Control of gene expression: tissue-specific expression Roger K. Patient Division of Biomolecular Sciences, King's College, London, WC2B 5RL, UK Current Opinion in Biotechnology 1990, 1:151-158

Introduction Tissue-specific gene expression is determined by the cellular environment. The major point at which control over expression is exerted is at transcription initiation; consequently, the primary focus of studies on the changing cellular environment during differentiation and development has been on transcription factors. Despite the ever-burgeoning number of factors being described in the literature, few have proved to be tissue-specific, leading to the belief that combinations of factors, rather than individual proteins, will prove to be critical to tissue-specific-expression. Receiving less attention, but probably no less important, is the role played by the precise chromatin structure of individual genes. For a given gene, this clearly varies between cell types and, although it is likely to be altered by some interaction with 'transcription factors', it may also determine which factors can gain access to the DNA template. The role of histones as generalized repressors is becoming clearer, with specifically positioned nucleosomes playing a particularly important part. The region over which chromatin structure is disrupted corresponds to the size of chromosome loops, and these domains appear to be under the control of DNA sequence elements at their boundaries; these are known, in the case of the human [3-globin locus, as the dominant control region (DCR) or locus activation region (LAR). In this review, I will attempt to highlight some recent developments in our understanding of the mechanisms by which transcription factors interact with their cognate DNA sequences in chromatin and with the basic transcriptional machinery. I will also review attempts to understand the mechanism by which the DCR/LAR works.

Transcription factors: tissue specificity and mechanism of action The number of transcription factors whose presence in the cell appears to be the sole determinant of the tissuespecific transcription of responding geneg is very small.

Because of the complexity of the system, it is not yet possible to give a clear description of the factors involved in many cases. One example of a single factor apparently determining the expression of a particular gene in a single cell type is the anterior pituitary transcription factor known as Pit-1 or growth hormone factor (GHF)-I. This factor is clearly responsible for growth hormone expression in the somatotrophic cells of the pituitary [1,2,3"']. Whether it also controls prolactin expression in the lactotrophic cells of the pituitary is still disputed [1,2,3 °°]. The timing of the appearance of Pit-1/GHF-1 during differentiation of the pituitary correlates precisely with growth hormone expression and suggests that it has a role in the specification of the somatotrophic cell lineage and commitment to terminal differentiation [3"']. The control of the expression of Pit-1/GHF-1 itself has now been examined [4. ]. This gene is stimulated by cAMP and its continued expression is ensured by autoregulation. However, its tissue-specific transcription appears to be determined by an as yet unidentified factor. Furthermore, during differentiation of the pituitary, the Pit-1/GHF-1 protein appears 3 days after its mRNA, suggesting that a translational control is operating [3 "']. Pit-1/GHF-1 contains a homeodomain and a so-called POU-specific domain, being one of the transcription factors that led to the identification of the POU domain (the others being Oct-l, Oct-2 and Unc-86 [5] ). Although the homeo domain is involved in DNA binding as in other homeobox proteins [6"], the POU-specific domain is also required for high-affinity, sequence-specific binding [7"]. The POU-specific domain is also involved in dimerization of Pit-1/GHF-1 on the DNA. The N-terminal serine-rich and threonine-rich region appears to be primarily responsible for the transcriptional activation function [6" ,7" ]. The mechanism by which this protein induces transcription is at present unclear, but studies of viral and ubiquitous transcription factors have begun to yield some mechanistic clues that are likely to apply to tissue-specific factors. Transcription factors generally contain distinct DNAbinding and activation domains [ 8 " , 9 " ] . The activation domains characterized so far, in addition to the serine-rich, threonine-rich region described above, include those rich in either acidic amino acids (e.g. VP16),

Abbreviations DCR~dominant control region; GHF~growth hormone factor; LAR--Iocus activation region; MMTV--mouse mammary tumour virus. (~) Current Biology Ltd ISSN 0958-1669

