The EMBO Journal vol.10 no.12 pp.3819-3827, 1991
A developmentally regulated and tissue-dependent transcription factor complexes with the retinoblastoma gene product
Janet F.Partridge and Nicholas B.La Thangue Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, UK
Communicated by D.P.Lane
DRTF1 is a cellular transcription factor which complexes with the retinoblastoma (Rb) gene product and cyclin A, an association modulated by certain viral oncogenes, such as adenovirus Ela. Complexed DRTF1, referred to as DRTFla, has similar DNA binding specificity and DNA binding polypeptides to DRTFlb, which lacks Rb. DRTFlb is abundant in both F9 embryonal carcinoma (EC) stem cells and pluripotent embryonic stem (ES) cells and is strongly down-regulated during the differentiation of both cell types, suggestive of a stem cell Ela-like activity. In contrast, DRTFla, which in F9 EC cells is much less abundant than the other activities, is induced as F9 cells begin to differentiate. Consistent with the relationship between EC, ES and inner cell mass cells, DRTFlb is present in blastocyst stage embryos although as embryogenesis progresses the levels of Rb-complexed DRTF1 increase. Certain tissues, such as liver and brain, contain high levels of DRTF1 during early embryonic stages but little in adult terminally differentiated tissue, in contrast to the thymus, which contains high levels of Rb-complexed DRTF1 but lacks associated cyclin A. These data show that DRTF1 is a group of transcription factors that share common DNA binding polypeptides and which complex with other non-DNA binding proteins, such as the Rb protein and cyclin A, in a developmentally regulated and tissue-dependent fashion. Key words: embryogenesis/retinoblastoma/transcription factor
Introduction During embryogenesis cells divide and differentiate in a defined and programmed fashion, being influenced by both spatial and temporal cues, a process which results in an organism with a wide variety of cell types and precise morphogenetic characteristics. This process requires that gene expression be exquisitely controlled since the appropriate gene product must be available in the right place and at the right time. The most frequent and widely used level for such control is mediated at the point of transcriptional initiation through proteins that bind to defined DNA sequences in the upstream control regions, which are usually described as either promoters or enhancers, bind a combination of transcription factors which confer on a given gene particular transcription properties (La Thangue and Rigby, 1988a; Mitchell and Tjian, 1989). Studies of the molecular mechanisms that regulate transcription during murine embryogenesis are practically Oxford University Press
difficult because of the the limited amount of material available from the embryo proper. This limitation can, however, be partly overcome by using embryonal carcinoma (EC) cells (Martin, 1980) or embryonic stem (ES) cells (Evans and Kaufman, 1981). EC cells, which are the transformed stem cells of teratocarcinomas, differentiate in vitro into defined cell types, a process that is thought to resemble one of the earliest differentiation events that inner cell mass (ICM) cells undergo (Rudnicki and McBurney, 1987). However, they are derived from tumours and therefore are transformed and often karyotypically abnormal. Alternatively, ES cells are derived directly from cultured blastocyst stage embryos and when re-introduced into blastocysts contribute to all tissues, including the germ line (Robertson, 1987); they therefore function as 'normal' pluripotent stem cells. Our previous studies have defined a cellular transcription factor the DNA binding activity of which is down-regulated as F9 EC cells differentiate to parietal endoderm-like cells (La Thangue and Rigby, 1987; La Thangue et al., 1990). This transcription factor, DRTF1 (for differentiationregulated transcription factor 1), exists as several discrete protein complexes when assayed by gel retardation in F9 EC cell extracts, and thus potentially represents a family of transcription factors. In F9 EC cells, DRTFIb is most abundant and functions as a binding site-dependent transcriptional activator when assayed by in vitro transcription (Shivji and La Thangue, 1991). In contrast to F9 EC cells, some cell types have increased amounts of DRTFla which can be dissociated to DRTFIb by the action of the adenovirus Ela protein, an effect mediated by the ability of Ela to sequester the tumour suppressor retinoblastoma (Rb) gene product from DRTFla (Bandara and La Thangue, 1991). In this respect, DRTF1 was the first cellular target defined for the Rb protein with known biological activity, and clearly argues that the Rb protein mediates its growth regulation by controlling transcription, and that it localizes to DNA indirectly by complexing with a sequence-specific transcription factor. The HeLa cell transcription factor E2F binds to a similar motif to DRTF1 and is also targeted by adenovirus Ela (Bagchi et al., 1990), although presently the exact relationship between DRTF1 and E2F is unclear. That DRTFlb is the predominant form in F9 EC cells is consistent with the presence of a cellular Ela-like activity in these cells, orginally defined because these cells allow the expression of an adenovirus that lacks a functional Ela gene product (Imperiale et al., 1984). DRTF1 can therefore complex with other non-DNA binding polypeptides, one being the Rb gene product, an association that may be important for altering the transcription activating properties of this transcription factor. In the present study, we have investigated the developmental regulation and tissue distribution of DRTF1. We found that DRTFlb is abundant in EC, ES and blastocyst
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J.F.Partridge and N.B.La Thangue
stage embryos, and moreover is down-regulated as ES cells begin to differentiate. During embryogenesis, the levels of DRTFlb decline whilst those of DRTFla increase. Moreover, the different DRTF1 complexes have characteristic tissue-dependent distributions, and we were able to define a novel form of the Rb-associated activity in the thymus. Our data show, for the first time, that complexing of the Rb protein with DRTF1 is developmentally regulated and tissue dependent. Both proteins, therefore, function as developmentally regulated transcription factors.
Results Differential regulation of DRTF1 during the differentiation of F9 EC stem cells In F9 EC stem cells DRTF1 exists as several distinct forms, referred to as la, lb and ic, which have distinct mobilities when DNA binding activity is assayed by gel retardation, where a is the slowest (Shivji and La Thangue, 1991). DRTF1a contains the Rb-gene product and is modulated by adenovirus Ela; DRTFlb and c lack associated Rb (Bandara and La Thangue, 1991). Previously, we characterized these activities in fractions derived from F9 EC cell extracts, where these activities were enriched and subsequent analysis much easier. We were, however, interested to determine the regulation of these activities as F9 cells differentiate, and to this end prepared extracts at various time points after F9 EC cells were induced to differentiate and assayed DRTF1 by gel retardation (Figure 1). Consistent with previous results, DRTFlb was the most abundant form in EC cells whereas la, which is a complex that includes the lb DNA binding polypeptides (see later), was rare (Figure 1, tracks 2 and 7). Although DRTFIc was less abundant than DRTFIb, it could be clearly resolved in crude whole cell extracts (Figure 1, tracks 2 and 7). As F9 EC cells began to differentiate, there was an immediate reduction in the activity of DRTF1b, whereas DRTFla increased (Figure 1; compare tracks 2 and 3, and 7 and 8). This trend continued during differentiation, so that after 7 days of treatment, when most cells in the culture were parietal endoderm-like (data not shown), DRTF1b was the least abundant activity whereas la and lc were about equal (Figure 1; tracks 5 and 10). During the differentiation process, the activity of DRTFla had increased whereas lb and Ic had decreased, although lb was more strongly regulated than lc. This high resolution analysis of DRTF1 during F9 differentiation indicated that the different forms of DRTF1 are differentially regulated, and suggested that they are subject to different regulatory cues. As a control for the integrity of these extracts, we analysed the activity of the ATF-site DNA binding activity, ECRE2, an activity that remains relatively constant during the differentiation of F9s (Tassios and La Thangue, 1990). As expected, ECRE2 was present throughout differentiation and showed little regulation (Figure 1; tracks 12- 15). We also studied DNA binding activities that recognize the octamer motif, since one of these, oct4, is present only in the pre-implantation embryo and derived stem cells, including EC cells (SchoTer et al., 1989; oct3 in Rosner et al., 1990). It therefore serves as a control for the phenotype of the cells, although it was also intrinsically interesting to compare the regulation of DRTFlb with that of oct4. With this in mind, we assayed octamer motif binding
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Fig. 1. Regulation of DRTF1 during F9 EC stem cell differentiation. F9 EC stem cells were grown as EC cells or differentiated for the indicated times, extracted and assayed for either DRTF1 (tracks I -10), ECRE2 (tracks 11 - 15) or octamer motif binding activities (tracks 16-20) using standard binding sites probes. Tracks 1, 6, 11 and 16 show probe alone. Note that DRTFIb and c were downregulated whereas the binding activity of DRTFla was enhanced by 3 days of differentiation (compare tracks 2 and 3). ECRE2 is constitutively active (tracks 12-15) whereas oct4 is detectable only in F9 stem cells (track 17). The specificity of each complex was confirmed (data not shown), and * indicates a non-specific complex. Tracks 1 -5 show an increased exposure time of tracks 6-10. About 12 jtg of each extract was assayed for DRTF1 and octamer binding activities and 6 yg for ECRE2.
