA RNA and is connected to the RRM3-RRM4 module by a short linker. In-vitro binding assays have shown that the association between SPEN and the SRA transcript involves both single stranded sequences and paired nucleotides, as can be found in stem-loop structures [72]. A similar docking platform might be required for SPEN binding to Xist RNA. Indeed, based on the findings of Chu et al [20], Spen most likely recognizes the repeat A region, which was previously suggested to adopt a structured stem-loop architecture [10]. This conclusion is further supported by EMSA assays showing that a purified fragment containing SPEN RRMs 2-3-4 binds to in-vitro transcribed repeat A motifs [24].
How might SPEN mediate transcriptional silencing? In mammals, SPEN has been shown to have essential roles in transcriptional repression [73-75]. In particular, SPEN is able to recruit transcriptional repressors, such as the silencing mediator for retinoid and thyroid receptor SMRT/nuclear receptor co-repressor (NCoR) [76-79], which are components of histone deacetylase (HDAC) complexes. The interaction between SPEN and SMRT is mediated by the SPOC domain, which recognises the serine-phosphorylated C-terminus of SMRT [79]. In addition, the SPOC domain of SPEN has also been shown to recruit HDAC1 and HDAC2 directly [76]. The ability of SPEN to recruit the SMRT/NCoR complex and thence HDAC activities may be critical for initiating the silencing cascade in the context of XCI. 15
McHugh et al [21] indeed demonstrated that knock-down of SMRT or HDAC3 mimics the silencing defects observed in the absence of SPEN. It is however notable that neither HDAC1/2/3 nor NCoR1/2 were identified in the genetic screens [23, 24]. Moreover, global histone hypoacetylation on Xi has previously been shown to occur independently of the repeat A element [52], arguing that the function of SPEN in gene repression may be more complex.
Further studies are required to clarify whether the HDAC complexes are
exclusively recruited by SPEN, and whether this constitutes the sole link between SPEN and transcriptional silencing.
SPEN may have a role in the recruitment of PRC1 and PRC2 complexes, either directly or indirectly, since McHugh et al [21] and Monfort et al [24] report reduced enrichment of PRC2 (Ezh2, Eed) and PRC1 (Ring1b), on Xi in the absence of SPEN. In contrast, H3K27me3 deposition was largely unaffected by SPEN knock-down in the study by Moindrot et al [23]. This apparent contradiction may stem from a threshold effect in which reduced levels of the complex are nevertheless sufficient to efficiently deposit the H3K27me3 modification. Regardless, it is unlikely that SPEN recruits Polycomb complexes directly as the critical region of Xist required for PRC2 recruitment is distinct from the repeat A region with which SPEN is proposed to interact. As noted above, differences in recruitment of Polycomb complexes could be a secondary consequence of ameliorated silencing due to antagonism by transcription-linked chromatin modifications.
A previous study, in which the SPEN gene was knocked out, demonstrated mouse embryo lethality around embryonic day (E)12.5 [73]. There was no evidence for preferential loss of female homozygous embryos, although the number of embryos analysed at earlier stages was insufficient to rule this out entirely.
This observation is perhaps surprising given the
critical role for SPEN in gene silencing by Xist RNA. By way of comparison, heterozygous 16
female embryos carrying a paternally inherited mutant Xist allele die early during embryonic development and exhibit growth defects from E6.5 [7]. One possible explanation for this disparity is that the SPEN knockout allele results in only partial loss of function: exon skipping, for example, may allow generation of a truncated but functional protein. Alternatively, there may be other proteins that function redundantly with SPEN in Xistmediated silencing, for example Rbm15.
RBM15 RBM15 is a homologue of SPEN that has three N-terminal RRM domains and a C-terminal SPOC domain (Figure 2). The protein is somewhat smaller than SPEN, approximately 900 aa, reflecting a much smaller spacer region between the RRMs and the SPOC domain. The RBM15 ortholog in human, sometimes named OTT1, is involved in a specific t(1;22) translocation
event
creating
the
RBM15-MKL1
fusion
associated
with
acute
megakaryoblastic leukaemia [80, 81].
Rbm15 was identified as an Xist interactor in proteomic [20-22] and genetic [23] screens (Figure 3). Detection of Rbm15 by in-vivo UV crosslinking indicates that it binds directly to Xist RNA, and indeed RBM15 was the second most highly enriched protein after SPEN [21]. Chu et al [20], also identified Rbm15 using the formaldehyde cross-link approach, but in contrast to SPEN, this association was not repeat A dependent. Moindrot et al found that Rbm15 is required for Xist-mediated silencing, with no obvious requirement for Xist localisation/domain formation [23]. Conversely, Rbm15 was not identified in the genetic screen of Montfort et al [24], and validation experiments by McHugh et al [21] found no effect of Rbm15 knockdown on Xist mediated silencing.
The reasons for these apparent
discrepancies are unclear. In the case of the Montfort et al screen [24], there may have been insufficient depth of coverage to detect Rbm15 (there was a twofold increase in Rbm15 17
mutations relative to uninduced control but this falls below the set threshold). Differences in the findings for Rbm15 by Moindrot et al [23] and McHugh et al [21] may reflect differences in the silencing assays that were employed.
Notably, Moindrot et al assessed silencing
directly by scoring nascent RNA foci for genes located in cis with Xist compared to control genes, a methodology widely employed in XCI studies. McHugh et al on the other hand employed a novel single mRNA FISH method which is less direct, and could, for example, be influenced by changes in levels of a given mRNA due to enhanced expression from the non-Xist associated allele.
Moindrot et al [23] reported a deficiency in deposition of H3K27me3 by PRC2 polycomb complexes following Rbm15 knockdown.
Rbm15 binding to Xist RNA apparently occurs
independent of the repeat A [20], and it is therefore conceivable that it interacts with the Xist sequence elements required for PRC2 recruitment (Figure 1). However, as argued above for SPEN, Rbm15 may instead affect Polycomb recruitment indirectly, and further studies are required to elucidate this point.
What role might RBM15 play in Xist mediated silencing? In mammals, RBM15 has been linked to two different functions: transcription regulation and mRNA metabolism. RBM15 transcriptional functions have mainly been characterized in the context of the fusion RBM15MKL1 associated with acute megakaryoblastic leukemia. This fusion protein can mediate gene activation, especially of Notch signalling target genes [82-84], possibly by the recruitment of the H3K4 methyltransferase Setd1b via the SPOC domain of the RBM15MKL1 fusion [85]. However, the endogenous RBM15 may also be associated with gene repression. Supporting this idea, endogenous Rbm15 but not the RBM15-MKL1 fusion, can interact with HDAC3 [83], a component of the NCoR/SMRT corepressor complex. In addition, gene expression analysis in embryos homozygous for RBM15XK135 (hypomorphic 18
low-expressed allele of RBM15 resulting from the insertion of LacZ-Neo cassette 20 nt upstream the initiator codon) revealed specific upregulation of transcripts that are normally expressed at low levels in wild-type embryos [86].
In addition to its role in repressing transcription, RBM15 has also been implicated in mRNA metabolism, mainly via its interaction with the RNA export factors Nxf1 and Nxt1, which are responsible for nuclear export of the majority of mRNAs [86, 87], and via its interaction with Dbp5, an ATPase docked at the nuclear pore [88]. Accordingly, the artificial tethering of RBM15 on reporter mRNA promotes nuclear export [86, 87, 89]. Again, the C-terminal SPOC domain of RBM15 appears to be required for the interaction with mRNA export factors [86, 87]. Interestingly, the role of RBM15 might be more complex than simply promoting nuclear export. Indeed, it has been reported that RBM15 can retain some mRNAs, instead of promoting their nuclear export, as exemplified by the nuclear accumulation of hyperpolyadenylated ORF59 RNA, a viral RNA coding for a DNA polymerase [90, 91]. In addition, RBM15b, a homolog of RBM15, has been shown to prevent the cytoplasmic export of an unspliced transcript generated from a reporter gene containing a cryptic splice site [92]. These two observations raise the possibility that RBM15 could be involved in RNA surveillance, selecting mRNAs that qualify for nuclear export, and consequently modulating gene expression at the post-transcriptional level.
While Rbm15 knock-down has no obvious effect on nuclear localisation of Xist, the link to regulation of RNA export gains importance in light of the finding that RNA export factors, notably Nxt1 but also Nxt2 and Nxf1, were identified by Moindrot et al in their genetic screen [23]. Further validation of Nxt1 was not possible as cell viability was strongly affected following gene knockdown. Proteomic screens by Chu et al [20] and McHugh et al [21] did not identify RNA export factors as being enriched, although this could reflect equivalent 19
binding to control RNAs. These considerations highlight some of the limitations of genetic and proteomic screening respectively. Regardless, further studies are required to determine the contribution of nuclear export and gene repression functions of RBM15 in the context of Xist-mediated silencing.