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glutamine (e.g. Spl), or proline (e.g. CTF/NF-1) [9"]. Although the popular view has been that these factors somehow contact the basic transcription machinery [10], dear evidence was lacking. In addition, it was unclear how the different activation domains might contact the same machinery. A flurry of recent papers has addressed these questions, albeit with some residual contradictions. The transcription machinery consists of RNA polymerase 1I and a number of ancillary proteins including TFIIA, TFnB, TFIID, TFIIE and TFIIF [11 ",12" ]. The ordered assembly of these proteins at the transcription initiation site is triggered by the binding of TFIID at the TATA box. DNA templates are thus committed to transcription by the binding of TFIID and it is this protein that the activators appear to contact, either directly or via an intermediary protein or 'adaptor/coactivator' (Fig. 1). The evidence for direct contact comes from the demonstration that an affinity column containing VP16, a viral acidic transactivator, can remove TFIID from a nuclear extract, and also bind cloned yeast TFIID [13"]. This rather convincing evidence is, however, contradicted by two types of transcription experiment, namely 'squelching' experiments and in vitro transcription assays in which the activation responses of cloned and partially purified TFIID proteins are compared. Squelching is the term used to describe the inhibition of transcription observed in the presence of excess transcriptional activators [14]. It has been suggested that the inhibition is caused by sequestration of the component of the transcriptional machinery that mediates the activation response. However, the failure to relieve VP16mediated inhibition by the addition of TFIID [15"'] sug-

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gests that TFIID is not the target of VP16 and implicates an adaptor/coactivator. In in vitro transcription assays, using extracts in which TFIID had been removed by fractionation, activation by Spl, CTF/NF-1 and VP16 was not rescued by the addition of cloned TFIID [16oo,17"o,18"]. (A contradictory result in the literature [19"'] may relate to the different extract fractionation procedures used.) In contrast, partially purified, native TFIID was able to complement these extracts. Furthermore, cloned TFIID was able to rescue extracts depleted of TFIID by heat treatment instead of by fractionation [17"']. The most plausible explanation for these observations is that the partially purified TFIID fractions contain a tightly associated adaptor/coactivator, and that this moiety is unaffected when extracts are heated to remove TFIID.

Squelching experiments have also provided evidence that, while some activators share common targets [ 1 5 " , 2 0 " ] , others have distinguishable targets [21 "']. In particular, although excess VP16 competes with EIA for its target in the transcription machinery, the reverse does not occur; that is, E1A does not squelch VP16. Thus, while these activators share a target in the transcriptional machinery, EIA has an additional target not required by VP16. The current reasonable interpretation of these observations is that even ifVP16 contacts TFIID directly, EIA does so via an adaptor/coactivator [21 ..]. It should be emphasized here that not all the proposed adaptors/coactivators need bind between the transactivators and TFIID. As pointed out by Ptashne and Gann [18.,], many of the observations could equally well be explained by the requirement for an adaptor/coactivator

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Fig. 1. Models for transcriptional activation. The activation domains of transcriptional activators may either contact TFIID directly (a) or via an intermediary adaptor/coactivator (b). In (a), an adaptor/coactivator may function by maximizing the efficiency of interaction with the rest of the transcription machinery. The DNA-binding and activation domains need not be part of the same polypeptide chain.

Control of gene expression: tissue-specific expression Patient to stimulate the rest of the transcription machinery maximally (Fig. 1). It remains to be seen whether adap:ors/coactivators display variability between cells, but such variability could, of course, confer tissue specificity on ubiquitous transcription factors.

Transcription factors: differentiation and cell division Although DNA replication may assist the gene reprogramming required for commitment [22,23"'], cell division and differentiation are clearly incompatible [24]. A couple of recent publications [25",26"°] suggest that some of this incompatibility may be determined by competitive transcription factor binding. One example of this, and another example of a single transcription factor specifying tissue type, is MyoD and its role in the muscle lineage. This factor binds to musclespecific enhancers [27] and induces muscle characteristics in a variety of cell types [28]. The MyoD protein contains a domain with homology to c-Myc, which is involved in the control of cell division [29]. Recently, it has been shown that this domain is responsible for the cessation of cell division in muscle differentiation, and that it acts independently of the transcriptional activation domain [25..]. Another link between differentiation and cell division may have been provided by recent data on the induction of bone differentiation by vitamin D 3 and the vitamin A metabolite retinoic acid [26.-]. The binding site for the receptors of these hormones upstream of the human osteocalcin gene overlaps an AP-1 binding site, which binds a Jun/Fos dimer. Furthermore, Jun/Fos, which is induced by serum and is found in dividing cells, suppresses osteocalcin expression. The competitive relationship between the hormone receptors and Jun/Fos may have a role in coordinating expression of the osteocalcin gene with cessation of cell division during bone differentiation.