activities during F9 EC differentiation and resolved both octI and oct4 (Figure 1; tracks 17-20). Octl was present throughout differentiation, whereas oct4 was only detectable in the EC cell extract and was rapidly down-regulated since it could not be detected 3 days after the differentiation process had begun (Figure 1, compare tracks 17 and 18). Oct4 and DRTF1b therefore have similar properties in this differentiation system, since both activities are quantitatively downregulated during the same time period. DRTF1 is regulated during the differentiation of embryonic stem cells Because EC cells are the transformed stem cells of teratocarcinomas which lose their transformed phenotype during differentiation, it was important to determine if the regulation of DRTF1 was influenced by this property. We approached this by studying ES cells, which are derived from pluripotent inner cell mass cells of 3.5 day old blastocyst stage embryos (Evans and Kaufman, 1981). In contrast to EC cells, ES cells are not transformed but retain their pluripotency in vitro since they can contribute to all embryonic germ layers when re-introduced into blastocysts. In vitro these cells can be maintained as ES cells or induced to differentiate (see Materials and methods). ES cells were differentiated by allowing them to form embryoid bodies, which are composed of a variety of differentiated cell types representing all three embryonic germ layers (Doetschman et al., 1985). After 2 days, the bodies had developed a Reichert membrane-like rind surrounding ectodermal-like cells, at 7 days they had usually become cystic, and at 8-10 days some were beating whilst others formed blood islands (data not shown). However, the frequency of these events varied from culture to culture despite consistent culture conditions. When ES cell extracts were assayed for DRTF1, the three
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Fig. 2. DRTF1 is regulated as ES cells differentiate. ES cells were grown as ES cells or differentiated for the indicated times, extracted and assayed for either DRTF1 (tracks 1-10; probe 71/50) or ECRE2 (tracks 11-15; probe P) in the presence of 50-fold excess 62/60* or Pml respectively. Note that DRTF1 is down-regulated within 2 days of differentiation although the activity fluctuates in differentiating cells, for example tracks 4 and 9. Tracks 6-10 show an increased exposure of tracks 1-5. About 12 yg and 6 /g of each extract was assayed for DRTF1 and ECRE2 respectively.
forms of activity were detected and, as in F9 EC stem cells, DRTFlb was most abundant (Figure 2; tracks 1 and 6). DRTF1 was always down-regulated within 2 days after differentiation had begun although the final phenotype of derived cells varied (Figure 2; compare tracks 1 and 6 with 2 and 7) and, as in F9 EC cells, DRTF lb was the most dramatically affected of the activities. After this initial regulation, which was consistent and not influenced by culture or differentiation conditions, the regulation of the different forms of DRTF1 varied with particular ES cell cultures. We show an example of the ES differentiation profile in Figure 2 where, after the initial early reduction in DRTFIb which occurred within 2 days, the levels of DRTF1 fluctuated as differentiation progressed (for example, day 6, where both DRTFla and b were induced), but never reached the much higher levels of DRTFIb in ES cells. This regulation occurred even in the presence of the detergent conditions assayed in Figure 8a (data not shown). The reasons for the fluctuating level of DRTF1 in differentiating ES cells are unclear but are probably related to the inherent variability in the growth rate and phenotype of the differentiating cells. Despite this, other transcription factors, such as GATA 1, a factor enriched in the haematopoietic lineage (Tsai et al., 1989), were induced in parallel with the appearance of blood islands (data not shown). These studies enabled us to make two important conclusions. First, DRTFlb is present at high levels in pluripotent ES cells, and secondly, the DNA binding activity is down-regulated as these cells differentiate. This situation resembles the regulation of DRTF1 during F9 EC differentiation, and therefore argues against the possibility that the regulation in EC cells is caused by reversion of the transformed phenotype, but rather that it reflects the decreasing potency that these cells acquire during differentiation per se. DRTF1 is expressed in blastocyst stage embryos Since both EC and ES cells are thought to resemble cells that comprise the inner cell mass (ICM) of the blastocyst, it was of interest to assess directly if DRTF1 was present at this stage of embryonic development. For this, whole cell extracts were prepared from blastocysts harvested from super-ovulated mice and assayed for DRTF1. Indeed, and
Fig. 3. DRTFIb is expressed in blastocyst stage embryos. About 40 blastocysts (3.5 d.p.c.) were harvested from super-ovulated mice, extracted and assayed for DRTF1 (track 2) or ECRE2 (track 4). For comparison, DRTF1 in F9 EC cell extracts is shown in track 3. The probe alone is shown in track 1. The arrow indicates DRTF1b in blastocyst and F9 EC cell extracts, and * indicates non-specific complexes; specificity was confirmed by assaying binding to mutant binding sites (data not shown). The complex migrating above DRTFIb in this blastocyst extract was not observed in other blastocyst extracts.
as predicted by the EC and ES studies, DRTF1b was present, as was ECRE2 (Figure 3; tracks 2 and 4 respectively). These activities were specific since there was no binding to a mutant DRTF1 or ECRE2 binding site (data not shown). Of course, because of the limited amounts of material that could be obtained from blastocysts, we could not exclude the possibility that, as in ES cells, DRTFla was also present. We conclude, therefore that DRTFIb is the most abundant form of DRTF 1 in blastocyst stage embryos and that this is a property that blastocysts share with EC and ES cells. DRTF1 is regulated during murine embryogenesis Both the EC and ES cell differentiation studies show that the different forms of DRTF1 are independently regulated because in F9 EC cells, for example, DRTFla is more abundant in differentiating cells than in EC stem cells, in contrast to DRTFIb, which has the opposite regulatory profile. With this in mind, we assessed the regulation of the different DRTF1 activities at later stages of early mouse development. Extracts were prepared from whole embryos at 8.5, 11.5, 14.5 and 17.5 days post coitum (d.p.c.) and assayed for DRTF1 in the presence of either a wild-type or
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from c in these assay conditions, but based on their mobility and our previous data (Shivji and La Thangue, 1991) we believe it reasonable to suggest that DRTF1c had the smaller molecular weight. Thus, DRTFla differs from DRTFla' by -35 000 molecular weight and from DRTFlb/c by 150 000. Also, these data establish that these multicomponent protein complexes associate before DNA binding because the gradient centrifugation was performed before the DNA binding assay, indicating that DRTF1 and the Rb protein exist as a complex in vivo and do not assemble in vitro during the gel retardation assay. We next determined the size of the DNA binding polypeptides in each of the complexes by cross-linking a radiolabelled DRTF1 binding site containing BUdR to the DNA-bound polypeptides (Shivji and La Thangue, 1991). After cross-linking, complexes were resolved in native gels by gel retardation, the appropriate complex excised and cross-linked polypeptides resolved in SDS denaturing gels. We could be confident, therefore, that we were assaying polypeptides in discrete complexes. Both DRTFla and b/c from F9 EC cells contained similar DNA binding polypeptides since a common polypeptide with 50 000 molecular weight was cross-linked (Figure 7, compare tracks 2 and 3). Likewise, a similar molecular weight polypeptide was cross-linked in adult thymus DRTFla' (Figure 7, track 4). A polypeptide with 30 000 molecular weight also cross-linked in all the complexes assayed (Figure 7, indicated by *, although the efficiency varied from experiment to experiment; this weak binding efficiency was also noted in our earlier experiments (Shivji and La Thangue, 1991). It is presently unclear why this is so, because this 30 000 molecular weight polypeptide is abundant in DRTF1 preparations affinity purified by bindingsite affinity chromatography (Shivji and La Thangue, 1991; and data not shown). However, these studies clearly 3824
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Fig. 7. The DRTF1 complexes have common DNA binding polypeptides. Gel retardation was performed with either F9 EC or adult extracts after cross-linking to the DRTF1 binding site in the presence of competing oligonucleotide 62/60*, and specific complexes were located, excised and cross-linked polypeptides resolved by SDS-gel electrophoresis. The specificity of each complex was confirmed in a parallel assay (data not shown). The polypeptides crosslinked in F9 EC DRTFlb/c and DRTFla are shown in tracks 2 and 3, and those in adult thymus DRTFIa' in track 4; standard molecular weights (x 1000) are shown in track 1. Note that the predominant polypeptide in each complex is p5O, and that p30 (indicated by *) cross-links inefficiently in these assay conditions.