Intriguingly, Spenito (nito), a drosophila homolog of RBM15, regulates the alternative splicing of Sex-lethal gene (Sxl), the master regulator of sex determination and dosage compensation in fly [93]. In addition, FPA, a homolog of RBM15 in A. thaliana, represses the expression of FLC, a gene required for controlling flowering time. The molecular mechanisms by which FPA regulates FLC expression have been investigated [94]. FPA appears to control the 3’-end cleavage and polyadenylation of an antisense transcript, which spans the FLC coding sequence and affects the rate of FLC sense transcription. Importantly, the RNA processing functions played by FPA may not be restricted to FLC transcript. Indeed, in the absence of FPA, several intergenic genomic segments became expressed, generally because of the alternative splicing or the alternative polyadenylation of adjacent transcripts [95]. This underlines the global role of the Rbm15 homolog in transcription regulation. These observations in drosophila and plants shed light on an intriguing parallel between RBM15 function in XCI on the one hand, and Spenito or FPA functions in sexual reproduction on the other, raising the possibility that Split-End proteins have evolved to modulate gender specific functions and reproduction.
Gene knockout of Rbm15 results in embryo lethality at E9.5-10.5 [96], somewhat earlier than observed for the SPEN knockout (E12.5) [73]. Similarly, mice homozygous for the poorly expressed hypomeorphic allele RBM15XK135 die during embryonic development by day E12.5, confirming the importance of RBM15 for mouse development [86]. No femalespecific phenotypes were reported in these studies. To gain insights into the developmental 20
function of RBM15, Raffel et al. made use of a Sox2-CRE strain to delete RBM15 in epiblast derived cells [97]. RBM15 null embryos were then identified throughout gestation at near the expected Mendelian frequencies and pups died only soon after birth [97]. At first glance, this observation seems to argue against an important role for RBM15 in XCI. However, in this experimental set-up, the Sox2-CRE deletes Rbm15 at E6.5 [98, 99], after establishment of both imprinted and random XCI [44]. Additionally, as argued in the case of SPEN knockout mice, redundancy with other SPOC domain proteins may account for the absence of femalespecific effects in single gene knockouts. The Rbm15 homologue, Rbm15b, may contribute functionally in this regard.
WTAP WTAP is a 400 aa protein that was first identified as an associated partner of Wilms’ tumor 1 protein in a yeast double-hybrid screen, and named thereafter as Wilms’ tumor 1associating protein [100]. Unlike SPEN and RBM15, WTAP protein does not contain annotated RNA-binding domains. However it has been implicated in the regulation of mRNA biogenesis, and moreover has been described as a regulatory component of the RNA methyltransferase complex, comprising WTAP, virilizer and the catalytic subunits METTL3 and METTL14, that together mediate formation of N6-methyladenosine (m6A) on RNA [101103] (see below). Using formaldehyde cross-linking, Chu et al [20] identified WTAP as an Xist-interacting protein and moreover as one of the three factors for which Xist association is dependent on the repeat A (Figure 3). WTAP was not identified using the UV cross-linking approach [21, 22], indicating that the protein does not interact with Xist RNA directly (Figure 3A). This could reflect association of WTAP with an enzymatic complex that interacts only transiently with substrate mRNAs.
Indeed Moindrot et al [23], identified WTAP and also
virilizer, a second regulatory subunit of the m6A methyltransferase complex, as high confidence hits in their genetic screen.
It is interesting to note that WTAP has been 21
reported to interact with both SPEN and RBM15 [104, 105], indicating a possible biochemical link between different factors implicated in Xist-mediated silencing. The second genetic screen, performed in haploid ES cells, did not identify WTAP [24], possibly reflecting limited coverage of this screen as discussed above.
Validation experiments have shown that, although WTAP knockdown does not affect Xist domain formation or H3K27me3 deposition, it noticeably impairs the silencing of the chromosome [23]. However, Chu et al [20] reached the opposite conclusion as no silencing defects were observed in differentiating ES cells treated with a WTAP siRNA (Figure 3B). Although the reasons for this discrepancy remain to be fully understood, it should be noted that silencing defects reported by Moindrot et al [23] in WTAP knockdown differentiated ES cells are milder than after RBM15 or SPEN knockdown.
How might WTAP participate in Xist mediated silencing? WTAP has been linked to different functions associated with transcript maturation and stability. For instance, WTAP prevents the degradation of cyclin A2 mRNA by stabilizing the transcript via a small RNA motif found in the 3’-UTR, and thereby regulates the G2/M transition [106]. WTAP is also found associated with splicing factors, within the spliceosome [107], or within interchromatin granule clusters where numerous splicing factors assemble [108]. Interestingly, WTAP can modulate the alternative splicing of its own transcript and thereby limit the synthesis of WTAP protein [104], which demonstrates that WTAP can negatively regulate gene expression at the post- or co-transcriptional level.
As mentioned above, WTAP is also a regulatory component of the RNA methyltransferase complex. It is currently unclear whether WTAP functions only in the context of the m6A complex or can also operate independently in aspects of mRNA regulation. The m6A 22
modification has been identified in many organisms, including mammals, plants and yeast, and is the most prevalent internal modification of mRNA and ncRNA. It is distributed throughout the transcript and enriched within the last exon of mammalian mRNAs [109-111], and has recently been associated with post-transcriptional gene regulation (reviewed in [112]). In short, m6A modification acts as a negative regulator of gene expression, probably by modulating the transcript stability since the depletion of the methyltransferase complex increases the quantity of the targeted m6A transcripts [101, 113-116]. This modulation of transcript stability may be mediated by a known m6A reader, YTHDF, which can target m6A modified transcripts to RNA decay bodies [113]. There is also evidence suggesting that m6A functions by modulating RNA structure and thereby facilitating binding of other RBPs during mRNA biogenesis, exemplified by studies on hnRNP C binding to mRNA [117, 118]. Indeed it is plausible that m6A function in regulating mRNA stability is a consequence of structural switches that modulate RBP binding.
Whether or not the m6A modification is important for Xist-mediated silencing remains an open question. It has been reported that human Xist RNA is methylated in exon 6 [109]. However, this analysis was performed in a male cancer cell line, HepG2, precluding any conclusion about where and when Xist RNA may get methylated during XCI. Two scenarios can nevertheless be considered. In the first one, the methylation status of Xist RNA would be modified at the onset of the XCI, which may in turn affect its physical properties (turnover, conformation etc.), and thereby the cis-silencing.
In the second scenario, the RNA
methylation complex would be attracted by Xist RNA to reinforce mRNA methylation of Xlinked gene products at the onset of XCI, and thereby mediate their degradation. Both possibilities would explain why WTAP knockdown alters Xist-mediated silencing. An important consideration is that Xist RNA localisation is not obviously affected by WTAP knockdown, precluding a major Xist stabilization defect, and also that METTL3 enzyme was 23
not identified in the genetic screen [23] (no shRNAs targeting METTL14 were included in the library used in this study).
WTAP knockout in mouse is embryonic lethal [106]. Developmental defects were reported from E6.5, with embryos lacking the characteristic morphology of endoderm and ectoderm at E8.5. So far, these observations were interpreted in the context of WTAP regulating the cyclin A2 transcript, rather than in the context of m6A modification. Similar to SPEN and RBM15 knockouts, no female-specific phenotypes were reported in this study, suggesting that at least partial dosage compensation can occur in the absence of WTAP. Of note, the homolog of WTAP in drosophila, fl(2)d, is implicated in sex determination and dosage compensation by controlling the splicing of Sxl transcript [119], suggesting that the molecular mechanisms regulating the equalization of gene expression between males and females might be, at least partially, evolutionary conserved.
LBR LBR (Lamin B receptor) is an approximately 600 aa protein, which lies embedded in the inner membrane of the nuclear envelope and was originally discovered as a B-type Lamin binding protein [120]. The LBR C-terminus is comprised of transmembrane domains, while the N-terminal region faces the nucleoplasm [121] (Figure 2). Heterozygous mutations in LBR are associated with the Pelger-Huet anomaly, a disorder in which neutrophil nuclei are hyposegmented and exhibit an atypical heterochromatin redistribution [122], underlying the role played by LBR in determining nuclear shape and tethering peripheral heterochromatin.
LBR, which does not contain a classical RNA recognition motif, was identified as a direct Xist RNA binder by McHugh et al [21] but not by Chu et al [20] (Figure 3A). Xist recognition by LBR might be mediated by an RS domain (arginine and serine) that has been reported 24
to have an RNA binding activity [123]. Validation experiments performed by McHugh et al [21] found that LBR depletion does not severely affect Xist RNA localisation/domain formation, but impairs Xist-mediated silencing, including in differentiating female ES cells, indicating that LBR intervenes in the downstream silencing cascade. An important feature of XCI is the exclusion of RNA polymerase II from the Xi territory, and gene internalization within the Xist domain [59]. Interestingly, LBR was found to be dispensable for the exclusion of RNA Polymerase II, and yet, the silencing of the chromosome is altered. This phenotype echoes what is observed with Xist RNA lacking the repeat A, suggesting that LBR does not repel the transcription machinery but instead, like the repeat A, could be implicated in the relocation of gene bodies within the Xi territory [21, 59].
It is interesting to note that LBR has also been linked to transcriptional silencing, for example via interaction with the transcriptional repressor HP1 [124, 125]. However, this interaction may be indirect and mediated though the core histones [126]. Accordingly, pull-down of LBR enriches for chromatin with repressive histone marks, such as H3K9me3 or H3K27me3 [127]. Importantly, LBR may actively contribute to transcriptional silencing. For instance, the N-terminal domain of LBR can induce the compaction of an in vitro reconstituted nucleofilament. This compaction can’t be explained solely by LBRs ability to self-associate since the same experiment performed with nucleosomes lacking the histone tails allows LBR binding but does not induce the compaction of the nucleofilament [128]. In addition, LBR can repress a luciferase reporter, whereas a mutant lacking the Tudor domain can’t [128]. Furthermore, the ectopic expression of LBR in olfactory sensory neurons, which normally do not express LBR, induces the overall silencing of olfactory receptor genes without affecting the global transcriptome, and impairs aggregation of olfactory receptor genes near the centre of the nucleus [129]. Taken together, these observations indicate that LBR can
25
mediate transcriptional silencing, most probably indirectly, by inducing compaction and/or tethering the chromatin in a repressive compartment.