in the presence of the appropriate transcription factors (TFIID and activator proteins) [22,23.-]. Altematively, some transcription factors may be able to bind to their targets even when they are wrapped around a nucleosome, and this binding may lead to nucleosome removal. The latter mechanism is presumably applicable to some cases of rapid induction of transcription, for example by steroid hormones. Zaret and Yamamoto [34] showed several years ago that the mouse mammary tumour virus (MMTV) promoter becomes nucleosomefree within minutes of glucocorticoid induction. Attempts to reproduce these events in vitro have, so far, been only partly successful. The MMTV promoter contains a strong nucleosome phasing signal such that a reconstituted nucleosome sits in a very specific position incorporating the glucocorticoid receptor binding sites and the NF-1 binding site [35,36], which must be filled by their cognate factors for transcription to occur [37-39]. When the glucocorticoid receptor was added to a mononucleosome in the presence of hormone, binding was observed and the nucleosome stayed iri place [36]. This appeared to imply that the glucocorticoid receptor was not responsible for nucleosome displacement. More recently, however, Pina et al. [40..] have shown that the binding of the glucocorticoid receptor does induce a conformational change in the nucleosome such that the NF-1 binding site, which was hitherto facing in towards the histone core and therefore inaccessible, rotates and may facilitate binding of NF-1. However, neither NF-1 binding nor nucleosome loss has yet been demonstrated in these in vitro experiments, leading to the suggestion that histone acetylation or the histone chaperones, nucleoplasmin or N1/N2, may also be required [40--]. Similarly, phased nucleosomes have been found over the [3-globin promoter in chickens [41], and also over the enhancer in chickens and the ~-globin promoter in humans (R Buckle et al., personal communication); this latter position overlaps a previously identiffed negative transcription element [42].

The role of nucleosomes

Chromosomal domains

Returning to Pit-1/GHF-1, the transcriptional elevation of the growth hormone gene conferred by this factor, either in vitro or in transfection assays, is orders of magnitude below that observed in vivo, where the difference in expression between, for instance, liver and pituitary is 108 [1,30]. As suggested 5 years ago by We'mtraub for globin gene expression [31], and more recently by Karin for growth hormone expression [1], the difference is probably a reflection of chromatin structure. The presence of nucleosomes on promoters inhibits transcription both in vitro [32" ] and in vivo [33]. In particular, the binding of TFIID is prevented, even in the presence of added transcriptional activators [32" ]. Therefore, to allow transcription, the nucleosome masking the TFUD binding site must be removed. One way of doing this in vivo might be to replicate the template

In addition to local effects, chromatin structure around active genes is known to alter throughout large domains, which correspond in size to chromosome loops [43]. The activity of these domains is thought to be specified by DNA sequences near their boundaries. For several gene systems these sequences (DCR and IAR in the case of the human [3-globin locus), have been shown to be the dominant influence on tissue-specific expression [44,45,46 "',47 .,48 "',49 "'], even overriding local factor binding sites [46..]. However, local sequences are thought to have critical roles in determining which members of a tissue-specific gene family are expressed during development. (O Hanscombe et al., personal communication) [ 5 0 " - 5 3 " ' ] . The most studied boundary sequence is the DCR/LAR found flanking the human ]3-globin gene locus. The re-

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gion is characterized by a cluster of four DNaseI hypersensitive sites (Fig. 2) which are specific to erythroid cells [44,45,54]. When linked to a number of genes, both erythroid and non-erythroid, this region specifies erythroid expression in stably transfected cells and in transgenic mice [44,45,46°°]. Furthermore, the level of expression approximates to that of the endogenous mouse [3-globin gene and is independent of the site of integration in the chromosome. These sequences therefore represent the single most important determinant of tissue-specific gene expression discovered so far.

