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Modification of Chromatin Structure by the Thyroid Hormone Receptor
Modification of Chromatin Structure by the Thyroid Hormone Receptor Qiao Li, Laurent Sachs, Yun-Bo Shi and Alan P. Wolffe
Pioneering experiments and recent observations have established the thyroid hormone receptor as a master manipulator of the chromosomal environment in targeting the activation and repression of transcription. Here we review how the thyroid hormone receptor is assembled into chromatin, where in the absence of thyroid hormone the receptor recruits histone deacetylase to silence transcription. On addition of hormone, the receptor undergoes a conformational change that leads to the release of deacetylase, while facilitating the recruitment of transcriptional coactivators that act as histone acetyltransferases. We discuss the biological importance of these observations for gene control by the thyroid hormone receptor and for oncogenic transformation by the mutated thyroid hormone receptor, v-ErbA. The thyroid hormone receptor (TR) regulates gene activity by alternatively silencing or activating transcription, depending on the absence or presence of thyroid hormone (T3)1. The TR plays an important role in development2,3, and mutations in the TR can lead to the clinical syndrome of generalized resistance to T3 in humans4. Interest in TR-regulated developmental pathways is enhanced by the existence of a closely related viral oncoprotein, v-ErbA. The differentiation of erythroblast progenitors in chickens is inhibited by the targeted repression of erythroidspecific genes mediated by v-ErbA, which in turn promotes cell proliferation. In contrast to the TR, v-ErbA does not bind T3 and cannot activate transcription5. An understanding of the molecular mechanisms through which the TR and v-ErbA control transcription is likely to provide insights into regulatory events in oncogenesis and development, while suggesting new avenues for therapeutic intervention in humans. Q. Li, L. Sachs, Y-B. Shi and A.P. Wolffe are at the Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, NIH, Bldg 18T, Rm 106, Bethesda, MD 20892-5431, USA.
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• The Thyroid Hormone Receptor as a Stable Component of Chromatin If a transcriptional regulator is to regulate gene expression through the targeted modification of chromatin, it has to be able to bind to DNA in a chromatin context. Endogenous chromosomes provide the most natural in vivo substrate to test for the binding of a particular transcription factor; however, chromosomal sites are difficult to manipulate. A solution to this problem is to transfect or microinject plasmid DNA into cells under conditions such that a fraction is assembled into chromatin6. However, a common problem with this approach is that large amounts of DNA either do not enter the cells or are degraded in the cytoplasm7. By contrast, plasmid DNA microinjected into the large nuclei of Xenopus oocytes is assembled efficiently into minichromosomes, thereby providing a surrogate and easily manipulated chromatin substrate8. The TR itself is relatively easy to detect within chromatin with radiolabeled T3, antibodies or genomic footprinting. By 1982, Samuels and colleagues had effectively used endogenous chromosomes to establish that the TR could form a stable complex with the
nucleosome9. Their strategy (Fig. 1) used controlled micrococcal nuclease digestion of chromatin isolated from a rat somatotrophic cell line with the presence of the TR in the chromatin digests being assayed by means of radiolabeled T3 (Ref. 10). Micrococcal nuclease preferentially digests the linker DNA between nucleosomes and prolonged digestion leads to the accumulation of a stable kinetic intermediate known as the nucleosome core that normally contains only the core histones and 146 base pairs (bp) of DNA. Samuels et al. discovered that within the chromatin digests released from cell nuclei, a significant fraction of the TR (10%) was stably associated with nucleosome cores as receptor–nucleosome complexes (Fig. 1B and C). The remainder of the TR was associated with linker DNA (Refs 9,10). Assuming one TR per nucleosome, the subset of nucleosomes associated with the receptor was 0.006% of the total or ~2000 per cell nucleus. As there are approximately 33107 nucleosomes in each mammalian cell nucleus, this implies that considerable specificity exists in the recognition of nucleosomal DNA by the TR. These pioneering experiments indicated that the TR can associate with chromatin through interactions with DNA, both within and outside the