How chromatin tethering at the nuclear periphery might affect XCI remains an open question. Because the Xi is often found near to the nuclear envelope [130], an attractive hypothesis would be that the Xi, once localised to the nuclear envelope, is then locked into this repressive compartment by LBR, thereby reinforcing transcriptional silencing. However, female-specific defects associated with impaired XCI have not been reported in two different mouse strains carrying mutations in Lbr gene [131, 132], indicating that XCI can occur in the absence of Lbr. Moreover, as already mentioned, LBR was not identified by Chu et al [20], nor in the two genetic screens performed in ES cells [23, 24] (Figure 3). Further studies would therefore be required to rationalise these observations in the context of the proposed role of LBR as a primary silencing factor in XCI.
Concluding remarks The recent studies discussed in this review have identified key factors involved in Xistmediated silencing. A synthesis of the different studies highlights factors implicated in transcriptional silencing through anchoring chromatin, chromatin modification, and posttranscriptional regulation (Figure 4). There is a broad agreement that SPEN plays a central and critical role in silencing, likely by binding to the repeat A sequence and recruiting NCoR/HDAC3 corepressors. Some details nevertheless remain to be confirmed. Other key factors that have emerged as strong candidates for Xist-mediated silencing are hnRNP K, Rbm15, Wtap and LBR. In these cases supporting evidence is less unanimous and further work is required to confirm involvement and elucidate their molecular mechanisms of action.
26
None of the novel factors significantly affect Xist RNA localisation, although the key role of hnRNP U in this process is confirmed.
Given that the recent studies have harnessed the power of both proteomic and genetic screening, it seems likely that we now know the identity of most of the factors required to establish Xist-mediated silencing. It should be noted that some candidates identified in one or more screen were not discussed here. These include several factors linked to mRNA biogenesis, for example Ptbp1/2, RALY, and CELF1. Indeed, Ptbp1, which has been shown to function in regulation of splicing (reviewed in [133]), was identified in McHugh et al [21] and Moindrot et al [23], and in both studies knockdown affected Xist-mediated silencing, at least to some degree. Conversely, some of the Xist interacting partners identified in the proteomics screen that are not required for gene-silencing, could contribute to other aspects of X-chromosome inactivation. RNF20/40, identified by Chu et al [20] as the third factor for which enrichment is dependent on the repeat A (Figure 3A), might fall into this category since it was found not to be required for silencing activity.
Further analysis of the novel factors should provide insights into long standing questions in the field. First, what is the basis of the developmental window of opportunity? Most of the key factors identified as having a role in establishing silencing were also seen in analysis of the Xist interactome in XX somatic cells [22] (Figure 3), suggesting that loss of Xist responsiveness may not be attributable simply to absence of key silencing factors. It is however plausible that developmentally regulated modification of these factors or regulation of absolute levels of the silencing factors, underlies Xist responsiveness. A second key question is to understand the link between primary silencing factors and maintenance factors. Association of factors such as macroH2A and Smchd1 is Xist dependent, yet Xi localisation occurs with a lag of several days following the onset of Xist expression [51, 55]. 27
A further question is to understand the link between primary silencing factors and chromatin modifications on Xi. As discussed above, features such as global hypoacetylation, loss of H3K4 methylation and depletion of RNA Polymerase II all appear to be established independently of the repeat A, at least in part [53, 59]. Additionally, several studies have shown that Xist mediated silencing occurs more efficiently on the X chromosome than on autosomes, an effect that may be linked to enrichment of LINE1 repeats on the X chromosome (reviewed in [134]). What is it about the function of primary silencing factors that limits their ability to repress autosomes compared to the X chromosome? Finally, how did Xist-mediated silencing evolve? Recent studies have demonstrated that marsupial mammals evolved a distinct lncRNA, Rsx, for dosage compensation of the X chromosome [135]. Do the factors found to mediate silencing by Xist RNA also play a role in Rsx mediated silencing, and are these factors utilised either in RNA mediated regulation of genes/gene clusters or in genome defence mechanisms, for example in relation to viruses and transposable elements? Thus, whilst we may have reached the end of the beginning, we certainly have not yet reached the beginning of the end.
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Figure legends
Figure 1: Structure of mouse Xist gene and mRNA. The Xist gene is composed of 8 exons. The processed RNA has several tandem repeat regions, named A to F. The critical regions for Xist function are indicated above and below the Xist transcript diagram. The asterisk denotes a small insertion downstream of the repeat A that partially disrupts Xist-mediated silencing [27].
Figure 2: Schematic drawing of HNRNPK, LBR, HNRNPU, RBM and SPEN. Known RNA binding domains are shown in orange. Wtap has no annotated functional domains. AAA: ATPases Associated domain; KH-1: K Homology domain 1; KOKNT: N-terminus of RNP K-like proteins; RID: Receptor Interaction Domain; RRM: RNA Recognition Motif; SPOC: Spen paralogue and orthologue SPOC, C-terminal domain; SPRY: SPla and the RYanodine Receptor domain; SR: Serine/Arginine-rich domain.
Figure 3: Comparison of the proteomic and genetic screen hits. A- Overlap between the Xist RNA binding proteins identified by McHugh et al [21], Chu et al [20] and Minajigi et al [22]. Using UV cross-linking in ES cells (McHugh et al [21]) or in somatic XX cells (Minajigi et al [22]), only direct Xist binders should be identified, whereas the formaldehyde cross-link used by Chu et al in stem cells [20] should detect direct and indirect interactors. For this analysis, only the candidates from Minajigi et al [22] having a ≥ twofold enrichment over the background were selected. SPEN, WTAP and RNF20, shown in ochre, are the only three proteins which depends on the repeat A for interacting with Xist RNA. WTAP and RNF20 were only detected by Chu et al [20]. B- Factors required for Xist-mediated silencing which have been validated by at least one of the two proteomic screens [20,21] or one of the two genetic screens [23,24]. All four studies 40
validated that SPEN knock-down or knock-out impair Xist-mediated silencing. A comparison is made for the other candidates. None of the candidates reviewed here were validated by Minajigi et al [22].
Figure 4: Possible implication of HNRNPU, LBR, SPEN, HNRNPK, RBM15 and WTAP in Xist-mediated silencing. A- We propose that HNRNPUand LBR are implicated in the silencing by anchoring Xist RNA to the nuclear matrix (HNRNPU) or to the nuclear envelope (LBR). The anchoring impacts on the transcription by an unknown mechanism. B- We propose that SPEN and HNRNPK are linked to transcriptional silencing by recruiting histone modifying complexes. SPEN can interact with SMRT/NCOR and might thereby direct HDAC3 to the chromatin. SPEN and HNRNPK are also involved in Polycomb recruitment. The deacetylation of histone tails combined with H3K27 trimethylation may then reduce transcription. C- RBM15 and WTAP may be implicated in the silencing at the post-transcriptional level. WTAP might impact on the stability and/or conformation of either Xist RNA or X-linked gene product via m6A. RBM15 could block the export of X-linked gene products at the onset of XCI or affect Xist RNA directly. Of note, RBM15 also seems to be implicated in the recruitment of Polycomb complex.
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S1084-9521(16)30029-5 http://dx.doi.org/doi:10.1016/j.semcdb.2016.01.029 YSCDB 1940
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
19-10-2015 20-1-2016 21-1-2016
Please cite this article as: Moindrot Benoit, Brockdorff Neil.RNA binding proteins implicated in Xist-mediated chromosome silencing.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.01.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
RNA binding proteins implicated in Xist-mediated chromosome silencing Benoit Moindrot and Neil Brockdorff Dept of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK email; [email protected]
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Abstract Chromosome silencing by Xist RNA occurs in two steps; localisation in cis within the nuclear matrix to form a domain that corresponds to the territory of the inactive X chromosome elect, and transduction of silencing signals from Xist RNA to the underlying chromatin. Key factors that mediate these processes have been identified in a series of recent studies that harnessed comprehensive proteomic or genetic screening strategies. In this review we discuss these findings in light of prior knowledge both of Xist-mediated silencing and known functions/properties of the novel factors.
Key words X chromosome inactivation Xist Chromatin non-coding RNA Epigenetic
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Introduction The X inactive specific transcript (Xist) gene was first identified as a candidate for the master switch locus required in cis for the process of X chromosome inactivation (XCI) [1-3]. Xist was shown to be a 15-17kb long non-coding (lnc) RNA that is retained in the nucleus, coating the inactive X chromosome (Xi) territory [4, 5]. Gene knockout and transgene studies demonstrated that Xist is both necessary and sufficient to establish chromosome silencing [6-9].
Key questions arising from these findings are what are the mechanisms for
developmental regulation of Xist expression that underpins establishment of appropriate XCI patterns in male and female embryos, and what is the basis for chromosome silencing by Xist RNA? Analysis of Xist RNA deletions indicates that the latter question can be divided into two parts, mechanisms that confer Xist RNA localisation in cis within the Xi territory, and mechanisms driving Xist RNA mediated chromatin modification and gene silencing [10]. Whilst our understanding of Xist gene regulation has advanced significantly (see review by ... in this issue), mechanisms governing Xist RNA localisation and establishment of chromosome silencing remain poorly characterised.