factor NFE-1 [61 "',62"']. In addition, the more locusproximal of the two middle sites has been shown to act as an erythroid enhancer in transient transfection assays [63 "]. This activity maps to two tandem AP-1 (Jun/Fos) binding sites [64-] which also bind another erythroid transcription factor, NFE-2 [62.-]. It is possible that, as for the human osteocalcin gene [26"] described above, Jun/Fos is a negative regulator of activity. Thus, mutually exclusive binding of NFE-2 and Jun/Fos may contribute to the coordination of DCWLARactivity and the cessation of cell division.

Much of the more recent work on these sequences has centred on their interaction with successive [3-globin family members during development. This family switches from e to 7 to [3 expression as development proceeds through embryonic, foetal and adult stages [55]. The emerging picture (Fig. 2) can be summarized as follows: the DCWLAR initially acts on the proximal e gene; during foetal development, a negative element upstream of the g gene becomes activated [42,52 °',53"',56 °], and so expression switches to the 7 genes; during adult development, the [3-globin gene enhancers become activated and confer a competitive advantage on the [3 promoter, so expression switches to the [3 gene (O Hanscombe et al, personal communication) [50"',51 "'].

The mechanism by which these regions activate the locus is still unknown. A favoured idea, that they function as nuclear scaffold attachment sites, was made more attractive by the recent discovery of similar transcriptional properties associated with a region upstream of the chicken lysozyme gene that was originally identified as a scaffold attachment site [48",49"]. However, the evidence in the human [3-globin locus is not yet compelling as, although a scaffold attachment site does map adjacent to the DCR/LAR, a number of others are present throughout the locus [65]. This 'excess' of sites may reflect the tendency of the available mapping methods to detect all potential sites, and not only those actually in use [66].

Attempts to define the critical sequences in the DCWLAR have revealed a hierarchy amongst the hypersensitive sites: the middle two are more important, and the site upstream of these two has more activity in transgenic mice than in stably transfected erythroid cell lines [57",58",59",60"]. Both the middle two sites contain at least three binding sites for the erythroid transcription

An altemative possible mechanism stems from the relationship between replication timing and transcription [67,68]. In particular, the whole of the [3-globin locus replicates early in S-phase in erythroid cells, but late in non-erythroid cells [69,70]. Thus, although early replication might be merely a consequence of the transcriptional activity of the locus, it is also possible that the I)CWLAR functions by specifying replication early in S-

Fig. 2. Developmental and tissue-specific expression of the human [[3-globingene locus. The locus is not drawn to scale and only the active genes are included, Horizontal arrows depict the direction of transcription; vertical arrows indicate either silencer/enhancer elements (thin arrows) or the DNasel hypersensitive sites (thick arrows) that define the dominant control region (DCR)/Iocus activation region (LAP,).

Control of gene expression: tissue-specific expression Patient 155 phase. This might then permit the binding of critical factors whose abundance or activity peaks in early Sphase, precipitating the chromatin structural alterations and subsequent transcriptional activation characteristic of erythroid cells. Consistent with this notion, deletion of the DCR/LAR is associated with a delay in the timing of replication of the whole locus [71 "']. Finally, it is worth reiterating an idea that was floated in a recent review by Jim Allan and myself [72 " ] . The loss of nucleosomes in the DCR/LAR region, detected as DNaseI hypersensitive sites, could lead to unfolding of the higher order fibre. Depending on the structure of the fibre, in particular the path of the linker DNA, such unfolding could release a wave of DNA supercoiling that could pass through the domain and lead to loss of the nucleosomes masking the transcription factor binding sites. This effect would be 'felt' first by the proximal gene, i.e. e, and may subsequendy be propagated to the next genes, i.e. 7 and finally [~.