nucleosome core. More recent studies using model systems have confirmed and extended these observations. • The Chromatin Assembly Pathway Influences Gene Regulation To understand the importance of the capacity of the TR to associate with chromatin it is important to use an experimentally manipulable system. The microinjection of DNA into Xenopus oocyte nuclei leads to the assembly of chromatin that can be alternately transcriptionally repressed or active11. These active or repressed minichromosomes provide a powerful tool to dissect transcriptional regulation in an in vivo context. In vivo chromatin assembly normally occurs during the S phase, when DNA and the histones are synthesized and when the molecular chaperones that direct the deposition of histones onto nascent DNA are maximally
Published by Elsevier Science Ltd. PII: S1043-2760(98)00141-6
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Figure 1. The thyroid hormone (T3) receptor (TR–RXR) functions as an integral component of the chromatin infrastructure. (A) Depending upon nucleosome positioning, a thyroid response element (TRE) could be within the linker DNA between two adjacent nucleosome cores (TRE in linker DNA) or it could be positioned within a nucleosome core itself (TRE in nucleosome). In the presence of thyroid hormone, the TR–RXR will recruit transcriptional coactivators that will modify nucleosomal arrays to make them more accessible to nuclease digestion. (B) Controlled micrococcal nuclease digestion of chromatin leads to the accumulation of mononucleosomes and dinucleosomes, together with the TR as a heterodimer with RXR bound either to fragments of linker DNA (receptor–DNA complex) or to nucleosome cores (receptor–nucleosome complex). (C) Isokinetic sucrose gradient centrifugation allows the fractionation of chromatin fragments from the micrococcal nuclease digestion. Free receptor has a sedimentation coefficient of 3.8 S, mononucleosomes of 11.5 S and dinucleosomes of 16 S. Their peaks of distribution in the fractionated sucrose gradient are indicated by the closed triangles. The receptor–DNA complex has a sedimentation coefficient of 6.5 S and the receptor–nucleosome complex of 12.5 S. Their distributions within a representative fractionated sucrose gradient are shown (see Refs 9 and 10 for examples of experimental data). The peaks of distribution for the receptor–DNA complex and the receptor–nucleosome complex are indicated by arrowheads.
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effective. Microinjection of singlestranded DNA into a Xenopus oocyte nucleus leads to second strand synthesis coupled to the rapid and efficient assembly of chromatin, which causes basal transcription to be repressed11. Repression occurs because the molecular mechanisms driving histone deposition outcompete those that direct the association of the basal transcriptional machinery with the promoter. Similar results in which appropriate chromatin assembly occurs on replicating templates are achieved with the use of viral episomes in mammalian cells7. A second pathway of chromatin assembly occurs when double-stranded DNA is microinjected into oocyte nuclei. This assembly pathway is not coupled to replication and resembles the consequence of transiently transfecting or microinjecting plasmid DNA molecules into mammalian tissue culture cells. Under these conditions, chromatin assembly will still occur on the fraction of DNA that is taken up into the nucleus, but does so with much slower kinetics in a process taking at least 4 h in the oocyte. This provides plenty of time for the basal transcriptional machinery to gain access to promoter elements and to assemble an active minichromosome. The T3- and TR-responsive X. laevis TRbA promoter is transcriptionally repressed when assembled into chromatin in a replicative pathway after injection of single-stranded templates into oocyte nuclei, whereas it is active when doublestranded templates are injected. In both instances, the final density of nucleosomes is equivalent, only the pathway of chromatin assembly differs11. This illustrates the potential importance of the chromatin context and assembly pathway on transcriptional control. • Determinants of TR Association with Chromatin The Xenopus oocyte lacks adequate TRs to regulate transcription in a T3-responsive manner. However, microinjection of TR mRNA into the oocyte cytoplasm leads to TR synthesis and the acquisition of T3-responsive transcription. Hormonal responsiveness is greatly enhanced in the presence of the retinoic X receptor RXR, which indicates that TEM Vol. 10, No. 4, 1999