An early study estimated that there are around 1000 Xist RNA molecules/cell [11], and it was also noted that levels vary considerably between different mouse strains [2]. More recent analysis using 3D-SIM super-resolution microscopy determined that Xist RNA localises to around 200 resolvable foci, likely corresponding to individual molecules, within the Xi (Xi) territory [12]. Similar estimates were obtained in a subsequent study using PALM/STORM– super resolution microscopy [13]. These observations indicate a ratio of one Xist RNA molecule for every 1Mb of DNA on the X chromosome, corresponding on average to around 5 genes.
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Xist RNA is tightly bound up within the insoluble nuclear matrix [14], and this has hampered efforts to apply biochemical approaches to identify the critical RNA binding proteins (RBPs) that interact directly with Xist RNA. A second factor that has limited progress towards identifying key factors in Xist-mediated silencing is that Xist function is normally limited to a window of opportunity during early development [15]. It follows that efforts to identify and validate candidate Xist interactors need ideally to be performed in Xist responsive cell types, for example embryonic stem (ES) cells, rather than in somatic cell lines. A further consideration is that multiple redundant pathways maintain XCI in somatic cells, and ongoing Xist expression is not required for maintenance of silencing [16-18].
It should be
noted that Xist RNA localisation/domain formation occurs normally when Xist is induced in differentiated cell types [15, 19], indicating that, unlike silencing, this process is not restricted to early developmental stages.
A recent series of studies that utilised either proteomic [20-22] or genetic [23, 24] screening strategies have led to a significant advance in the identification of key RBPs that interact with Xist RNA. Several, but not all, of the factors were identified independently in more than one study, lending added confidence to their candidacy as critical factors acting in the XCI pathway.
The proteomic screens used Xist antisense oligonucleotides to capture Xist RNA/protein complexes from soluble cell extracts. Chu et al [20] performed their analysis following formaldehyde cross-linking, which covalently links nucleic acid -protein interactions and protein-protein interactions, and therefore identifies factors that bind Xist RNA either directly or indirectly. Mass spectrometry data were filtered by comparing results obtained for Xist RNA and other nuclear non-coding (nc) RNAs, and further by comparing data from different cell types, including ES cells, providing a list of 81 candidate interactors. A further 4
comparison of full length Xist RNA with Xist RNA deleted for the repeat A element, a region required for chromosome silencing (see below), highlighted three key factors (SPEN, WTAP and RNF20). The study by McHugh et al [21], in contrast, used UV cross-linking in ES cells, and thereby identified only candidate direct interactors/RBPs.
Again mass spectrometry
data were filtered by comparing results obtained for other nuclear ncRNAs, resulting in the identification of ten candidate Xist interactors, all except one of which had protein domains commonly found in classical RBPs. Minajigi et al [22], also used UV cross-linking but their experiments were performed in a somatic cell line. They identified more than 200 proteins as direct Xist interactors, the majority of which did not have domains commonly found in RBPs.
Validation experiments focused on the role of factors identified in maintenance of
XCI, specifically on the role of cohesin complexes in topological organisation of the Xi in somatic cells, which is discussed in detail in another review in this series (see review by....in this issue).
In the genetic screens, Moindrot et al [23] set out to identify the primary factors involved in Xist localisation/silencing by using a reporter ES cell line with a GFP ORF knocked-in in cis with an inducible Xist transgene. The reporter line was screened with a pooled shRNA library targeting each of 5,000 nuclear factors with at least 9 independent hairpins. A ranking list of around 250 genes was determined based on enrichment in GFP-high cells and number of independent hairpins for which there was significant enrichment. Within the top ranked hits they identified multiple subunits of known complexes, including those linked to RNA and chromatin silencing. Monfort et al [24] used a different strategy, namely insertional mutagenesis in haploid ES cells in which inducible Xist RNA was engineered into the single X chromosome to screen for Xist silencing/localisation factors. Silencing of the single X chromosome in the reporter cell line results in cell lethality and it was therefore possible to screen for mutagenesis events that allowed cells to survive following Xist induction. The 5
frequency of mutagenesis events at individual loci was determined for induced and noninduced cells in order to identify candidate silencing factors.
Proteomic and genetic screens each have different benefits and limitations. Thus, for example, proteomic screens can identify direct interactors, but may miss factors with low abundance or for which there is a paucity of peptides suitable for detection in mass spectrometry. Additionally, the filtering process discards factors that bind equally well to Xist and control ncRNAs, some of which could have important functions in Xist-mediated silencing.
Genetic screens on the other hand have the advantage of potentially defining
multiple factors within an RBP complex, reinforcing the verity of the observation. Genetics can also identify factors that function more indirectly in the pathway of interest. Limitations on the other hand include not detecting factors because of functional redundancy or because loss of function is detrimental to cell survival, independent of the role of the factor in XCI. With these considerations in mind, in this review we compare and contrast the findings of the proteomic and genetic screening studies. Additionally, we examine how prior knowledge of the function of the key candidate RBPs identified in these studies provides clues as to their possible role in Xist localisation and/or chromosome silencing.
State of the Art Before discussing the recently discovered candidate silencing factors we will provide an overview of prior knowledge relevant to understanding establishment of XCI by Xist RNA. Xist is a large molecule, 15-17kb, and therefore has the capacity to interact with many factors simultaneously.
As illustrated in Figure 1, there are several tandemly arranged simple
repeats, labelled A-F, within the Xist sequence. Some of these repeats, and also some unique sequence regions, show a relatively high degree of sequence conservation [4, 5, 25, 26]. Key functional elements within Xist RNA have been defined in genetic experiments 6
(Figure 1).
The repeat A element at the 5’ end of the transcript, is uniquely required for
Xist mediated silencing, as determined by analysis of cell lethality in XY ES cells expressing Xist RNA transgenes with deletions of overlapping regions [10]. This same study further concluded that several elements, contribute in a redundant fashion to Xist RNA localisation, referring to the ability of the RNA to form a coherent cloud or domain in interphase nuclei following transgene induction. There is evidence pointing to a greater complexity. A small insertion downstream of the repeat A was found to disrupt Xist-mediated silencing, at least in part [27], and a distinct element, also in Xist exon 1, has been found to be critical for recruitment of the Polycomb Repressive complex 2 (PRC2), one of the known silencing factors that is recruited in an Xist dependent manner [28]. Additional Xist localisation elements, for example the repeat C, have been defined in studies that disrupt Xist localisation using short targeted LNA or PNA probes [29, 30], and using gene knockout analysis [31].
Developmental context is a further important consideration. The window of opportunity for Xist-mediated silencing (but not localisation) points to the involvement of factors that are themselves developmentally regulated. However, the reality may again be more complicated as some somatic cell lines, and adult cell types retain Xist responsiveness [32, 33]. Moreover, factors/chromatin modifications that are hallmarks of the Xi, for example recruitment of the PRC2 complex and associated histone H3 lysine 27 methylation (H3K27me3) [34, 35], occur in the absence of silencing, i.e. in response to expression of an Xist transgene lacking the repeat A element [19, 28]. Linked to this, XCI is faithfully maintained in somatic cells in which Xist is conditionally deleted, despite the fact that several Xist dependent factors/chromatin modifications, including PRC2 and H3K27me3, are lost [17, 35].
7
The nuclear matrix proteins SATB1/2 were found to be important for conferring Xist responsiveness in tumour cells and in adult somatic cell types [36], although apparently not in early embryos [37]. Nevertheless, the link between SATB1/2 and Xist could be important, especially in light of the evidence that Xist RNA domains are retained in insoluble nuclear matrix preparations. More recent studies have reinforced the link between Xist RNA function and the nuclear matrix. Notably, 3D-SIM super-resolution microscopy analysis demonstrated that Xist RNA is confined within perichromatin spaces, rather than on chromatin [12, 38], colocalising with the nuclear matrix protein hnRNP U (also referred to as SAF-A) [12]. hnRNP U was previously shown to have a role in Xist RNA localisation [39], and until recently represented the only XCI associated factor with a bona fide RNA recognition domain. Indeed UV cross-linking qRT-PCR analysis suggested that hnRNP U interacts directly with Xist RNA [39].