Conclusion A knowledge of the components and mechanisms controlling tissue-specific gene expression is invaluable for optimizing expression systems in cultered cells and transgenic animals and, ultimately, for gene therapy. In particular, locus controls, such as the DCWLAR from the human [3-globin gene locus may be utilized to maximise the expression of genes intergrated into chromosomes. In the absence of homologous recombination/allelic replacement, the locus-controlling sequences may be required to obtain expression of the integrant regardless of the surrounding chromosome.

3.

DOLLEP, CASTRILLOJ-L, THEILL LE, DEERINCK T, ELLISMANM, KARINM: Expression of GHF-1 protein in m o u s e pituitaries correlates b o t h temporally and spatially w i t h the onset of g r o w t h h o r m o n e gene activity. Cell 1990, 60:809-820. In situ hybridization patterns and immunohistochemistry of Pit-i/ GHF-1 were compared with that of growth hormone and prolactin in developing mouse embryo sections. Growth hormone and Pit-1/GHF-1 expression correlate, whereas prolactin and Pit-1/GHF-1 expression do not. Pit-1/GHF-1 protein appearance lags behind that of its mRNA by 3 days. . .

4. •

MCCORMICK A, BRADY H, THE1LL LE, KARIN M: Regulation of the pituitary-specific h o m e o b o x gene GHF-1 by cell-autonomous and environmental cues. Nature 1990, 345:829-832. Analysis of factors which bind to the Pit-1/GHF-1 promoter and regulate transcription; cAMP and Pit-1/GHF-1 itself have a positive effect. 5.

HERE W, STURM RA, CLERC RG, CORCORAN LM, BALTIMORED, SHARP PA, INGRAHAMHA, ROSENFELDMG, FINNEY M, RUVKUNG, HORVIqZ HR: T h e POU domain: a large conserved region in t h e mammalian pit-l, oct-l, oct-2, and Caenorhabditis elegans unc-86 g e n e products. Genes Dev 1988, 2:1513-1516.

6. •

THEILLLE, CASTPaLLOJ-L, Wu D, KARIN M: Dissection of functional domains of the pituitary-specfiic transcription factor GHF-1. Nature 1989, 342:945-948. The homeodomain of Pit-1/GHF-1 is sufficient for sequence-specific DNA binding, but activity is stimulated by the POU-specific domain. The transcriptional activation region is rich in hydroxylated serine and threonine residues, 7. •

INGRAHAMHA, FLYNN SE, VOSS JW, ALBERTVR, KAPILOFF MS, WILSONL, ROSENFELDMG: T h e POU-specific domain of Pit-1 is essential for sequence-specific, high-aflLrfity DNA binding and DNA-dependent Pit-l-Pit-1 interactions. Cell 1990, 61:1021-1033. Identification of DNA-binding, dimerization and transcriptional activation domains in Pit-1/GHF-1. The POU-specific domain is essential for recognition of natural response elements. 8. JOHNSONPF, McKNIGHT SL: Eukaryotic transcriptional regu•• latory proteins. Annu Rev Biochem 1989, 58:799-839. Predictably comprehensive review, focussing on DNA binding, target site or protein degeneracy, heterodimerization and mechanisms of latency. 9. ••

Acknowledgements I would like to thank the Medical Research Council and the Wellcome Trust for their support. I would also like to thank Dave Greaves and Frank Grosveld, Mark Groudine, Tariq Enver and Jim Allan for communicating their results to me ahead of publication. I am also grateful to Jim Allan, Martin Cullen, Geoff Partington and Angus Wilson for critical reading of the manuscript.

References and recommended reading • ••

Of interest Of outstanding interest

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11. ••

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SALTZMANAG, WEINMANN R: Promoter specificity and modulation of RNA polymerase II transcription. FASEB J 1989, 3:1723-1733. A detailed summary of the properties of the basic transcription machinery which consists of RNA polymerase II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and SII. 13. ••

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SCHULER, UMESONO K, MANGELSDORFnJ, BOLADOJ, PIKE JW, EVANSRM: Jun-fos and receptors for vitamins A and D recognize a c o m m o n response e l e m e n t in the h u m a n osteocalcin gene. Cell 1990, 61:497-504. The binding sites for the vitamin D receptor, the retinoic acid receptor and AP-1 are shown to be coincident. Co-transfection assays show that Jun/Fos suppresses both basal and hormone-induced transcription.