the primary functional form of the TR in the oocyte is as a heterodimer with RXR (Ref. 12). In the absence of T3, TR–RXR will repress transcription from the active minichromosome and will further reduce transcription from the already repressed minichromosome assembled during replication. Consistent with these functional studies, in vivo DNase I footprinting indicates that TR–RXR will bind to a thyroid response element (TRE) in the TRbA gene, independent of the prior assembly of this element into chromatin and independent of the presence of T3 (Ref. 11). The phenomenon of nucleosome positioning determines the access of TR–RXR to TREs within chromatin. Nucleosome positioning occurs when a particular DNA sequence is organized in a non-random manner when wrapped around the core histones. Within a translationally positioned nucleosome, histone–DNA contacts begin and end at a particular sequence. In the rotational positioning of DNA in a nucleosome, a particular major or minor groove of the double helix is facing out towards the solution, while another is facing the histones. The translational positioning of nucleosomes and the rotational positioning of DNA on the surface of the histones are important contributory factors in transcriptional regulation of chromatin templates by nuclear receptors13–15. DNA sequences within the first 500 bp of the TRbA gene have the capacity to direct the translational positioning of histone octamers16–18. The type of thyroid receptor recognition element present in the TRbA promoter is a direct repeat separated by 4 bp. The rotational position adopted by the TRE on the surface of the nucleosome assembled on the wild-type TRbA gene is optimal for continued access of the TR–RXR within the chromatin (Fig. 2). The TR–RXR heterodimer is able to bind to the TRE present in the TRbA gene in a nucleosome without significant impediment. The importance of rotational positioning for the continued capacity of the TR–RXR to bind to nucleosomal DNA is shown by a small 3 bp alteration in placement of the TRE, leading to an approximate 102º change in orientation with respect to the histone surface. This TEM Vol. 10, No. 4, 1999
Figure 2. A model for the nucleosome assembled on the thyroid hormone receptor bA (TRbA) gene that contains the thyroid response element (TRE). The TRE is represented by the hatched DNA and the boxed sequences. The dyad position in the nucleosome is indicated, as are integral turns of DNA away from the dyad axis (negative to 25, positive to 12). The histones are shown in the cartoon (see Ref. 42 for details).
rotation leads to a reduction in the capacity of the TR–RXR to bind to nucleosomal DNA in vitro, to a reduction in the capacity of the TR–RXR to bind to polynucleosomal arrays in vivo, and to a reduction in the capacity of the TR–RXR to activate transcription16–18. By contrast, the manipulation of the translational position of nucleosomes over the TRbA promoter is without major consequences for TR–RXR association. Thus, considerable flexibility might exist in terms of exactly where in the nucleosome the TRE is positioned for transcriptional regulation, as long as the TRE is oriented on the histone surface with the appropriate rotational organization. There are several unanticipated consequences of TR–RXR binding to
chromatin. In the absence of T3, the underlying nucleosomal array with which the TR–RXR is associated remains very regular upon binding of the receptors, with no increase in accessibility to micrococcal nuclease16,17. This regularity is surprising because the TR–RXR generates a major site of preferential cleavage by DNase I. We interpret these results to indicate that the unliganded TR–RXR binds to the minichromosome and functions to repress transcription within a regular nucleosomal infrastructure. DNase Ihypersensitive sites are useful markers for chromatin-bound TRs and also potentially for v-ErbA bound in chromatin. Indeed, several transcriptional silencers that contain v-ErbA sites are hypersensitive to DNase I in vivo, 159
Figure 3. A working model for the function of the thyroid hormone (T3) receptor in chromatin. Normal chromatin has a basal level of histone acetylation and transcriptional activity. The binding of the thyroid hormone receptor (TR–RXR) to a thyroid response element (TRE) on a positioned nucleosome in the absence of T3 leads to the recruitment of a corepressor complex (NCoR, Sin3, RPD3) to direct histone deacetylation and transcriptional repression. The binding of the TR–RXR to a TRE in the presence of ligand leads to the recruitment of the coactivator complex (SRC-1, p300/CBP, PCAF) that directs histone acetylation and facilitates transcription.