To date, the best studied factors with a role in Xist-mediated silencing are the Polycomb repressive complexes PRC2 [34, 35, 40], canonical PRC1 [41] and variant PRC1 [42, 43]. These complexes catalyse the histone modifications H3K27me3 and mono-ubiquitylation of histone H2A lysine 119 (H2AK119u1) respectively, both of which are hallmarks of the Xi. Polycomb complexes are recruited rapidly in response to Xist RNA expression and their association with the Xi is strictly dependent on ongoing Xist expression [19, 35, 42-44], suggesting that they might be primary silencing factors recruited by Xist RNA. In the case of PRC2 there is evidence for a direct interaction between the PRC2 subunit Ezh2 and the repeat A region of Xist RNA [45]. Ezh2 does not have a known RNA binding domain, and this conclusion has been challenged in other studies [28, 38]. Regardless of the mechanism of Polycomb recruitment, it needs to be borne in mind that Xist RNA, in which the repeat A region is deleted, does recruit Polycomb complexes [19], arguing that Polycomb is probably not sufficient for the establishment of chromosome silencing. 8
Studies aimed at
understanding Polycomb recruitment and function in X inactivation have been extensively covered in recent reviews [46-48], and we will instead focus on the possible roles of newly identified factors in XCI. Several other factors have been implicated in Xist-mediated silencing. Examples include the variant histone macroH2A [49], the chromosomal protein Smchd1 [50], the de novo DNA methyltransferase Dnmt3b [51], hnRNP U [39], the trithorax protein Ash2l [52], as well as HP1 [53] and Cdyl [54] proteins that are thought to bind directly to histone modifications enriched on Xi. There is evidence to suggest that most if not all of these factors function in maintenance rather than establishment of XCI. This conclusion is based in part on observations indicating a significant lag in the recruitment of these factors following the onset of Xist expression in an in vitro model system, differentiating XX ES cells [51, 52, 55-57]. Additionally, as mentioned above, none of these factors has a characterised RNA binding domain, with the exception of hnRNP U. Together, these considerations indicate that the critical RBPs required to initiate Xist-mediated silencing remain to be identified. Indeed, factors mediating well characterised Xi chromatin features, such as global histone hypoacetylation [58], depletion of histone H3K4 methylation [56] and of RNA polymerase II [59], are not known. These Xi features are established rapidly in response to the onset of Xist RNA expression [56, 57, 59], and as such may play a key role in initiating chromosome silencing.
hnRNP U hnRNP U is an approximately 800 aa protein with several conserved domains: an Nterminally located SAP domain, implicated in DNA binding, centrally located SPRY and ATP binding domains of unknown function, and an N-terminal RGG box linked to RNA binding (Figure 2). hnRNP U was identified in the proteomic screens [20-22], consistent with a previous study in which UV cross-linking analysis indicated a direct interaction between 9
hnRNP U and Xist RNA [39]. hnRNP U therefore served as a positive control that validated the proteomic screening strategies. Chu et al [20] used UV cross-linking RBP immunoprecipitation followed by qRT-PCR to determine binding sites for hnRNP U on Xist RNA. Binding was seen to occur broadly over much of Xist RNA. This is similar to prior findings from Hasegawa et al [39], although in this latter case there was evidence for preferred binding towards the 5’ end of the transcript. Genome wide analysis of hnRNP U binding has been determined using UV-cross-linking immunoprecipitation (CLiP) [60], although in cells that do not express Xist RNA. Further studies are therefore required to better define sites of interaction of hnRNP U with Xist RNA.
The genetic screen studies did not report hnRNP U amongst the top ranked hits (Figure 3). The reason for this is currently unclear. hnRNP U shRNAs were included in the library used by Moindrot et al [23], but none were enriched, indicating that knockdown may have been inefficient or alternatively, that knockdown had a strong effect on cell viability. The latter explanation seems unlikely given that other studies were successful in assaying Xist localisation following hnRNP U knockdown in ESCs. HnRNP U was not detected in Monfort et al either [24], maybe because the probability of having a mutational insertion in a small gene like hnRNP U (9kb) is relatively low. This lack of saturation of the screen may also explain why some other factors validated in the parallel studies, were not seen.
Knockdown of hnRNP U by both Chu et al [20] and McHugh et al [21], confirmed its important role in Xist RNA localisation. Specifically, Xist RNA showed a diffuse localisation in the nucleus, contrasting with the focal localisation observed in the parental cell lines. Perhaps surprisingly, validation of other factors found in the genetic and proteomic screens did not reveal detectable deficiencies in formation of Xist RNA domains, indicating that hnRNP U is likely to be the principal determinant for localising Xist RNA within the nuclear matrix 10
compartment. However, it cannot be ruled out that factors in the screens that were not validated contribute to this pathway. In this regard, it is interesting to note that Matrin3, another nuclear matrix protein which also binds RNA [61], was identified by Chu et al [20] and Moindrot et al [23]. The fact that McHugh et al [21] failed to find this protein using the high stringency UV cross-linking approach may indicate that it is an indirect interactor, perhaps functioning in cooperation with hnRNP U in defining the nuclear matrix environment in which Xist RNA is entrapped.
An unresolved issue regarding the role of hnRNP U in XCI is the paradoxical observation that enrichment of the protein within Xi domains is seen only late in the XCI cascade, as determined by analysis of differentiating XX ESCs [53]. It is not obvious how to reconcile this with the requirement for hnRNP U for Xist localisation at the onset of XCI in ES cell models [20, 21]. One possibility is that low levels of hnRNP U, below that required for detection by immunolocalisation but sufficient for Xist RNA localisation, interact with Xist RNA at the onset of XCI and that hnRNP U then accumulates in the Xi domain progressively thereafter.
Alternatively, it may be that a more general role of hnRNP U in nuclear
organisation is important for correct Xist RNA localisation and it is the disruption of this function in ES cells, rather than direct binding, that affects Xist domain formation.
hnRNP K hnRNP K is an approximately 450 aa protein that has three KH domains that mediate interaction with RNA (Figure 2) [62]. hnRNP K was identified by Chu et al [20] as one of two highly abundant proteins in the formaldehyde cross-linked Xist interactome, the other being hnRNP U. Like hnRNP U, UV cross-linking RBP immunoprecipitation followed by qRT-PCR suggested hnRNP K binds at similar levels across the Xist transcript. However, McHugh et al [21] did not identify hnRNP K using UV cross-linking mass spectrometry (Figure 3). 11
Further analysis, for example using CLiP, is therefore required both to confirm that hnRNP K interacts directly with Xist RNA, and to identify putative preferred binding sites. At present there are no published CLiP analyses of hnRNP K, although the protein is known to show a strong preference for cytosine rich RNA sequences, similar to the related proteins PCBP1/2 (reviewed in [63]).
Chu et al [20] tested the role of hnRNP K in Xist-mediated silencing in ESCs by assaying silencing of genes located in cis to an inducible Xist transgene. hnRNP K siRNA abrogated Xist-mediated silencing to a similar degree to that seen with hnRNP U knockdown. Moreover, it was found that Xist induced domains for the PRC1 and PRC2 mediated histone modifications H2AK119u1 and H3K27me3, were also reduced, suggesting a role for hnRNP K in chromatin modification on the Xi.
hnRNP K is found as a major component of nuclear matrix preparations [64], consistent with it having a role in Xist-mediated silencing. The protein has been suggested to act as a docking platform or hub for several complexes that mediate nucleic acid linked processes, notably gene activation and gene silencing, translation and RNA processing (reviewed in [65]). More recently hnRNP K has been implicated in coordinating transcriptional silencing by the histone modifying complex SETDB1 [66].
Whilst the findings of Chu et al [20] support hnRNP K direct role in Xist-mediated silencing, there are reasons to consider this with caution. As mentioned above, McHugh et al [21] did not identify hnRNP K as a direct Xist interactor, and neither of the genetic screening studies identified hnRNP K as a candidate silencing factor (Figure 3). The latter may indicate limitations in identifying silencing factors by genetic screening, but nevertheless needs to be taken into consideration. It should also be noted that hnRNP K is a common contaminant 12
in mass spectrometry experiments due to non-specific binding to agarose and magnetic beads used to purify protein complexes from extracts [67].
What then of the proposed role of hnRNP K in Xist-mediated chromatin modifications, notably recruitment of Polycomb repressors? As discussed above, Polycomb repressor complexes are implicated in maintenance rather that establishment of gene silencing in XCI, and as such hnRNP K may play a role in their recruitment regardless of its role in gene silencing in cis. Indeed, there is some evidence that hnRNP K can interact with the PRC2 protein EED [68]. However, it is important to consider the converse argument, that hnRNP K affects gene silencing, either directly or indirectly, and that this in turn reduces establishment of Polycomb mediated chromatin modifications, due to antagonism by chromatin modifications associated with gene activation (reviewed in [69]).
A similar
argument can be applied to Rbm15 and Spen, two other candidate factors identified in the genetic and proteomic screens, that were also found to have a role in Polycomb recruitment (see below). Mapping interaction sites for these factors in relation to elements implicated in silencing versus Polycomb recruitment should help to resolve this issue.
In relation to Polycomb recruitment by Xist RNA it should be noted that none of the proteomic studies identified core proteins uniquely found in PRC2 complexes, arguing against the prevailing model that PRC2 interacts directly with Xist RNA. Minajigi et al [22], did report the RbAp46/48 PRC2 subunit as a direct interactor, but this protein is also found in several other chromatin modification complexes, and in itself cannot be considered as representative of PRC2. PRC1 proteins on the other hand were identified by Chu et al [20] (and Minijigi et al [22]), but not by McHugh et al [21], indicating that they are recruited to Xist RNA either directly or via an RBP co-factor. Moindrot et al [23] also identified PRC1 proteins in their genetic screen, albeit with a relatively low ranking, whilst Montfort et al [24] identified 13
the PRC2 protein Eed. The fact that Polycomb is not sufficient for establishment of silencing by Xist RNA (see above), indicates that these findings are most likely due to indirect genetic interactions. Thus, in summary, whilst hnRNP K is an important candidate protein, further experiments are required to confirm its function both in Xist-mediated silencing and in chromatin modification by Polycomb repressor proteins.