demonstrate that all the DRTF1 complexes contain a common DNA binding polypeptide of 50 000 molecular weight, and since each complex had identical sequence specificity, it is very likely that this DNA binding polypeptide is common to all DRTF1 complexes and that the different native molecular weights result from the association of other non-DNA binding proteins with these common polypeptides. To confirm this idea, we tested if DRTFla could be dissociated by treating it with a variety of agents and assayed the effect by gel retardation. There was some dissociation of DRTFla and a significant enhancement of the lb/c binding activity when treated with detergent (Figure 8a, compare tracks 2, 3 and 4). In F9 EC cell extracts this effect could also be mediated by certain viral oncogenes, such as adenovirus Ela and SV40 large T antigen (Bandara and La Thangue, 1991; L.R.Bandara and N.B.La Thangue, data not shown), an effect mediated by the ability of these proteins to dissociate DRTFla. Adenovirus Ela behaved in a similar fashion in tissue extracts because it dissociated thymus DRTF1a' to DRTF1b (Figure 8b, compare tracks 2 and 3). In this respect, DRTF1 behaves in a similar fashion to E2F (Bagchi et al., 1990). Since DRTFla' has a molecular weight of 165 000 and DRTF1b/c one of 50 000, this effect most probably results from the ability of Ela to sequester the Rb protein from DRTFla', a dissociation that results in the minimal DNA binding unit. The combined conclusion from these data is that DRTF 1 can complex with the Rb protein to form a multicomponent complex, an association which is regulated during development and one which is targeted by viral Ela. -
Discussion DRTF1 complexes with the retinoblastoma tumour product The data presented in this study show that DRTF1 is a transcription factor that is developmentally regulated and tissue dependent, and that its association with the Rb protein is also under similar control. Although the Rb gene product
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Fig. 8. (a) DRTFI DNA binding is enhanced in the presence of detergent. F9 EC whole cell extracts were treated with either NP40 (tracks 3 and 7) NP40 and deoxycholate together (tracks 4 and 8) and assayed for either DRTF1 (tracks 3 and 4) or ECRE2 (tracks 7 and 8); tracks 2 and 6 show untreated F9 EC cell extract, and tracks and 5 the probe alone. Note that in track 4 DRTFIb/c DNA binding activity is enhanced. * indicates a non-specific complex. (b) Thymus DRTFla' can be dissociated in vitro by adenovirus Ela. An adenovirus 12S Ela cDNA was transcribed and translated in vitro and added to a fraction from adult thymus extract containing DRTFIa' (taken from the glycerol gradient analysis shown in Figure 6) and DRTF1 assayed by gel retardation in the presence of competing oligonucleotide 62/60*. The effect of in vitro translated Ela (track 2) was compared with reticulocyte lysate alone (track 3); the binding activity in the fraction alone is shown in track 1. * indicates a non-specific complex. or an
is known to complex with a variety of cellular polypeptides through which it may mediate its effects as a negative regulator of cellular proliferation (Kaelin et al., 1991), DRTF1 was the first target identified with known biological function. Since then, others have reported that the Rb protein binds to E2F (Bagchi et al., 1991; Chellappan et al., 1991; Chittenden et al., 1991). That the Rb protein is able to complex with such transcription factors clearly argues that the Rb protein mediates some of its growth-regulating effects by altering the transcriptional activating properties of target transcription factors such as DRTF1. In F9 EC and ES cells DRTFIb was more abundant than DRTFIa. Since the Rb protein is sequestered from DRTFla by the adenovirus Ela protein and SV40 large T antigen (L.R.Bandara and La Thangue, 1991; Bandara and N.B.La Thangue, data not shown), we believe that this is further evidence for a cellular Ela-like activity which acts to prevent the Rb protein from complexing with cellular proteins, such as DRTF1, in stem cells of early embryogenesis. Consistent with this model, as embryonic development progressed Rb-complexed DRTF1 became more abundant whereas the levels of Rb-free DRTF1 declined. We suggest, therefore, that the transcriptional activating properties of DRTF1 are regulated during embryonic development, and that part of this regulation is mediated through complexing with the Rb protein. DRTFlb was abundant in F9 EC cells and down-regulated during differentiation, whereas DRTFla was regulated in the converse fashion. Since the abundance of induced DRTF1a was much less than the reduction in Ib, association with the Rb protein cannot therefore be the only mechanism responsible for regulating DRTFlb, and other as yet undefined mechanisms must also be operational. DRTF1 complexes are tissue-dependent Each tissue examined has a characteristic pattern of DRTF 1 DNA binding activity. In general, there were high levels
of DRTF 1 when the tissue concerned underwent maximum proliferation and differentiation. For example, in embryonic liver this occurred between days 11.5 and 17.5, when cells in this tissue are actively dividing and proliferating. This suggests that DRTF1 is involved in regulating stem cell proliferation, perhaps by targeting genes whose protein products are required for progression through the cell cycle. This idea is also supported from the EC and ES cell studies because DRTFlb was abundant in stem cells, but not in their differentiated derivatives, and is consistent with the fact that it is the transcriptionally active form of DRTF1 (Shivji and La Thangue, 1991). We believe, therefore, that DRTF1 has an important role in regulating the transcription of genes whose protein products are required for progression through the cell cycle. It was also clear during brain development that DRTFlc predominated, a situation that contrasts with liver development. We do not know how DRTF lb and lc differ, but we have found that DRTFlb has a larger size than DRTFlc (Shivji and La Thangue, 1991), an effect that could result from a post-translational modification or another complexing protein. Our previous data also indicate that DRTFlc is transcriptionally active, so we suggest again that its function during brain development is to positively regulate stem cell proliferation. Testis, thymus and embryo extracts from late stages of embryogenesis contained a novel form of DRTF1, which we referred to as DRTFla', because it had a smaller native molecular weight than DRTFla but was still very much larger than DRTFlb; we believe that DRTFla' is the same activity that we were able to generate in our previous analysis by detergent treatment (Shivji and La Thangue, 1991). Both DRTFla and la' are sensitive to the action of adenovirus Ela. However, DRTFla contains an additional molecule, cyclin A (Bandara et al., 1991), which is probably responsible for the different native molecular weights of DRTFla and la'. Like the Rb protein, cyclin A is also sequestered by adenovirus Ela (Giordano et al., 1989). That 3825
J.F.Partridge and N.B.La Thangue
DRTFIa' rather than DRTFla exists in certain tissues and also during the later stages of murine development argues that the association of cyclin A with the DRTF1 complex is also developmentally regulated. DRTF1 as a universal regulator of stem cell proliferation DRTF1 is expressed both in vivo and in vitro in early embryonic stem cells, and during the early developmental stages of certain tissues, when they undergo maximum proliferation, whereas in terminally differentiated cells and tissues it is present, with the exception of the thymus, at low levels. These data suggest a role for DRTF1 in regulating the progression of cells into the cell cycle and hence proliferation, a model consistent with its interaction with the Rb protein, which is a negative regulator of cell growth (Marshall, 1991). The Rb protein may control the transcriptional activity of DRTF1 by directly complexing with the DNA-bound transcription factor and through this association regulate genes required for cell cycle progression. That DRTF1 involves an association with cyclin A is consistent with these ideas since cyclin A undergoes periodic accumulation and destruction during each cell cycle and is required for normal cell cycle progression (Lehner and O'Farrell, 1990; Pines and Hunter, 1990). These data not only demonstrate that DRTF1 is developmentally regulated, but also for the first time suggest that the Rb protein is under developmental control. However, this is not at the level of gene expression, since the Rb protein is expressed constitutively during murine development (Bernards et al., 1989), but rather at the level of its association with its target proteins. For DRTF1, this association may have important consequences for transcriptional control. We believe that an understanding of the mechanisms that control this interaction will provide important insights into the regulation of stem cell proliferation during embryonic development.