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26. ••

• •

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21. ••

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PINA B, BRUGGEMEIERU, BEATO M: N u c l e o s o m e positioning modulates accessibility of regulatory proteins to t h e m o u s e m a m m a r y t u r n o u t virus promoter, Cell 1990, 60:719-731. Hormone receptors bind to the MMTV promoter with similar affinities regardless of the presence or absence of the phased nucleosome, but NF-1 only binds to naked DNA. However, the conformational alteration to the nucleosome brought about by hormone receptor binding may facilitate NF-1 binding.

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LINDENBAUMMH, GROSVELD F: An in vitro globin gene switching model based o n differentiated embryonic s t e m cells. Genes Dev 1990, 4:2075-2085. Mouse embryonic stem cells differentiated in culture mimic haematop oietic development. The transfected h u m a n c-globin gene linked to the DCR/LAR is correctly regulated developmentally, regardless of the presence of other globin genes on the same fragment, suggesting that although the 7-fl switch depends on competition, the e switch-off does not. ..

40. •,

41.

KEFALASP, GRAYFC, ALLANJ: Precise n u c l e o s o m e positioning in the p r o m o t e r of the c h i c k e n ~A globin gene. Nucleic Acids Res 1988, 16:501-517.

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LAMB P, WATT P, PROUDFOOT N: Negative regulation of t h e h u m a n embryonic globin g e n e s zeta and epsilon. In Hemoglobin Switching, Part A. Transcriptional Regulation. Alan R. Liss Inc, 1989, pp 269-277.

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GASSERSM, LAEMMLIUK: A glimpse at c h r o m o s o m a l order. Trends Genet 1987, 3:16-22.

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RAICHR, ENVER T, JOSEPHSON B, NAKAMOTO B, PAPAYANNOPOULOU T, STAMATOYANNOPOULOSG: A u t o n o m o u s c o n t r o l of h u m a n embryonic globin gene switching in transgenic mice. Science, in press. The h u m a n g-globin gene linked to the DCR/LAR is expressed only in embryonic erythroid cells in transgenic mice. Thus, the control of g-globin expression is independent of linkage to the 7- and ~-globin genes. 54.

TtlAN D, SOLOMON W, LI Q, LONDON IM: T h e '~-like-globin' g e n e domain in h u m a n erythroid cells. Proc Natl Acad Sci USA 1985, 82:63844388.

GROSVELDF, BLOM VAN ASSEMDELFTM, GREAVESD, KOLLIASG: Position-independent, high level expression of t h e h u m a n ~-giobin g e n e in transgeulc mice. Cell 1987, 51:21-31.

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COLLINS F, WEISSMAN S: T h e molecular genetics of hum a n haemoglobin. Prog Nucleic Acid Res Mol Biol, 1984, 32:315-462.

FORRESTERW, TAKEGAWAS, PAPAYANNOPOULOUT, STAMATOYANNOPOULOS G, GROUDINE M: Evidence for a locus activating region: t h e formation of developmentally stable hypersensitive sites in globin expressing hybrids. Nucleic Acids Res 1987, 15:10159-10177.

56. •

46. ••

BLOMVAN ASSEMDELFTM, HANSCOMBEO, GROSVELDF, GREAVES DR: T h e 13-giobin dominant control region activates homologous and heterologous p r o m o t e r s in a tissue-specific manner. Cell 1989, 56:969-977. DCR/LAR override local sequences in specifying expression of the Tcell-specific T / ~ I gene. 47.

GREAVESDR, WILSON FD, LANG G, KIOUSSIS D: H u m a n CD2 3'-flanking s e q u e n c e s confer high-level, T cell-specific, position-independent g e n e expression in transgenic mice. Cell 1989, 56:97°/986. Identification in a gene system other than globin, of a region with functional characteristics similar to the [3-globin DCR/LAR. •

CAO SX, GUTMAN PD, DAVE HPG, SCHECHTERAN: Identifica, i o n of a transcriptional silencer in the 5' flanking region of t h e h u m a n ~-globin gene. Proc Natl Acad Sci USA 1989, 86:5306-5309. Deletion analysis in transfection assays revealed a negative element between 177 and 392 bp upstream of the ~-globin gene transcription start site, which was more active in non-erythroid cells than in erythroid cells. 57. .