SHARP/SPEN/MINT SPEN is an exceptionally large protein, approximately 3500 aa, and is the founding member of the Split-End family of proteins, first discovered in drosophila [70]. Members of this family are characterized by the presence of 3/4 RNA Recognition Motifs (RRMs) at the N-terminal and a SPEN paralog and ortholog conserved (SPOC) domain at the C-terminus (Figure 2). SPEN is also known as SMRT/HDAC1 Associated Repressor Protein (SHARP) in human, and Msx2-Interacting Nuclear Target (MINT) in mouse.
SPEN was identified and validated as a high confidence hit in the proteomic [20-22] and genetic [23, 24] screens (Figure 3). In the work of Chu et al, SPEN was one of only three factors found to be enriched only in the presence of the repeat A, suggesting that it may bind to this region [20]. McHugh et al, using the UV crosslinking approach, identified SPEN as the factor most highly enriched for Xist compared to control RNAs, further indicating that SPEN directly interacts with Xist RNA [21]. Moreover, Moindrot et al [23], using superresolution 3D-SIM, demonstrated that SPEN co-localizes with Xist RNA in the interchromatin compartment, consisting of a network of channels relatively devoid of DNA [71]. All four studies validated SPEN extensively, demonstrating that loss of function interferes with Xistmediated silencing but has no effect on Xist RNA localisation/domain formation (Figure 3). Given the large size of SPEN, it is possible to envisage that the protein bridges Xist
14
RNA/nuclear matrix and the underlying chromatin by binding via its N-terminal RRMs and C-terminal SPOC domain respectively.
SPEN, a well characterised RBP, is known to bind to the SRA transcript (steroid receptor RNA activator), and this interaction has been explored in detail [72]. The RNA recognition region of SPEN is organized into two modules. The first comprises the RRM3 and RRM4 domains, which both engage with the H12-H13 fragment of the SRA transcript. The second module comprises the RRM2 domain that is not essential for interacting with the SRA RNA and is connected to the RRM3-RRM4 module by a short linker. In-vitro binding assays have shown that the association between SPEN and the SRA transcript involves both single stranded sequences and paired nucleotides, as can be found in stem-loop structures [72]. A similar docking platform might be required for SPEN binding to Xist RNA. Indeed, based on the findings of Chu et al [20], Spen most likely recognizes the repeat A region, which was previously suggested to adopt a structured stem-loop architecture [10]. This conclusion is further supported by EMSA assays showing that a purified fragment containing SPEN RRMs 2-3-4 binds to in-vitro transcribed repeat A motifs [24].
How might SPEN mediate transcriptional silencing? In mammals, SPEN has been shown to have essential roles in transcriptional repression [73-75]. In particular, SPEN is able to recruit transcriptional repressors, such as the silencing mediator for retinoid and thyroid receptor SMRT/nuclear receptor co-repressor (NCoR) [76-79], which are components of histone deacetylase (HDAC) complexes. The interaction between SPEN and SMRT is mediated by the SPOC domain, which recognises the serine-phosphorylated C-terminus of SMRT [79]. In addition, the SPOC domain of SPEN has also been shown to recruit HDAC1 and HDAC2 directly [76]. The ability of SPEN to recruit the SMRT/NCoR complex and thence HDAC activities may be critical for initiating the silencing cascade in the context of XCI. 15
McHugh et al [21] indeed demonstrated that knock-down of SMRT or HDAC3 mimics the silencing defects observed in the absence of SPEN. It is however notable that neither HDAC1/2/3 nor NCoR1/2 were identified in the genetic screens [23, 24]. Moreover, global histone hypoacetylation on Xi has previously been shown to occur independently of the repeat A element [52], arguing that the function of SPEN in gene repression may be more complex.
Further studies are required to clarify whether the HDAC complexes are
exclusively recruited by SPEN, and whether this constitutes the sole link between SPEN and transcriptional silencing.
SPEN may have a role in the recruitment of PRC1 and PRC2 complexes, either directly or indirectly, since McHugh et al [21] and Monfort et al [24] report reduced enrichment of PRC2 (Ezh2, Eed) and PRC1 (Ring1b), on Xi in the absence of SPEN. In contrast, H3K27me3 deposition was largely unaffected by SPEN knock-down in the study by Moindrot et al [23]. This apparent contradiction may stem from a threshold effect in which reduced levels of the complex are nevertheless sufficient to efficiently deposit the H3K27me3 modification. Regardless, it is unlikely that SPEN recruits Polycomb complexes directly as the critical region of Xist required for PRC2 recruitment is distinct from the repeat A region with which SPEN is proposed to interact. As noted above, differences in recruitment of Polycomb complexes could be a secondary consequence of ameliorated silencing due to antagonism by transcription-linked chromatin modifications.
A previous study, in which the SPEN gene was knocked out, demonstrated mouse embryo lethality around embryonic day (E)12.5 [73]. There was no evidence for preferential loss of female homozygous embryos, although the number of embryos analysed at earlier stages was insufficient to rule this out entirely.
This observation is perhaps surprising given the
critical role for SPEN in gene silencing by Xist RNA. By way of comparison, heterozygous 16
female embryos carrying a paternally inherited mutant Xist allele die early during embryonic development and exhibit growth defects from E6.5 [7]. One possible explanation for this disparity is that the SPEN knockout allele results in only partial loss of function: exon skipping, for example, may allow generation of a truncated but functional protein. Alternatively, there may be other proteins that function redundantly with SPEN in Xistmediated silencing, for example Rbm15.
RBM15 RBM15 is a homologue of SPEN that has three N-terminal RRM domains and a C-terminal SPOC domain (Figure 2). The protein is somewhat smaller than SPEN, approximately 900 aa, reflecting a much smaller spacer region between the RRMs and the SPOC domain. The RBM15 ortholog in human, sometimes named OTT1, is involved in a specific t(1;22) translocation
event
creating
the
RBM15-MKL1
fusion
associated
with
acute
megakaryoblastic leukaemia [80, 81].
Rbm15 was identified as an Xist interactor in proteomic [20-22] and genetic [23] screens (Figure 3). Detection of Rbm15 by in-vivo UV crosslinking indicates that it binds directly to Xist RNA, and indeed RBM15 was the second most highly enriched protein after SPEN [21]. Chu et al [20], also identified Rbm15 using the formaldehyde cross-link approach, but in contrast to SPEN, this association was not repeat A dependent. Moindrot et al found that Rbm15 is required for Xist-mediated silencing, with no obvious requirement for Xist localisation/domain formation [23]. Conversely, Rbm15 was not identified in the genetic screen of Montfort et al [24], and validation experiments by McHugh et al [21] found no effect of Rbm15 knockdown on Xist mediated silencing.
The reasons for these apparent
discrepancies are unclear. In the case of the Montfort et al screen [24], there may have been insufficient depth of coverage to detect Rbm15 (there was a twofold increase in Rbm15 17
mutations relative to uninduced control but this falls below the set threshold). Differences in the findings for Rbm15 by Moindrot et al [23] and McHugh et al [21] may reflect differences in the silencing assays that were employed.
Notably, Moindrot et al assessed silencing
directly by scoring nascent RNA foci for genes located in cis with Xist compared to control genes, a methodology widely employed in XCI studies. McHugh et al on the other hand employed a novel single mRNA FISH method which is less direct, and could, for example, be influenced by changes in levels of a given mRNA due to enhanced expression from the non-Xist associated allele.
Moindrot et al [23] reported a deficiency in deposition of H3K27me3 by PRC2 polycomb complexes following Rbm15 knockdown.
Rbm15 binding to Xist RNA apparently occurs
independent of the repeat A [20], and it is therefore conceivable that it interacts with the Xist sequence elements required for PRC2 recruitment (Figure 1). However, as argued above for SPEN, Rbm15 may instead affect Polycomb recruitment indirectly, and further studies are required to elucidate this point.
What role might RBM15 play in Xist mediated silencing? In mammals, RBM15 has been linked to two different functions: transcription regulation and mRNA metabolism. RBM15 transcriptional functions have mainly been characterized in the context of the fusion RBM15MKL1 associated with acute megakaryoblastic leukemia. This fusion protein can mediate gene activation, especially of Notch signalling target genes [82-84], possibly by the recruitment of the H3K4 methyltransferase Setd1b via the SPOC domain of the RBM15MKL1 fusion [85]. However, the endogenous RBM15 may also be associated with gene repression. Supporting this idea, endogenous Rbm15 but not the RBM15-MKL1 fusion, can interact with HDAC3 [83], a component of the NCoR/SMRT corepressor complex. In addition, gene expression analysis in embryos homozygous for RBM15XK135 (hypomorphic 18
low-expressed allele of RBM15 resulting from the insertion of LacZ-Neo cassette 20 nt upstream the initiator codon) revealed specific upregulation of transcripts that are normally expressed at low levels in wild-type embryos [86].
In addition to its role in repressing transcription, RBM15 has also been implicated in mRNA metabolism, mainly via its interaction with the RNA export factors Nxf1 and Nxt1, which are responsible for nuclear export of the majority of mRNAs [86, 87], and via its interaction with Dbp5, an ATPase docked at the nuclear pore [88]. Accordingly, the artificial tethering of RBM15 on reporter mRNA promotes nuclear export [86, 87, 89]. Again, the C-terminal SPOC domain of RBM15 appears to be required for the interaction with mRNA export factors [86, 87]. Interestingly, the role of RBM15 might be more complex than simply promoting nuclear export. Indeed, it has been reported that RBM15 can retain some mRNAs, instead of promoting their nuclear export, as exemplified by the nuclear accumulation of hyperpolyadenylated ORF59 RNA, a viral RNA coding for a DNA polymerase [90, 91]. In addition, RBM15b, a homolog of RBM15, has been shown to prevent the cytoplasmic export of an unspliced transcript generated from a reporter gene containing a cryptic splice site [92]. These two observations raise the possibility that RBM15 could be involved in RNA surveillance, selecting mRNAs that qualify for nuclear export, and consequently modulating gene expression at the post-transcriptional level.