consistent with stable association of the v-ErbA protein with recognition elements in a chromatin context19. • Transcriptional Coactivators and Corepressors of TR–RXR and v-ErbA that Modify Chromatin and Regulate Transcription Biochemical approaches using the ligand-binding domains of nuclear
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receptors to screen for interacting factors have identified diverse coactivators and corepressors that bind the TR–RXR in a manner that is dependent on the presence or absence of T3 (Ref. 20). In the absence of ligand, the TR has been shown to interact with the corepressors NCoR and SMRT (Refs 20–22). These corepressors also interact with the mammalian proteins
mSin3A and mSin3B (Refs 23,24). The Sin3 proteins associate with histone deacetylase (RPD3)25. These interconnections suggested that the TR–RXR might target transcriptional repression by recruiting histone deacetylase to modify the local chromatin environment. However, it is also possible that components of the recruited corepressor complex such as NCoR, SMRT or Sin3 might exert repressive activities on components of the basal machinery or organize repressive chromatin structures directly without the requirement for histone deacetylase26. Discrimination between these models was possible through the microinjection of doublestranded DNA templates into Xenopus oocyte nuclei. The slow kinetics of chromatin assembly on doublestranded DNA were especially advantageous for following the role of nucleosomes in transcriptional silencing. Expression of histone deacetylase could repress transcription of the Xenopus TRbA gene, depending on the assembly of a nucleosomal template, and this repression could be overcome by hormone-bound TR–RXR. The role of histone deacetylase could also be examined using a highly specific inhibitor trichostatin A (TSA). Incubation of oocytes containing a repressed TRbA gene assembled into nucleosomes with TSA relieved repression of the TRbA promoter to the same extent as the presence of hormone-bound TR–RXR (Ref. 27). This indicates that the activity of histone deacetylase on chromatin has a major role in transcriptional silencing (Fig. 3). A unifying feature of several of the diverse coactivator proteins described earlier is their capacity to function as histone acetyltransferases20,28. Thus, a common theme for transcriptional activation by ligand-bound nuclear receptors is the recruitment of histone acetyltransferase activity. This recruitment might be expected to counteract the repressive influence of histone deacetylase. Mutant forms of the human TR that cannot recruit transcriptional coactivators are responsible for the clinical syndrome of generalized resistance to T3 (Ref. 4). Later we discuss the biological roles of the TEM Vol. 10, No. 4, 1999
oncoprotein v-ErbA, which is also deficient in coactivator recruitment. The dynamics of histone acetylation provide an attractive mechanistic foundation for the reversible activation and repression of transcription. Histone acetylation states provide an example of the dynamic properties of chromatin, the TR itself provides another. Even though the TR–RXR complex can stably associate with chromatin, coactivators and corepressors, the TR is continually being degraded and replaced in vivo. Raaka and Samuels monitored the stability of TRs within the chromatin of mammalian cells through the labeling of TRs with dense amino acids29. Newly synthesized proteins are of higher density than pre-existing proteins and can be separated with gradient centrifugation techniques. The use of radioactive T3 allows the position of normal and dense receptors to be assayed independently in sucrose gradients. By quantifying the abundance of normal receptors at different times after the addition of dense amino acids to the cells, the receptor half-life can be assayed. The accumulation of dense receptors allows the rate of receptor synthesis to be determined. The TR has a half-life of 4.5 h in rat somatotrophic cells cultured in the absence of T3, and of 3 h in the presence of hormone. Importantly, most (95%) of the receptor decays with an identical half-life, indicating that no appreciable nuclear-associated pools exist that have distinct stabilities (Fig. 4). Moreover, the presence or absence of T3 does not influence the rate of TR decay. This suggests that the TR is exchanged from chromatin with equivalent efficiency whether it is engaged in repressing or activating transcription. In addition, more than 1700 molecules of TR were synthesized per hour in each cell to replace those being degraded. These kinetics imply that more than 95% of the receptor will be replaced within chromatin in 24 h. This capacity of a protein to be released from chromatin, degraded and then replaced with newly synthesized molecules is a common feature of both structural and regulatory chromosomal components. This release–replenishment cycle offers conTEM Vol. 10, No. 4, 1999
Figure 4. The thyroid hormone (T3) receptor (TR–RXR) is subject to continual turnover within a single cell cycle. TR is synthesized at a rate of 1700 molecules per cell per hour. The TR is then engaged in repression or activation of transcription as indicated. It is also probable that other pools of TRs exist in the nucleus as sites of storage or degradation. The TR decays with a half-life of 3–5 h independent of gene activity29.