COLLISP, ANTONIOU M, GROSVELDF: Definition of t h e minimal r e q u i r e m e n t s within t h e h u m a n ~-globin gene and the d o m i n a n t control region for high level expression. EMBO J 1990, 9:233-240. Identifies the middle two hypersensitive sites of the DCR/LAR as the most important in stable MEL cell transfections. The second intron, but not the 3' enhancer, of the J3-globin gene is required for a response to the DCR/LAR.

STIEF A, WINTER DM, STRATLINGWH, SIPPEL AE: A nuclear DNA a t t a c h m e n t element mediates elevated and positioni n d e p e n d e n t g e n e activity. Nature 1989, 341:343-345. Nuclear scaffold attachment sims at the 5' boundary of the chicken lysozyme locus act in concert with the gene enhancer to confer positionindependent expression in stably transfected pro-macrophage cell lines.

FRASERP, HURSTJ, ColMs P, GROSVem F: DNaseI hypersensitive sites 1, 2 and 3 of t h e h u m a n ~-globin dominant control region direct position-independent expression. Nucleic Acids Res 1990, 18:3503-3508. Shows the importance of the middle two hypersensitive sites of the DCR/LAR in transgenic mice, in agreement with the MEL cell results. In addition, the site upstream of these two has significant activity which was not so apparent in stable transfections.

49.

59.

48. ••

58. •

BONIFER C, VIDAL M, GROSVELD F, SIPPEL AE: Tissue specific and p o s i t i o n i n d e p e n d e n t expression of t h e complete g e n e domain for chicken lysozyme in transgenic mice. EMBO J 1990, 9:2843-2848. The chicken lysozyme domain, as defined by the boundary nuclear scaffold attachment sites, confers position independence and tissue specificity on expression of the lysozyme gene in transgenic mice.

RYANTiM, BEHRINGER RR, MARTIN NC, TOWNES TM, PALMITER RD, BRINSTER RL: A single erythroid-specific DNaseI superhypersensitive site activates high levels of h u m a n 13-globin g e n e expression in transgenic mice. Genes Dev 1989, 3:314-323. This paper and [60"] identify major activity in the second most locusproximal hypersensitive site of the DCR/LAR.

50. ••

60. •

• •

ENVERT, RAICH N, EBENS AJ, PAPAYANNOPOULOUT, COSTANTINI F, STAMATOYANNOPOULOSG: Developmental regulation of hum a n foetal-to-adult giobin g e n e switching in transgenic mice. Nature 1990, 344:309-313. This paper (and [51 . • ] describes how linkage of the DCR/LAR to either the foetal 7-globin gene or the adult [~-globin gene alone results in expression throughout development. In contrast, linkage of the two genes on the same DNA fragment under the control o( the DCR/LAR results in correct developmental expression, thereby implying compe tition between the two genes for the DCR/LAR. 51. ••

BEHRINGERRR, RYAN TM, PALM1TERR[), BRINSTER RL, TOWNES TM: H u m a n gamma- to beta-globin g e n e switching in transgenic mice. Genes Dev 1990, 4:380-389.

.

CURTINPT, LIU D, LIE W, CHANG JC, KAN YW: H u m a n J3globin gene expression in transgenic mice is e n h a n c e d by a distant DNase I hypertensive site: Proc Natl Acad Sci USA 1989, 86:7082-7086. This paper and [59 ° ] identify major activity in the second most locusproximal hypersensitive site of the DCP,/LAR. 61.

PHILIPSENS, TALBOT D, FRASER P, GROSVELD F: The ~-globin d o m i n a n t c o n t r o l region: hypersensitive site 2. EMBO J 1990, 9:2159-2167. The activity of the more locus-distal of the middle two hypersensitive sims of the DCR/LAR is localized to a 255 bp fragment. DNaseI footprinting reveals a repeamd set of binding sims for NFE-1 and a GGTGGbinding protein. •.