While Rbm15 knock-down has no obvious effect on nuclear localisation of Xist, the link to regulation of RNA export gains importance in light of the finding that RNA export factors, notably Nxt1 but also Nxt2 and Nxf1, were identified by Moindrot et al in their genetic screen [23]. Further validation of Nxt1 was not possible as cell viability was strongly affected following gene knockdown. Proteomic screens by Chu et al [20] and McHugh et al [21] did not identify RNA export factors as being enriched, although this could reflect equivalent 19
binding to control RNAs. These considerations highlight some of the limitations of genetic and proteomic screening respectively. Regardless, further studies are required to determine the contribution of nuclear export and gene repression functions of RBM15 in the context of Xist-mediated silencing.
Intriguingly, Spenito (nito), a drosophila homolog of RBM15, regulates the alternative splicing of Sex-lethal gene (Sxl), the master regulator of sex determination and dosage compensation in fly [93]. In addition, FPA, a homolog of RBM15 in A. thaliana, represses the expression of FLC, a gene required for controlling flowering time. The molecular mechanisms by which FPA regulates FLC expression have been investigated [94]. FPA appears to control the 3’-end cleavage and polyadenylation of an antisense transcript, which spans the FLC coding sequence and affects the rate of FLC sense transcription. Importantly, the RNA processing functions played by FPA may not be restricted to FLC transcript. Indeed, in the absence of FPA, several intergenic genomic segments became expressed, generally because of the alternative splicing or the alternative polyadenylation of adjacent transcripts [95]. This underlines the global role of the Rbm15 homolog in transcription regulation. These observations in drosophila and plants shed light on an intriguing parallel between RBM15 function in XCI on the one hand, and Spenito or FPA functions in sexual reproduction on the other, raising the possibility that Split-End proteins have evolved to modulate gender specific functions and reproduction.
Gene knockout of Rbm15 results in embryo lethality at E9.5-10.5 [96], somewhat earlier than observed for the SPEN knockout (E12.5) [73]. Similarly, mice homozygous for the poorly expressed hypomeorphic allele RBM15XK135 die during embryonic development by day E12.5, confirming the importance of RBM15 for mouse development [86]. No femalespecific phenotypes were reported in these studies. To gain insights into the developmental 20
function of RBM15, Raffel et al. made use of a Sox2-CRE strain to delete RBM15 in epiblast derived cells [97]. RBM15 null embryos were then identified throughout gestation at near the expected Mendelian frequencies and pups died only soon after birth [97]. At first glance, this observation seems to argue against an important role for RBM15 in XCI. However, in this experimental set-up, the Sox2-CRE deletes Rbm15 at E6.5 [98, 99], after establishment of both imprinted and random XCI [44]. Additionally, as argued in the case of SPEN knockout mice, redundancy with other SPOC domain proteins may account for the absence of femalespecific effects in single gene knockouts. The Rbm15 homologue, Rbm15b, may contribute functionally in this regard.
WTAP WTAP is a 400 aa protein that was first identified as an associated partner of Wilms’ tumor 1 protein in a yeast double-hybrid screen, and named thereafter as Wilms’ tumor 1associating protein [100]. Unlike SPEN and RBM15, WTAP protein does not contain annotated RNA-binding domains. However it has been implicated in the regulation of mRNA biogenesis, and moreover has been described as a regulatory component of the RNA methyltransferase complex, comprising WTAP, virilizer and the catalytic subunits METTL3 and METTL14, that together mediate formation of N6-methyladenosine (m6A) on RNA [101103] (see below). Using formaldehyde cross-linking, Chu et al [20] identified WTAP as an Xist-interacting protein and moreover as one of the three factors for which Xist association is dependent on the repeat A (Figure 3). WTAP was not identified using the UV cross-linking approach [21, 22], indicating that the protein does not interact with Xist RNA directly (Figure 3A). This could reflect association of WTAP with an enzymatic complex that interacts only transiently with substrate mRNAs.
Indeed Moindrot et al [23], identified WTAP and also
virilizer, a second regulatory subunit of the m6A methyltransferase complex, as high confidence hits in their genetic screen.
It is interesting to note that WTAP has been 21
reported to interact with both SPEN and RBM15 [104, 105], indicating a possible biochemical link between different factors implicated in Xist-mediated silencing. The second genetic screen, performed in haploid ES cells, did not identify WTAP [24], possibly reflecting limited coverage of this screen as discussed above.
Validation experiments have shown that, although WTAP knockdown does not affect Xist domain formation or H3K27me3 deposition, it noticeably impairs the silencing of the chromosome [23]. However, Chu et al [20] reached the opposite conclusion as no silencing defects were observed in differentiating ES cells treated with a WTAP siRNA (Figure 3B). Although the reasons for this discrepancy remain to be fully understood, it should be noted that silencing defects reported by Moindrot et al [23] in WTAP knockdown differentiated ES cells are milder than after RBM15 or SPEN knockdown.
How might WTAP participate in Xist mediated silencing? WTAP has been linked to different functions associated with transcript maturation and stability. For instance, WTAP prevents the degradation of cyclin A2 mRNA by stabilizing the transcript via a small RNA motif found in the 3’-UTR, and thereby regulates the G2/M transition [106]. WTAP is also found associated with splicing factors, within the spliceosome [107], or within interchromatin granule clusters where numerous splicing factors assemble [108]. Interestingly, WTAP can modulate the alternative splicing of its own transcript and thereby limit the synthesis of WTAP protein [104], which demonstrates that WTAP can negatively regulate gene expression at the post- or co-transcriptional level.
As mentioned above, WTAP is also a regulatory component of the RNA methyltransferase complex. It is currently unclear whether WTAP functions only in the context of the m6A complex or can also operate independently in aspects of mRNA regulation. The m6A 22
modification has been identified in many organisms, including mammals, plants and yeast, and is the most prevalent internal modification of mRNA and ncRNA. It is distributed throughout the transcript and enriched within the last exon of mammalian mRNAs [109-111], and has recently been associated with post-transcriptional gene regulation (reviewed in [112]). In short, m6A modification acts as a negative regulator of gene expression, probably by modulating the transcript stability since the depletion of the methyltransferase complex increases the quantity of the targeted m6A transcripts [101, 113-116]. This modulation of transcript stability may be mediated by a known m6A reader, YTHDF, which can target m6A modified transcripts to RNA decay bodies [113]. There is also evidence suggesting that m6A functions by modulating RNA structure and thereby facilitating binding of other RBPs during mRNA biogenesis, exemplified by studies on hnRNP C binding to mRNA [117, 118]. Indeed it is plausible that m6A function in regulating mRNA stability is a consequence of structural switches that modulate RBP binding.
Whether or not the m6A modification is important for Xist-mediated silencing remains an open question. It has been reported that human Xist RNA is methylated in exon 6 [109]. However, this analysis was performed in a male cancer cell line, HepG2, precluding any conclusion about where and when Xist RNA may get methylated during XCI. Two scenarios can nevertheless be considered. In the first one, the methylation status of Xist RNA would be modified at the onset of the XCI, which may in turn affect its physical properties (turnover, conformation etc.), and thereby the cis-silencing.
In the second scenario, the RNA
methylation complex would be attracted by Xist RNA to reinforce mRNA methylation of Xlinked gene products at the onset of XCI, and thereby mediate their degradation. Both possibilities would explain why WTAP knockdown alters Xist-mediated silencing. An important consideration is that Xist RNA localisation is not obviously affected by WTAP knockdown, precluding a major Xist stabilization defect, and also that METTL3 enzyme was 23
not identified in the genetic screen [23] (no shRNAs targeting METTL14 were included in the library used in this study).
WTAP knockout in mouse is embryonic lethal [106]. Developmental defects were reported from E6.5, with embryos lacking the characteristic morphology of endoderm and ectoderm at E8.5. So far, these observations were interpreted in the context of WTAP regulating the cyclin A2 transcript, rather than in the context of m6A modification. Similar to SPEN and RBM15 knockouts, no female-specific phenotypes were reported in this study, suggesting that at least partial dosage compensation can occur in the absence of WTAP. Of note, the homolog of WTAP in drosophila, fl(2)d, is implicated in sex determination and dosage compensation by controlling the splicing of Sxl transcript [119], suggesting that the molecular mechanisms regulating the equalization of gene expression between males and females might be, at least partially, evolutionary conserved.
LBR LBR (Lamin B receptor) is an approximately 600 aa protein, which lies embedded in the inner membrane of the nuclear envelope and was originally discovered as a B-type Lamin binding protein [120]. The LBR C-terminus is comprised of transmembrane domains, while the N-terminal region faces the nucleoplasm [121] (Figure 2). Heterozygous mutations in LBR are associated with the Pelger-Huet anomaly, a disorder in which neutrophil nuclei are hyposegmented and exhibit an atypical heterochromatin redistribution [122], underlying the role played by LBR in determining nuclear shape and tethering peripheral heterochromatin.