siderable regulatory opportunities and would benefit from further investigation in easily manipulated model systems such as the Xenopus oocyte. Thus, just as histone acetylation states are dynamic, it would also appear that the TR, which targets chromatin modification, will be exchanged from corepressor and coactivator complexes within a given cell cycle. Together, these dynamic qualities should provide ample opportunity to reset patterns of gene expression rapidly in response to physiological demands. • Chromatin Disruption by the Ligand-bound TR–RXR We have discussed how the TR–RXR can bind to nucleosomal DNA to target both transcriptional repression and
activation. The TR–RXR silences transcription effectively in the context of a positioned nucleosomal array11,16. On the addition of ligand this regular nucleosomal array is disrupted. The requirements for this disruption offer additional insight into the potential roles for histone acetylation and chromatin modification in gene control. The TR–RXR alone and its recognition element are sufficient to initiate a process of chromatin disruption over an extended segment of DNA sequence, as assayed by micrococcal nuclease cleavage16. Each TRE can target the loss of topological constraint found in three to four nucleosomes, provided the TREs are separated by at least 400 bp of DNA. Transcription is not required for chromatin disruption, and additional 161
proximal promoter elements are necessary to facilitate hormone-dependent activation of transcription by the TR–RXR. The T3-dependent chromatin disruption depends on the transcription activation domain at the C-terminus of the TR. An important, yet unresolved, question concerns the role of histone acetyltransferases in this disruption process. At this time, the roles of transcriptional coactivators such as PCAF, p300 and SRC-1 in chromatin disruption have not been assayed; however, the structural and functional consequences of histone acetylation have been investigated intensively. Chromatin disruption targeted by ligand-bound TR–RXR leads to major topological changes in minichromosomes consistent with a loss of wrapping of DNA around the histones. Although histone acetyltransferases are recruited to the ligand-bound TR–RXR, it is difficult to account for the large topological change observed through acetylation alone30. In fact, transcription can be activated without significant topological change from a TRbA promoter complexed in chromatin with unliganded TR–RXR simply by the addition of the deacetylase inhibitor TSA (Ref. 27). Thus, histone hyperacetylation in isolation seems unlikely to account for chromatin disruption. More importantly, the chromatin disruption phenomenon in a minichromosomal context, as assayed by topological change and increases in micrococcal nuclease digestion, is not necessary for transcriptional activation. Chromatin disruption, as assayed by topological change and increases in micrococcal nuclease cleavage, has generally been interpreted as essential for transcriptional activation. However, transcriptional activation can occur with minimal changes in DNA topology31,32. This would be anticipated if histone acetylation were the only alteration to chromatin structure necessary for transcriptional activation. It is possible that the pathways directing chromatin disruption in the oocyte nucleus in response to ligand-bound TR–RXR do not involve histone acetylation, and that alternative means of disrupting histone–DNA contacts are employed,
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leading to similar transcriptional consequences. Candidate mechanisms include recruitment of molecular machines such as vertebrate chromatin remodeling complexes33,34. • Biological Implications of TR and v-ErbA Function in Chromatin Investigation of the requirements for the TR in animal development reveal relatively minor determinative roles, but more extensive requirements for the TR in adaptive responses and differentiation2,3,35. These observations might be explained by the TR exerting gene control predominantly by binding in chromatin and repressing gene activity. Thus, ablation of the TR will have the same consequence as adding hormone, in that repression will be relieved and transcription will return to high basal levels. If