Preparation of whole cell extract Whole cell extracts were prepared from 5 ml of packed cells, as previously described (Manley et al., 1980; La Thangue and Rigby, 1988b), for the experiment in Figure 1. For all other experiments, whole cell extracts from small numbers of cells or small quantities of tissue (micro-extracts) were prepared essentially as described previously (Scholer et al., 1989) but were extracted by freeze-thawing; protein concentrations were determined by the Bradford assay using the Bio-Rad protein assay. Oligonucleotides Oligonucleotides containing DRTF1 and ECRE2 binding sites, 71/50 and P respectively, were as previously described as were the mutant sites 62/60*, 63* and Pml (La Thangue et al., 1990; Tassios and La Thangue, 1990). The octamer binding site probe contained a consensus octamer taken from the immunoglobulin heavy chain enhancer (sequences -554 to 536). The oligonucleotide used for UV cross-linking experiments, -50/-82 CREM, encompassed a consensus ATF site, in addition to the DRTF1 binding site, but was mutated to prevent binding of ATF activities. Gel retardation assays Gel retardation was performed as previously described (La Thangue et al., 1990) in a buffer containing 135 mM NaCl, in 50 mM Tris, 0.2 mM EDTA, 1 mM DTT, 15% glycerol and electrophoresed at 150 V for 2 h. In experiments using blastocyst extracts, the amount of salmon sperm DNA was reduced to 0.5 jig, and the extraction buffer was supplemented with 1 mg/mi BSA. In experiments where Ela (12S) was added, the efficiency of translation was assessed by standard procedures and the translate was added to the binding reaction during pre-incubation. UV cross-linking studies Binding reactions were set up essentially as described for gel retardation assays in the presence of a 50-fold excess of mutant oligonucleotide competitor 62/60* using the -82/- 50 CRE-M probe. Before gel retardation samples were irradiated for 20 min. After loading and electrophoresis, complexes were located, excised, boiled in SDS loading buffer, and electrophoresed in a 7.5% SDS-polyacrylamide gel. Glycerol gradient sedimentation Gradients from 10 to 40% glycerol in 450 mM Nacl, 20 mM HEPES pH 7.9, 1 mM DTT, 0.2 mM EDTA were prepared and after loading the sample were centrifuged at 55 000 r.p.m. for 8 h at 4°C. Aliquots were assayed by gel retardation using probe 71/50. In each experiment, molecular weight markers were sedimented in parallel and fractions assayed by SDS electrophoresis. The protein size markers used were carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa) and amylase (200 kDa).
Materials and methods
Acknowledgements
Cell culture F9 EC cells were cultured as adherent monolayers in Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Cells were replated at a density of 4 x 104/ml every 3 days. Stem cells were induced to differentiate to parietal endoderm-like cells by treating them with 0.05 AM retinoic acid, 1 mM dibutyryl-adenosine-3'-5'-monophosphate, and 0.1 mM isobutyl methyl xanthine for up to 7 days (Strickland et al., 1980). Cells were harvested at 3, 5 and 7 days after treatment. CCE ES cells (Robertson et al., 1987) were plated at 1 x 105/mI on to mitomycin-treated STO feeders, and cultured in DMEM supplemented with 20% fetal calf serum. ES cells were separated from STOs for differentiation by trypsinization (Gossler et al., 1986) and were subsequently cultured as aggregates in 10% serum.
We thank Panayotis Tassios and Lan Bandara for critically reading the manuscript, David Latchman and our colleagues in the Laboratory of Eukaryotic Molecular Genetics for their helpful comments, Steve Jackson for the anti-Spl antibodies, Robin Lovell-Badge and Blanche Capel for help with embryo dissections, Randy Rossi for ES cell cultures and Nic Jones for the Ela 12S cDNA. J.F.P. was supported by a SERC CASE award and N.B.L.T. is a Jenner Fellow of the Lister Institute of Preventive Medicine.
Isolation of mouse tissues Tissues were isolated from adult mice (6 week old 129 mice), and from 11.5, 14.5 and 17.5 day old embryos (Parkes). Whole 8.5, 11.5, 14.5 and 17.5 day embryos (Parkes) were also isolated. All embryos and tissues were snap frozen on dry ice. Frozen whole embryos and large pieces of tissue were ground in liquid nitrogen and stored at -70°C. Isolation of blastocysts Six week old female outbred MF1 mice were super-ovulated (Hogan et al., 1986) and mated with (C57 Bllo x CBA) Fl studs. 3.5 days later, blastocysts were collected from the uterine horns of pregnant mice by dissecting out the uterus, and flushing it with 1 mg/mi BSA in PBS. Blastocysts were pooled and frozen in a minimum volume of flushing solution.
3826
References Bagchi,S., Raychaudhuri,P. and Nevins,J.R. (1990) Cell, 62, 659-669. Bagchi,S., Weinmann, R. and Raychaudhuri,P. (1991) Cell, 65, 1063-1072. Bandara,L.R. and La Thangue,N.B. (1991) Nature, 351, 494-497. Bandara,L.R., Adamczewski,J.P., Hunt,T. and La Thangue,N.B. (1991) Nature, 352, 249-251. Bernards,R., Schackleford,G.M., Gerber,M.R., Horowitz,J.M., Friend,S.H., Schartl,M., Bogenmann,E., Rapaport,J.M., McGee,T., Dryja,T.P. and Weinberg,R.A. (1989) Proc. Natl Acad. Sci. USA, 86, 6474-6478. Briggs,B., Kadonaga,J.T., Bell.S.P. and Tjian,R. (1986) Science, 234, 47-52. Chellappan,S.P., Hiebert,S., Mudryi,M., Horowitz,J.M. and Nevins,J.R. (1991) Cell, 65, 1053-1061. Chittenden,T., Livingston,D.M. and Kaelin,W.G. (1991) Cell, 65, 1073-1082.