LBR, which does not contain a classical RNA recognition motif, was identified as a direct Xist RNA binder by McHugh et al [21] but not by Chu et al [20] (Figure 3A). Xist recognition by LBR might be mediated by an RS domain (arginine and serine) that has been reported 24
to have an RNA binding activity [123]. Validation experiments performed by McHugh et al [21] found that LBR depletion does not severely affect Xist RNA localisation/domain formation, but impairs Xist-mediated silencing, including in differentiating female ES cells, indicating that LBR intervenes in the downstream silencing cascade. An important feature of XCI is the exclusion of RNA polymerase II from the Xi territory, and gene internalization within the Xist domain [59]. Interestingly, LBR was found to be dispensable for the exclusion of RNA Polymerase II, and yet, the silencing of the chromosome is altered. This phenotype echoes what is observed with Xist RNA lacking the repeat A, suggesting that LBR does not repel the transcription machinery but instead, like the repeat A, could be implicated in the relocation of gene bodies within the Xi territory [21, 59].
It is interesting to note that LBR has also been linked to transcriptional silencing, for example via interaction with the transcriptional repressor HP1 [124, 125]. However, this interaction may be indirect and mediated though the core histones [126]. Accordingly, pull-down of LBR enriches for chromatin with repressive histone marks, such as H3K9me3 or H3K27me3 [127]. Importantly, LBR may actively contribute to transcriptional silencing. For instance, the N-terminal domain of LBR can induce the compaction of an in vitro reconstituted nucleofilament. This compaction can’t be explained solely by LBRs ability to self-associate since the same experiment performed with nucleosomes lacking the histone tails allows LBR binding but does not induce the compaction of the nucleofilament [128]. In addition, LBR can repress a luciferase reporter, whereas a mutant lacking the Tudor domain can’t [128]. Furthermore, the ectopic expression of LBR in olfactory sensory neurons, which normally do not express LBR, induces the overall silencing of olfactory receptor genes without affecting the global transcriptome, and impairs aggregation of olfactory receptor genes near the centre of the nucleus [129]. Taken together, these observations indicate that LBR can
25
mediate transcriptional silencing, most probably indirectly, by inducing compaction and/or tethering the chromatin in a repressive compartment.
How chromatin tethering at the nuclear periphery might affect XCI remains an open question. Because the Xi is often found near to the nuclear envelope [130], an attractive hypothesis would be that the Xi, once localised to the nuclear envelope, is then locked into this repressive compartment by LBR, thereby reinforcing transcriptional silencing. However, female-specific defects associated with impaired XCI have not been reported in two different mouse strains carrying mutations in Lbr gene [131, 132], indicating that XCI can occur in the absence of Lbr. Moreover, as already mentioned, LBR was not identified by Chu et al [20], nor in the two genetic screens performed in ES cells [23, 24] (Figure 3). Further studies would therefore be required to rationalise these observations in the context of the proposed role of LBR as a primary silencing factor in XCI.
Concluding remarks The recent studies discussed in this review have identified key factors involved in Xistmediated silencing. A synthesis of the different studies highlights factors implicated in transcriptional silencing through anchoring chromatin, chromatin modification, and posttranscriptional regulation (Figure 4). There is a broad agreement that SPEN plays a central and critical role in silencing, likely by binding to the repeat A sequence and recruiting NCoR/HDAC3 corepressors. Some details nevertheless remain to be confirmed. Other key factors that have emerged as strong candidates for Xist-mediated silencing are hnRNP K, Rbm15, Wtap and LBR. In these cases supporting evidence is less unanimous and further work is required to confirm involvement and elucidate their molecular mechanisms of action.
26
None of the novel factors significantly affect Xist RNA localisation, although the key role of hnRNP U in this process is confirmed.
Given that the recent studies have harnessed the power of both proteomic and genetic screening, it seems likely that we now know the identity of most of the factors required to establish Xist-mediated silencing. It should be noted that some candidates identified in one or more screen were not discussed here. These include several factors linked to mRNA biogenesis, for example Ptbp1/2, RALY, and CELF1. Indeed, Ptbp1, which has been shown to function in regulation of splicing (reviewed in [133]), was identified in McHugh et al [21] and Moindrot et al [23], and in both studies knockdown affected Xist-mediated silencing, at least to some degree. Conversely, some of the Xist interacting partners identified in the proteomics screen that are not required for gene-silencing, could contribute to other aspects of X-chromosome inactivation. RNF20/40, identified by Chu et al [20] as the third factor for which enrichment is dependent on the repeat A (Figure 3A), might fall into this category since it was found not to be required for silencing activity.
Further analysis of the novel factors should provide insights into long standing questions in the field. First, what is the basis of the developmental window of opportunity? Most of the key factors identified as having a role in establishing silencing were also seen in analysis of the Xist interactome in XX somatic cells [22] (Figure 3), suggesting that loss of Xist responsiveness may not be attributable simply to absence of key silencing factors. It is however plausible that developmentally regulated modification of these factors or regulation of absolute levels of the silencing factors, underlies Xist responsiveness. A second key question is to understand the link between primary silencing factors and maintenance factors. Association of factors such as macroH2A and Smchd1 is Xist dependent, yet Xi localisation occurs with a lag of several days following the onset of Xist expression [51, 55]. 27
A further question is to understand the link between primary silencing factors and chromatin modifications on Xi. As discussed above, features such as global hypoacetylation, loss of H3K4 methylation and depletion of RNA Polymerase II all appear to be established independently of the repeat A, at least in part [53, 59]. Additionally, several studies have shown that Xist mediated silencing occurs more efficiently on the X chromosome than on autosomes, an effect that may be linked to enrichment of LINE1 repeats on the X chromosome (reviewed in [134]). What is it about the function of primary silencing factors that limits their ability to repress autosomes compared to the X chromosome? Finally, how did Xist-mediated silencing evolve? Recent studies have demonstrated that marsupial mammals evolved a distinct lncRNA, Rsx, for dosage compensation of the X chromosome [135]. Do the factors found to mediate silencing by Xist RNA also play a role in Rsx mediated silencing, and are these factors utilised either in RNA mediated regulation of genes/gene clusters or in genome defence mechanisms, for example in relation to viruses and transposable elements? Thus, whilst we may have reached the end of the beginning, we certainly have not yet reached the beginning of the end.
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Figure legends
Figure 1: Structure of mouse Xist gene and mRNA. The Xist gene is composed of 8 exons. The processed RNA has several tandem repeat regions, named A to F. The critical regions for Xist function are indicated above and below the Xist transcript diagram. The asterisk denotes a small insertion downstream of the repeat A that partially disrupts Xist-mediated silencing [27].
Figure 2: Schematic drawing of HNRNPK, LBR, HNRNPU, RBM and SPEN. Known RNA binding domains are shown in orange. Wtap has no annotated functional domains. AAA: ATPases Associated domain; KH-1: K Homology domain 1; KOKNT: N-terminus of RNP K-like proteins; RID: Receptor Interaction Domain; RRM: RNA Recognition Motif; SPOC: Spen paralogue and orthologue SPOC, C-terminal domain; SPRY: SPla and the RYanodine Receptor domain; SR: Serine/Arginine-rich domain.
Figure 3: Comparison of the proteomic and genetic screen hits. A- Overlap between the Xist RNA binding proteins identified by McHugh et al [21], Chu et al [20] and Minajigi et al [22]. Using UV cross-linking in ES cells (McHugh et al [21]) or in somatic XX cells (Minajigi et al [22]), only direct Xist binders should be identified, whereas the formaldehyde cross-link used by Chu et al in stem cells [20] should detect direct and indirect interactors. For this analysis, only the candidates from Minajigi et al [22] having a ≥ twofold enrichment over the background were selected. SPEN, WTAP and RNF20, shown in ochre, are the only three proteins which depends on the repeat A for interacting with Xist RNA. WTAP and RNF20 were only detected by Chu et al [20]. B- Factors required for Xist-mediated silencing which have been validated by at least one of the two proteomic screens [20,21] or one of the two genetic screens [23,24]. All four studies 40
validated that SPEN knock-down or knock-out impair Xist-mediated silencing. A comparison is made for the other candidates. None of the candidates reviewed here were validated by Minajigi et al [22].
Figure 4: Possible implication of HNRNPU, LBR, SPEN, HNRNPK, RBM15 and WTAP in Xist-mediated silencing. A- We propose that HNRNPUand LBR are implicated in the silencing by anchoring Xist RNA to the nuclear matrix (HNRNPU) or to the nuclear envelope (LBR). The anchoring impacts on the transcription by an unknown mechanism. B- We propose that SPEN and HNRNPK are linked to transcriptional silencing by recruiting histone modifying complexes. SPEN can interact with SMRT/NCOR and might thereby direct HDAC3 to the chromatin. SPEN and HNRNPK are also involved in Polycomb recruitment. The deacetylation of histone tails combined with H3K27 trimethylation may then reduce transcription. C- RBM15 and WTAP may be implicated in the silencing at the post-transcriptional level. WTAP might impact on the stability and/or conformation of either Xist RNA or X-linked gene product via m6A. RBM15 could block the export of X-linked gene products at the onset of XCI or affect Xist RNA directly. Of note, RBM15 also seems to be implicated in the recruitment of Polycomb complex.
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