TR function is not to activate transcription much above basal levels in chromatin then removing the receptor will provide a phenotype similar to that of an animal constitutively exposed to T3. v-ErbA lacks the capacity to activate transcription and to interact with thyroid hormone as a consequence of small deletions in the ligand-binding domain. Neoplastic transformation by v-ErbA appears to be a result, in part, of the inhibition of genes regulated by the TR and the retinoic acid receptor (RAR)5. v-ErbA interacts with corepressors constitutively21 and requires histone deacetylase activity to silence transcription36. Transformation-defective variants of v-ErbA that fail to repress transcription also fail to interact with corepressors21. The capacity of v-ErbA to repress specific transcription, presumably via the recruitment of histone deacetylase, inhibits cell differentiation and allows cell proliferation to proceed. This mode of action of nuclear receptors appears to be co-opted by diverse oncoproteins containing nuclear receptor corepressor-binding domains37,38. Recent results demonstrate that in media depleted of T3 and retinoids, overexpressed TRa functions exactly like v-ErbA (Ref. 36). These experiments establish that unliganded TRa could impede differentiation, associate with histone deacetylase and repress
carbonic anhydrase and c-myb. These phenomena were reversed on the addition of hormone. These authors further suggest that TRa can function as a ligand-operated molecular switch, regulating the balance between erythroblast self-renewal and differentiation. It is attractive to speculate that TRa in Xenopus might have a similar function during tadpole development before the differentiation of a thyroid gland and the synthesis of thyroid hormone. Unliganded Xenopus TRa accumulates early in larval development, when it may serve to repress the expression of genes that are not required until metamorphosis. Thus, the TR might facilitate the proliferation of adult stem cell lineages in the absence of differentiation. Once thyroid hormone is synthesized, the same TRa bound within chromatin will serve to activate gene expression, including that of the TRb gene. The surge in TRb gene expression may be important in facilitating metamorphic climax by activating genes. In this scenario, TRb is the immediate-early gene whose transcription factor product initiates the cascade of gene activity associated with metamorphosis39. • Summary and Conclusions The TR and the related oncoprotein vErbA provide useful tools to investigate the functional capabilities of chromatin architecture and modification. The TR has the capacity to undergo regulated interactions with a rich variety of transcriptional coactivators and corepressors. A unifying feature of these interactions is the capacity of coactivators to acetylate histones and of corepressors to deacetylate histones. The TR also facilitates other structural modifications to chromatin, with as yet unknown functional consequences. For the vast majority of genes regulated by the TR, acetylation of proteins in the vicinity of the TRE appears to activate transcription, while deacetylation represses. Exactly how this comes about is unknown at present. However, it is clear that chromatin structure is destabilized as a consequence of acetylating the N-termini of the core histones, and that this modification facilitates the accessibility of DNA in TEM Vol. 10, No. 4, 1999
chromatin to the transcriptional machinery. Evidence also exists for direct contacts between the TR and components of the basal transcriptional machinery40,41. An understanding of nucleoprotein architecture within which the basal transcriptional machinery functions is undoubtedly essential to interpret the regulatory roles of the TR. However, the key point is that the activation or repression of a particular promoter will depend on the recruitment of a molecular machinery that modifies or disrupts the chromatin infrastructure. Large gaps in our knowledge as to how this occurs exist at both the structural and functional levels. Filling these gaps will contribute greatly to our understanding of the molecular mechanisms during cell transformation and also in generating targeted therapeutic strategies for intervention in human clinical disorders.