Embryonic regulation of a transcription factor Doetschman,T.C., Eistetter,H., Katz,M., Schmidt,W. and Kemler,R. (1985) J. Embryol. Exp. Morphol., 87, 27-45. Evans,M.J. and Kaufman,M.H. (1981) Nature, 292, 154-156. Giordano,A., Whyte,P., Harlow,E., Franza,B.R., Beach,D. and Draetta,G. (1989) Cell, 58, 981-990. Gossler,A., Doetschman,T., Korn,R., Serfling,E. and Kemler,R. (1986) Proc. Natl, Acad. Sci. USA, 83, 9065-9069. Hogan,B.L.M., Constantini,F. and Lacy,E. (1986) Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Imperiale,M.J., Kao,H.T., Feldman,L.T., Nevins,J.R. and Strickland,S. (1984) Mol. Cell. Biol., 4, 867-874. Kaelin,W.G., Pallas,D.C., De Caprio,J.A., Kaye.F.J. and Livingston,D.M. (1991) Cell, 64, 521-532. La Thangue,N.B. and Rigby,P.W.J. (1987) Cell, 49, 507-513. La Thangue,N.B. and Rigby,P.W.J. (1988a) In Hames,B.D. and Glover, D.M. (eds), Transcription and Splicing. IRL Press, Oxford, pp. 1-43. La Thangue,N.B. and Rigby,P.W.J. (1988b) Nucleic Acids Res., 16, 11417-11430. La Thangue,N.B., Thimmappaya,B. and Rigby,P.W.J. (1990) Nucleic Acids Res., 18, 2929-2938. Lehner,C.F. and O'Farrell,P.H. (1990) Cell, 61, 535-547. Manley,J.L., Fire,A., Cano,A., Sharp,P.A. and Gefter,M.L. (1980) Proc. Natl Acad. Sci. USA, 77, 3855-3859. Marshall,C.J. (1991) Cell, 64, 313-326. Martin,G.R. (1980) Science, 209, 768-776. Mitchell,P.J. and Tjian,R. (1989) Science, 245, 371 -378. Pines,J. and HunterT. (1990) Nature, 346, 760-763. Robertson,E.J. (1987) In Robertson,E.J. (ed.), Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford, pp. 71-112. Rosner,M.H., Vigano,M.A., Ozato,K., Timmons,P.M., Poirier,F., Rigby,P.W.J. and Staudt,L.M. (1990) Nature, 345, 686-692. Rudnicki,M. and McBurney,M. (1987) In Roberston,E.J. (ed.), Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford, pp. 19-49. Rugh,R. (1968) The Mouse: Its Reproduction and Development. Oxford University Press, Oxford. Scholer,H.R., Hatzopoulos,A.K., Balling,R., Susuki,N. and Gruss,P. (1989) EMBO J., 8, 2543-2550. Shivji,M.K. and La Thangue,N.B. (1991) Mol. Cell. Biol., 11, 1686-1695. Strickland,S., Smith,K.K. and Marotti,H.R. (1980) Cell, 21, 347-355. Tassios,P.T.T. and La Thangue,N.B. (1990) New Biologist, 2, 1123 -1134. Tsai,S.-F., Martin,D.I.K., Zon,L.I., D'Andrea,A.D., Wong,G.G. and Orkin,S.H. (1989) Nature, 339, 446-451.
Received on June 4, 1991; revised on July 31, 1991
Note added in proof We have recently demonstrated using antibodies that the Rb protein is in thymus DRTFIa'.
3827
A developmentally regulated and tissue-dependent transcription factor complexes with the retinoblastoma gene product
Janet F.Partridge and Nicholas B.La Thangue Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, UK
Communicated by D.P.Lane
DRTF1 is a cellular transcription factor which complexes with the retinoblastoma (Rb) gene product and cyclin A, an association modulated by certain viral oncogenes, such as adenovirus Ela. Complexed DRTF1, referred to as DRTFla, has similar DNA binding specificity and DNA binding polypeptides to DRTFlb, which lacks Rb. DRTFlb is abundant in both F9 embryonal carcinoma (EC) stem cells and pluripotent embryonic stem (ES) cells and is strongly down-regulated during the differentiation of both cell types, suggestive of a stem cell Ela-like activity. In contrast, DRTFla, which in F9 EC cells is much less abundant than the other activities, is induced as F9 cells begin to differentiate. Consistent with the relationship between EC, ES and inner cell mass cells, DRTFlb is present in blastocyst stage embryos although as embryogenesis progresses the levels of Rb-complexed DRTF1 increase. Certain tissues, such as liver and brain, contain high levels of DRTF1 during early embryonic stages but little in adult terminally differentiated tissue, in contrast to the thymus, which contains high levels of Rb-complexed DRTF1 but lacks associated cyclin A. These data show that DRTF1 is a group of transcription factors that share common DNA binding polypeptides and which complex with other non-DNA binding proteins, such as the Rb protein and cyclin A, in a developmentally regulated and tissue-dependent fashion. Key words: embryogenesis/retinoblastoma/transcription factor
Introduction During embryogenesis cells divide and differentiate in a defined and programmed fashion, being influenced by both spatial and temporal cues, a process which results in an organism with a wide variety of cell types and precise morphogenetic characteristics. This process requires that gene expression be exquisitely controlled since the appropriate gene product must be available in the right place and at the right time. The most frequent and widely used level for such control is mediated at the point of transcriptional initiation through proteins that bind to defined DNA sequences in the upstream control regions, which are usually described as either promoters or enhancers, bind a combination of transcription factors which confer on a given gene particular transcription properties (La Thangue and Rigby, 1988a; Mitchell and Tjian, 1989). Studies of the molecular mechanisms that regulate transcription during murine embryogenesis are practically Oxford University Press
difficult because of the the limited amount of material available from the embryo proper. This limitation can, however, be partly overcome by using embryonal carcinoma (EC) cells (Martin, 1980) or embryonic stem (ES) cells (Evans and Kaufman, 1981). EC cells, which are the transformed stem cells of teratocarcinomas, differentiate in vitro into defined cell types, a process that is thought to resemble one of the earliest differentiation events that inner cell mass (ICM) cells undergo (Rudnicki and McBurney, 1987). However, they are derived from tumours and therefore are transformed and often karyotypically abnormal. Alternatively, ES cells are derived directly from cultured blastocyst stage embryos and when re-introduced into blastocysts contribute to all tissues, including the germ line (Robertson, 1987); they therefore function as 'normal' pluripotent stem cells. Our previous studies have defined a cellular transcription factor the DNA binding activity of which is down-regulated as F9 EC cells differentiate to parietal endoderm-like cells (La Thangue and Rigby, 1987; La Thangue et al., 1990). This transcription factor, DRTF1 (for differentiationregulated transcription factor 1), exists as several discrete protein complexes when assayed by gel retardation in F9 EC cell extracts, and thus potentially represents a family of transcription factors. In F9 EC cells, DRTFIb is most abundant and functions as a binding site-dependent transcriptional activator when assayed by in vitro transcription (Shivji and La Thangue, 1991). In contrast to F9 EC cells, some cell types have increased amounts of DRTFla which can be dissociated to DRTFIb by the action of the adenovirus Ela protein, an effect mediated by the ability of Ela to sequester the tumour suppressor retinoblastoma (Rb) gene product from DRTFla (Bandara and La Thangue, 1991). In this respect, DRTF1 was the first cellular target defined for the Rb protein with known biological activity, and clearly argues that the Rb protein mediates its growth regulation by controlling transcription, and that it localizes to DNA indirectly by complexing with a sequence-specific transcription factor. The HeLa cell transcription factor E2F binds to a similar motif to DRTF1 and is also targeted by adenovirus Ela (Bagchi et al., 1990), although presently the exact relationship between DRTF1 and E2F is unclear. That DRTFlb is the predominant form in F9 EC cells is consistent with the presence of a cellular Ela-like activity in these cells, orginally defined because these cells allow the expression of an adenovirus that lacks a functional Ela gene product (Imperiale et al., 1984). DRTF1 can therefore complex with other non-DNA binding polypeptides, one being the Rb gene product, an association that may be important for altering the transcription activating properties of this transcription factor. In the present study, we have investigated the developmental regulation and tissue distribution of DRTF1. We found that DRTFlb is abundant in EC, ES and blastocyst
3819
J.F.Partridge and N.B.La Thangue
stage embryos, and moreover is down-regulated as ES cells begin to differentiate. During embryogenesis, the levels of DRTFlb decline whilst those of DRTFla increase. Moreover, the different DRTF1 complexes have characteristic tissue-dependent distributions, and we were able to define a novel form of the Rb-associated activity in the thymus. Our data show, for the first time, that complexing of the Rb protein with DRTF1 is developmentally regulated and tissue dependent. Both proteins, therefore, function as developmentally regulated transcription factors.