157

158

Mammalian gene studies 62. ••

TALBOTD, PHILIPSEN S, FRASER P, GROSVELD F: Detailed analysis of t h e site 3 region o f t h e h u m a n ~-globin dominant control region. EMBO J 1990, 9:2169-2178. The activity of the more locus-proximal of the middle two hypersensitive sites of the DCR/LAR is localized to a 300 bp fragment. DNaseI footprinting reveals binding sites for NFE-1 and NFE-2. Multimerized NFE-2 binding sites fail to give position-independent high levels of expression in transgenic mice or stably transfected MEL cells. 63. •

TUANDYH, SOLOMONWB, LONDON I i , LEE DP: An erythroidspecific developmental stage i n d e p e n d e n t e n h a n c e r far upstream of the h u m a n '~l-like globin' genes. Proc Natl Acad Sci USA 1989, 86:2554-2558. The more locus-proximal of the middle two hypersensitive sites in the DCR/LAR acts as an enhancer in transient transfection assays. 64. .

NEY PA, SORRENTINOBP, MCDONAGH KT, NmNHUIS AW: Tand e m AP-l-binding sites w i t h i n t h e h u m a n ~-giobin dominant control region function as an inducible e n h a n c e r in erythroid cells. Genes Dev 1990, 4:993-1006. Enhancer activity of the m o r e locus-proximal of the two middle hypersensitive sites of the DCR/LAR localizes to an 18 bp segment containing two AP1 (Jun/Fos) binding sites. 65.

JARMANAP, I-I~GGS DR: Nuclear scaffold a t t a c h m e n t sites in t h e h u m a n globin g e n e complexes. ~ B O J 1988, 7:3337-3344.

66.

GARRARDWT: Chromatin remodelling during immunoglobul i n g e n e activation. In Tissue Specific Gene Expression edited by Renkawitz R. Weinheim: VCH Publications, 1989, pp 13-31.

67.

GOLDMANMA, HOLMQUIST GP, GRAY MC, CASTON LA, NAG A: Replication timing of g e n e s and middle repetitive sequences. Science 1984, 224:686-692.

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HOLMQUIST GP: Role of replication timing in t h e control of tissue-specific g e n e expression. A m J H u m Genet 1987, 40:151-173.

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EPNER ER, FORRESTERWC, GROUDINE M: A s y n c h r o n o u s DNA replication within the h u m a n I]-giobin gene locus. Proc Natl Acad Sci USA 1988, 85:8081-8085.

70.

DHAR V, MAGERD, IGBALA, SCHILDKRAUTCL: T h e coordinate replication of t h e h u m a n l]-globin g e n e domain reflects its transcriptional activity and nuclease hypersensitivity. Mol Cell Biol 1988, 8:4958-4965.

71.

FORRESTERWC, EPNER E, DRISCOLL MC, ENVER T, BRICE M, PAPAYANNOPOULOU T, GROUDINE M: A deletion of t h e h u m a n 13-globin locus activation region (LAR) causes a major alteration in chromatin structure and replication across t h e entire J3-globin locus. Genes Dev 1990, 4:1637-1649. A 25 Kb deletion in human chromosome 11, which removes elements of the DCR/LAR, led to late replication of the entire 13-globin locus, even though the erythroid background of the somatic cell hybrid was maintained. This was accompanied by a DNaseI-resistant chromatin structure lacking the normal hypersensitive sites. This paper highlights the fact that although the chromatin effects of the DCP,/LAR are polar, the effects on replication are bidirectional and encompass the normally 'dead' chromatin upstream. • •

72. PATIENTRK, ALLANJ: Active chromatin. Curr Opin Cell Biol •• 1989, 1:454-459. A review focussing on the mechanism by which DNaseI hypersensitive sites are formed, perturbations of the chromatin fibre by transcription and DNA supercoiling. A mechanism for DCR/LAR action is proposed.