Results Differential regulation of DRTF1 during the differentiation of F9 EC stem cells In F9 EC stem cells DRTF1 exists as several distinct forms, referred to as la, lb and ic, which have distinct mobilities when DNA binding activity is assayed by gel retardation, where a is the slowest (Shivji and La Thangue, 1991). DRTF1a contains the Rb-gene product and is modulated by adenovirus Ela; DRTFlb and c lack associated Rb (Bandara and La Thangue, 1991). Previously, we characterized these activities in fractions derived from F9 EC cell extracts, where these activities were enriched and subsequent analysis much easier. We were, however, interested to determine the regulation of these activities as F9 cells differentiate, and to this end prepared extracts at various time points after F9 EC cells were induced to differentiate and assayed DRTF1 by gel retardation (Figure 1). Consistent with previous results, DRTFlb was the most abundant form in EC cells whereas la, which is a complex that includes the lb DNA binding polypeptides (see later), was rare (Figure 1, tracks 2 and 7). Although DRTFIc was less abundant than DRTFIb, it could be clearly resolved in crude whole cell extracts (Figure 1, tracks 2 and 7). As F9 EC cells began to differentiate, there was an immediate reduction in the activity of DRTF1b, whereas DRTFla increased (Figure 1; compare tracks 2 and 3, and 7 and 8). This trend continued during differentiation, so that after 7 days of treatment, when most cells in the culture were parietal endoderm-like (data not shown), DRTF1b was the least abundant activity whereas la and lc were about equal (Figure 1; tracks 5 and 10). During the differentiation process, the activity of DRTFla had increased whereas lb and Ic had decreased, although lb was more strongly regulated than lc. This high resolution analysis of DRTF1 during F9 differentiation indicated that the different forms of DRTF1 are differentially regulated, and suggested that they are subject to different regulatory cues. As a control for the integrity of these extracts, we analysed the activity of the ATF-site DNA binding activity, ECRE2, an activity that remains relatively constant during the differentiation of F9s (Tassios and La Thangue, 1990). As expected, ECRE2 was present throughout differentiation and showed little regulation (Figure 1; tracks 12- 15). We also studied DNA binding activities that recognize the octamer motif, since one of these, oct4, is present only in the pre-implantation embryo and derived stem cells, including EC cells (SchoTer et al., 1989; oct3 in Rosner et al., 1990). It therefore serves as a control for the phenotype of the cells, although it was also intrinsically interesting to compare the regulation of DRTFlb with that of oct4. With this in mind, we assayed octamer motif binding
3820
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Fig. 1. Regulation of DRTF1 during F9 EC stem cell differentiation. F9 EC stem cells were grown as EC cells or differentiated for the indicated times, extracted and assayed for either DRTF1 (tracks I -10), ECRE2 (tracks 11 - 15) or octamer motif binding activities (tracks 16-20) using standard binding sites probes. Tracks 1, 6, 11 and 16 show probe alone. Note that DRTFIb and c were downregulated whereas the binding activity of DRTFla was enhanced by 3 days of differentiation (compare tracks 2 and 3). ECRE2 is constitutively active (tracks 12-15) whereas oct4 is detectable only in F9 stem cells (track 17). The specificity of each complex was confirmed (data not shown), and * indicates a non-specific complex. Tracks 1 -5 show an increased exposure time of tracks 6-10. About 12 jtg of each extract was assayed for DRTF1 and octamer binding activities and 6 yg for ECRE2.
activities during F9 EC differentiation and resolved both octI and oct4 (Figure 1; tracks 17-20). Octl was present throughout differentiation, whereas oct4 was only detectable in the EC cell extract and was rapidly down-regulated since it could not be detected 3 days after the differentiation process had begun (Figure 1, compare tracks 17 and 18). Oct4 and DRTF1b therefore have similar properties in this differentiation system, since both activities are quantitatively downregulated during the same time period. DRTF1 is regulated during the differentiation of embryonic stem cells Because EC cells are the transformed stem cells of teratocarcinomas which lose their transformed phenotype during differentiation, it was important to determine if the regulation of DRTF1 was influenced by this property. We approached this by studying ES cells, which are derived from pluripotent inner cell mass cells of 3.5 day old blastocyst stage embryos (Evans and Kaufman, 1981). In contrast to EC cells, ES cells are not transformed but retain their pluripotency in vitro since they can contribute to all embryonic germ layers when re-introduced into blastocysts. In vitro these cells can be maintained as ES cells or induced to differentiate (see Materials and methods). ES cells were differentiated by allowing them to form embryoid bodies, which are composed of a variety of differentiated cell types representing all three embryonic germ layers (Doetschman et al., 1985). After 2 days, the bodies had developed a Reichert membrane-like rind surrounding ectodermal-like cells, at 7 days they had usually become cystic, and at 8-10 days some were beating whilst others formed blood islands (data not shown). However, the frequency of these events varied from culture to culture despite consistent culture conditions. When ES cell extracts were assayed for DRTF1, the three
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forms of activity were detected and, as in F9 EC stem cells, DRTFlb was most abundant (Figure 2; tracks 1 and 6). DRTF1 was always down-regulated within 2 days after differentiation had begun although the final phenotype of derived cells varied (Figure 2; compare tracks 1 and 6 with 2 and 7) and, as in F9 EC cells, DRTF lb was the most dramatically affected of the activities. After this initial regulation, which was consistent and not influenced by culture or differentiation conditions, the regulation of the different forms of DRTF1 varied with particular ES cell cultures. We show an example of the ES differentiation profile in Figure 2 where, after the initial early reduction in DRTFIb which occurred within 2 days, the levels of DRTF1 fluctuated as differentiation progressed (for example, day 6, where both DRTFla and b were induced), but never reached the much higher levels of DRTFIb in ES cells. This regulation occurred even in the presence of the detergent conditions assayed in Figure 8a (data not shown). The reasons for the fluctuating level of DRTF1 in differentiating ES cells are unclear but are probably related to the inherent variability in the growth rate and phenotype of the differentiating cells. Despite this, other transcription factors, such as GATA 1, a factor enriched in the haematopoietic lineage (Tsai et al., 1989), were induced in parallel with the appearance of blood islands (data not shown). These studies enabled us to make two important conclusions. First, DRTFlb is present at high levels in pluripotent ES cells, and secondly, the DNA binding activity is down-regulated as these cells differentiate. This situation resembles the regulation of DRTF1 during F9 EC differentiation, and therefore argues against the possibility that the regulation in EC cells is caused by reversion of the transformed phenotype, but rather that it reflects the decreasing potency that these cells acquire during differentiation per se. DRTF1 is expressed in blastocyst stage embryos Since both EC and ES cells are thought to resemble cells that comprise the inner cell mass (ICM) of the blastocyst, it was of interest to assess directly if DRTF1 was present at this stage of embryonic development. For this, whole cell extracts were prepared from blastocysts harvested from super-ovulated mice and assayed for DRTF1. Indeed, and
Fig. 3. DRTFIb is expressed in blastocyst stage embryos. About 40 blastocysts (3.5 d.p.c.) were harvested from super-ovulated mice, extracted and assayed for DRTF1 (track 2) or ECRE2 (track 4). For comparison, DRTF1 in F9 EC cell extracts is shown in track 3. The probe alone is shown in track 1. The arrow indicates DRTF1b in blastocyst and F9 EC cell extracts, and * indicates non-specific complexes; specificity was confirmed by assaying binding to mutant binding sites (data not shown). The complex migrating above DRTFIb in this blastocyst extract was not observed in other blastocyst extracts.
as predicted by the EC and ES studies, DRTF1b was present, as was ECRE2 (Figure 3; tracks 2 and 4 respectively). These activities were specific since there was no binding to a mutant DRTF1 or ECRE2 binding site (data not shown). Of course, because of the limited amounts of material that could be obtained from blastocysts, we could not exclude the possibility that, as in ES cells, DRTFla was also present. We conclude, therefore that DRTFIb is the most abundant form of DRTF 1 in blastocyst stage embryos and that this is a property that blastocysts share with EC and ES cells. DRTF1 is regulated during murine embryogenesis Both the EC and ES cell differentiation studies show that the different forms of DRTF1 are independently regulated because in F9 EC cells, for example, DRTFla is more abundant in differentiating cells than in EC stem cells, in contrast to DRTFIb, which has the opposite regulatory profile. With this in mind, we assessed the regulation of the different DRTF1 activities at later stages of early mouse development. Extracts were prepared from whole embryos at 8.5, 11.5, 14.5 and 17.5 days post coitum (d.p.c.) and assayed for DRTF1 in the presence of either a wild-type or
3821
J.F.Partridge and N.B.La Thangue
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from c in these assay conditions, but based on their mobility and our previous data (Shivji and La Thangue, 1991) we believe it reasonable to suggest that DRTF1c had the smaller molecular weight. Thus, DRTFla differs from DRTFla' by -35 000 molecular weight and from DRTFlb/c by 150 000. Also, these data establish that these multicomponent protein complexes associate before DNA binding because the gradient centrifugation was performed before the DNA binding assay, indicating that DRTF1 and the Rb protein exist as a complex in vivo and do not assemble in vitro during the gel retardation assay. We next determined the size of the DNA binding polypeptides in each of the complexes by cross-linking a radiolabelled DRTF1 binding site containing BUdR to the DNA-bound polypeptides (Shivji and La Thangue, 1991). After cross-linking, complexes were resolved in native gels by gel retardation, the appropriate complex excised and cross-linked polypeptides resolved in SDS denaturing gels. We could be confident, therefore, that we were assaying polypeptides in discrete complexes. Both DRTFla and b/c from F9 EC cells contained similar DNA binding polypeptides since a common polypeptide with 50 000 molecular weight was cross-linked (Figure 7, compare tracks 2 and 3). Likewise, a similar molecular weight polypeptide was cross-linked in adult thymus DRTFla' (Figure 7, track 4). A polypeptide with 30 000 molecular weight also cross-linked in all the complexes assayed (Figure 7, indicated by *, although the efficiency varied from experiment to experiment; this weak binding efficiency was also noted in our earlier experiments (Shivji and La Thangue, 1991). It is presently unclear why this is so, because this 30 000 molecular weight polypeptide is abundant in DRTF1 preparations affinity purified by bindingsite affinity chromatography (Shivji and La Thangue, 1991; and data not shown). However, these studies clearly 3824
3
Fig. 7. The DRTF1 complexes have common DNA binding polypeptides. Gel retardation was performed with either F9 EC or adult extracts after cross-linking to the DRTF1 binding site in the presence of competing oligonucleotide 62/60*, and specific complexes were located, excised and cross-linked polypeptides resolved by SDS-gel electrophoresis. The specificity of each complex was confirmed in a parallel assay (data not shown). The polypeptides crosslinked in F9 EC DRTFlb/c and DRTFla are shown in tracks 2 and 3, and those in adult thymus DRTFIa' in track 4; standard molecular weights (x 1000) are shown in track 1. Note that the predominant polypeptide in each complex is p5O, and that p30 (indicated by *) cross-links inefficiently in these assay conditions.
demonstrate that all the DRTF1 complexes contain a common DNA binding polypeptide of 50 000 molecular weight, and since each complex had identical sequence specificity, it is very likely that this DNA binding polypeptide is common to all DRTF1 complexes and that the different native molecular weights result from the association of other non-DNA binding proteins with these common polypeptides. To confirm this idea, we tested if DRTFla could be dissociated by treating it with a variety of agents and assayed the effect by gel retardation. There was some dissociation of DRTFla and a significant enhancement of the lb/c binding activity when treated with detergent (Figure 8a, compare tracks 2, 3 and 4). In F9 EC cell extracts this effect could also be mediated by certain viral oncogenes, such as adenovirus Ela and SV40 large T antigen (Bandara and La Thangue, 1991; L.R.Bandara and N.B.La Thangue, data not shown), an effect mediated by the ability of these proteins to dissociate DRTFla. Adenovirus Ela behaved in a similar fashion in tissue extracts because it dissociated thymus DRTF1a' to DRTF1b (Figure 8b, compare tracks 2 and 3). In this respect, DRTF1 behaves in a similar fashion to E2F (Bagchi et al., 1990). Since DRTFla' has a molecular weight of 165 000 and DRTF1b/c one of 50 000, this effect most probably results from the ability of Ela to sequester the Rb protein from DRTFla', a dissociation that results in the minimal DNA binding unit. The combined conclusion from these data is that DRTF 1 can complex with the Rb protein to form a multicomponent complex, an association which is regulated during development and one which is targeted by viral Ela. -
Discussion DRTF1 complexes with the retinoblastoma tumour product The data presented in this study show that DRTF1 is a transcription factor that is developmentally regulated and tissue dependent, and that its association with the Rb protein is also under similar control. Although the Rb gene product
suppressor gene
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8
Fig. 8. (a) DRTFI DNA binding is enhanced in the presence of detergent. F9 EC whole cell extracts were treated with either NP40 (tracks 3 and 7) NP40 and deoxycholate together (tracks 4 and 8) and assayed for either DRTF1 (tracks 3 and 4) or ECRE2 (tracks 7 and 8); tracks 2 and 6 show untreated F9 EC cell extract, and tracks and 5 the probe alone. Note that in track 4 DRTFIb/c DNA binding activity is enhanced. * indicates a non-specific complex. (b) Thymus DRTFla' can be dissociated in vitro by adenovirus Ela. An adenovirus 12S Ela cDNA was transcribed and translated in vitro and added to a fraction from adult thymus extract containing DRTFIa' (taken from the glycerol gradient analysis shown in Figure 6) and DRTF1 assayed by gel retardation in the presence of competing oligonucleotide 62/60*. The effect of in vitro translated Ela (track 2) was compared with reticulocyte lysate alone (track 3); the binding activity in the fraction alone is shown in track 1. * indicates a non-specific complex. or an
is known to complex with a variety of cellular polypeptides through which it may mediate its effects as a negative regulator of cellular proliferation (Kaelin et al., 1991), DRTF1 was the first target identified with known biological function. Since then, others have reported that the Rb protein binds to E2F (Bagchi et al., 1991; Chellappan et al., 1991; Chittenden et al., 1991). That the Rb protein is able to complex with such transcription factors clearly argues that the Rb protein mediates some of its growth-regulating effects by altering the transcriptional activating properties of target transcription factors such as DRTF1. In F9 EC and ES cells DRTFIb was more abundant than DRTFIa. Since the Rb protein is sequestered from DRTFla by the adenovirus Ela protein and SV40 large T antigen (L.R.Bandara and La Thangue, 1991; Bandara and N.B.La Thangue, data not shown), we believe that this is further evidence for a cellular Ela-like activity which acts to prevent the Rb protein from complexing with cellular proteins, such as DRTF1, in stem cells of early embryogenesis. Consistent with this model, as embryonic development progressed Rb-complexed DRTF1 became more abundant whereas the levels of Rb-free DRTF1 declined. We suggest, therefore, that the transcriptional activating properties of DRTF1 are regulated during embryonic development, and that part of this regulation is mediated through complexing with the Rb protein. DRTFlb was abundant in F9 EC cells and down-regulated during differentiation, whereas DRTFla was regulated in the converse fashion. Since the abundance of induced DRTF1a was much less than the reduction in Ib, association with the Rb protein cannot therefore be the only mechanism responsible for regulating DRTFlb, and other as yet undefined mechanisms must also be operational. DRTF1 complexes are tissue-dependent Each tissue examined has a characteristic pattern of DRTF 1 DNA binding activity. In general, there were high levels
of DRTF 1 when the tissue concerned underwent maximum proliferation and differentiation. For example, in embryonic liver this occurred between days 11.5 and 17.5, when cells in this tissue are actively dividing and proliferating. This suggests that DRTF1 is involved in regulating stem cell proliferation, perhaps by targeting genes whose protein products are required for progression through the cell cycle. This idea is also supported from the EC and ES cell studies because DRTFlb was abundant in stem cells, but not in their differentiated derivatives, and is consistent with the fact that it is the transcriptionally active form of DRTF1 (Shivji and La Thangue, 1991). We believe, therefore, that DRTF1 has an important role in regulating the transcription of genes whose protein products are required for progression through the cell cycle. It was also clear during brain development that DRTFlc predominated, a situation that contrasts with liver development. We do not know how DRTF lb and lc differ, but we have found that DRTFlb has a larger size than DRTFlc (Shivji and La Thangue, 1991), an effect that could result from a post-translational modification or another complexing protein. Our previous data also indicate that DRTFlc is transcriptionally active, so we suggest again that its function during brain development is to positively regulate stem cell proliferation. Testis, thymus and embryo extracts from late stages of embryogenesis contained a novel form of DRTF1, which we referred to as DRTFla', because it had a smaller native molecular weight than DRTFla but was still very much larger than DRTFlb; we believe that DRTFla' is the same activity that we were able to generate in our previous analysis by detergent treatment (Shivji and La Thangue, 1991). Both DRTFla and la' are sensitive to the action of adenovirus Ela. However, DRTFla contains an additional molecule, cyclin A (Bandara et al., 1991), which is probably responsible for the different native molecular weights of DRTFla and la'. Like the Rb protein, cyclin A is also sequestered by adenovirus Ela (Giordano et al., 1989). That 3825
J.F.Partridge and N.B.La Thangue
DRTFIa' rather than DRTFla exists in certain tissues and also during the later stages of murine development argues that the association of cyclin A with the DRTF1 complex is also developmentally regulated. DRTF1 as a universal regulator of stem cell proliferation DRTF1 is expressed both in vivo and in vitro in early embryonic stem cells, and during the early developmental stages of certain tissues, when they undergo maximum proliferation, whereas in terminally differentiated cells and tissues it is present, with the exception of the thymus, at low levels. These data suggest a role for DRTF1 in regulating the progression of cells into the cell cycle and hence proliferation, a model consistent with its interaction with the Rb protein, which is a negative regulator of cell growth (Marshall, 1991). The Rb protein may control the transcriptional activity of DRTF1 by directly complexing with the DNA-bound transcription factor and through this association regulate genes required for cell cycle progression. That DRTF1 involves an association with cyclin A is consistent with these ideas since cyclin A undergoes periodic accumulation and destruction during each cell cycle and is required for normal cell cycle progression (Lehner and O'Farrell, 1990; Pines and Hunter, 1990). These data not only demonstrate that DRTF1 is developmentally regulated, but also for the first time suggest that the Rb protein is under developmental control. However, this is not at the level of gene expression, since the Rb protein is expressed constitutively during murine development (Bernards et al., 1989), but rather at the level of its association with its target proteins. For DRTF1, this association may have important consequences for transcriptional control. We believe that an understanding of the mechanisms that control this interaction will provide important insights into the regulation of stem cell proliferation during embryonic development.
Preparation of whole cell extract Whole cell extracts were prepared from 5 ml of packed cells, as previously described (Manley et al., 1980; La Thangue and Rigby, 1988b), for the experiment in Figure 1. For all other experiments, whole cell extracts from small numbers of cells or small quantities of tissue (micro-extracts) were prepared essentially as described previously (Scholer et al., 1989) but were extracted by freeze-thawing; protein concentrations were determined by the Bradford assay using the Bio-Rad protein assay. Oligonucleotides Oligonucleotides containing DRTF1 and ECRE2 binding sites, 71/50 and P respectively, were as previously described as were the mutant sites 62/60*, 63* and Pml (La Thangue et al., 1990; Tassios and La Thangue, 1990). The octamer binding site probe contained a consensus octamer taken from the immunoglobulin heavy chain enhancer (sequences -554 to 536). The oligonucleotide used for UV cross-linking experiments, -50/-82 CREM, encompassed a consensus ATF site, in addition to the DRTF1 binding site, but was mutated to prevent binding of ATF activities. Gel retardation assays Gel retardation was performed as previously described (La Thangue et al., 1990) in a buffer containing 135 mM NaCl, in 50 mM Tris, 0.2 mM EDTA, 1 mM DTT, 15% glycerol and electrophoresed at 150 V for 2 h. In experiments using blastocyst extracts, the amount of salmon sperm DNA was reduced to 0.5 jig, and the extraction buffer was supplemented with 1 mg/mi BSA. In experiments where Ela (12S) was added, the efficiency of translation was assessed by standard procedures and the translate was added to the binding reaction during pre-incubation. UV cross-linking studies Binding reactions were set up essentially as described for gel retardation assays in the presence of a 50-fold excess of mutant oligonucleotide competitor 62/60* using the -82/- 50 CRE-M probe. Before gel retardation samples were irradiated for 20 min. After loading and electrophoresis, complexes were located, excised, boiled in SDS loading buffer, and electrophoresed in a 7.5% SDS-polyacrylamide gel. Glycerol gradient sedimentation Gradients from 10 to 40% glycerol in 450 mM Nacl, 20 mM HEPES pH 7.9, 1 mM DTT, 0.2 mM EDTA were prepared and after loading the sample were centrifuged at 55 000 r.p.m. for 8 h at 4°C. Aliquots were assayed by gel retardation using probe 71/50. In each experiment, molecular weight markers were sedimented in parallel and fractions assayed by SDS electrophoresis. The protein size markers used were carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa) and amylase (200 kDa).
Materials and methods
Acknowledgements
Cell culture F9 EC cells were cultured as adherent monolayers in Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Cells were replated at a density of 4 x 104/ml every 3 days. Stem cells were induced to differentiate to parietal endoderm-like cells by treating them with 0.05 AM retinoic acid, 1 mM dibutyryl-adenosine-3'-5'-monophosphate, and 0.1 mM isobutyl methyl xanthine for up to 7 days (Strickland et al., 1980). Cells were harvested at 3, 5 and 7 days after treatment. CCE ES cells (Robertson et al., 1987) were plated at 1 x 105/mI on to mitomycin-treated STO feeders, and cultured in DMEM supplemented with 20% fetal calf serum. ES cells were separated from STOs for differentiation by trypsinization (Gossler et al., 1986) and were subsequently cultured as aggregates in 10% serum.
We thank Panayotis Tassios and Lan Bandara for critically reading the manuscript, David Latchman and our colleagues in the Laboratory of Eukaryotic Molecular Genetics for their helpful comments, Steve Jackson for the anti-Spl antibodies, Robin Lovell-Badge and Blanche Capel for help with embryo dissections, Randy Rossi for ES cell cultures and Nic Jones for the Ela 12S cDNA. J.F.P. was supported by a SERC CASE award and N.B.L.T. is a Jenner Fellow of the Lister Institute of Preventive Medicine.
Isolation of mouse tissues Tissues were isolated from adult mice (6 week old 129 mice), and from 11.5, 14.5 and 17.5 day old embryos (Parkes). Whole 8.5, 11.5, 14.5 and 17.5 day embryos (Parkes) were also isolated. All embryos and tissues were snap frozen on dry ice. Frozen whole embryos and large pieces of tissue were ground in liquid nitrogen and stored at -70°C. Isolation of blastocysts Six week old female outbred MF1 mice were super-ovulated (Hogan et al., 1986) and mated with (C57 Bllo x CBA) Fl studs. 3.5 days later, blastocysts were collected from the uterine horns of pregnant mice by dissecting out the uterus, and flushing it with 1 mg/mi BSA in PBS. Blastocysts were pooled and frozen in a minimum volume of flushing solution.
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Received on June 4, 1991; revised on July 31, 1991
Note added in proof We have recently demonstrated using antibodies that the Rb protein is in thymus DRTFIa'.
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