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Role of Pax Genes in Endoderm-derived Organs Ahmed Mansouri, Luc St-Onge and Peter Gruss
Pax genes, which encode a family of transcription factors, are essentially required for the formation of several tissues from all germ layers in the mammalian embryo. Specifically, in organogenesis, they are involved in triggering early events of cell differentiation. The differentiation of endodermderived endocrine pancreas is mediated through Pax4 and Pax6. In the thyroid gland, Pax8 is essential for the formation of thyroxine-producing follicular cells, also of endodermal origin. The analysis of loss-of-function mutants revealed a common function of Pax genes in organogenesis. The Pax gene family of transcription factors has been isolated on the basis of their sequence homology with Drosophila segmentation genes1–3. They share a common conserved DNA-binding domain of 128 amino acids (reviewed in Ref. 4). During embryogenesis, Pax genes exhibit highly restricted temporal and spatial expression patterns4. Functional studies of mouse mutants revealed that these genes are required for early events to generate a variety of tissues of all germ layers4. Specifically, in the endoderm, Pax genes are essential for the differentiation of endocrine cells in the pancreas and follicular cells in the mature thyroid gland. In this review, we will emphasize the role of Pax4 and Pax6 in the endocrine pancreas and Pax8 in the generation of thyroid hormone-producing follicular cells. The three genes are necessary to establish the differentiation of endoderm progenitors in A. Mansouri, L. St-Onge and P. Gruss are at the Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg, D-37077 Göttingen, Germany.
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specific cell types and indicate a common mechanism in the role of Pax genes in organogenesis. • Pax4 and Pax6 in the Developing Pancreas The mammalian pancreas is derived from the endoderm5,6. In the mouse, the first morphological appearance of the developing pancreas is evident at embryonic day (e) 9.5 as a dorsal, and later as a ventral, bud between the foreand midgut, in the region of the future duodenum. During further differentiation, a ductal branching system is formed, giving rise to the adult pancreas7–9. The major part consists of exocrine cells (acini) that produce the digestive enzymes. The second population (endocrine cells) is organized in units called islets of Langerhans6,9. Four endocrine cell types are found in the adult pancreas, a, b, d and PP, which produce glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. The genes encoding several transcription factors are expressed in the pancreas9–15, including Pax4 and Pax6.
mone receptor coactivator complex. Proc. Natl. Acad. Sci. U. S. A. 93, 8329–8333 42 Pruss, D., Hayes, J.J. and Wolffe, A.P. (1995) Nucleosomal anatomy – where are the histones? BioEssays 17, 161–170
Expression of Pax4 and Pax6 in the Developing Pancreas Pax4 and Pax6 expression occurs as early as e9.0 (15 somites) in the foregut of the mouse embryo16–18. At e10.5, Pax4 gene expression is observed in the dorsal and, by e11.0, in the ventral pancreatic bud. Already, at these early stages, Pax4 expression is restricted to a few cells, which also express the insulin gene. This pattern is maintained during further development and, in newborns, Pax4 is only detected in insulin-producing b cells16. Thus, Pax4 is expressed selectively in b cells. Pax6, on the other hand, is also first localized in a few cells, including cells coexpressing glucagon, but from e15.5 on its expression expands to all pancreatic endocrine cells. In newborn animals, the Pax6 protein is detected in all cells of the islets of Langerhans17,18. Taken together, these expression data show that at early stages of pancreatic development Pax4 and Pax6 gene expression is confined to endocrine precursor cells. Later, Pax4 gene expression continues to be restricted to insulinproducing b cells, but Pax6 expression is found in all pancreatic endocrine cells16–18. However, expression is never seen in the exocrine tissue. This suggests that Pax4 and Pax6 are required during early steps to generate specific endocrine cells of the pancreas. Role of Pax4 and Pax6 in the Pancreas An analysis of Pax4 function in pancreas development has been performed in mice generated by replacing the DNA-binding domain of genes with the bacterial β-galactosidase (lacZ) gene via homologous recombination in embryonic stem cells. In these embryos, Pax4expressing cells can be imaged by staining for LacZ (Ref. 16). Newborn mice homozygous for the mutated Pax4 gene develop to term but die soon after birth. Histological analysis
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Pioneering experiments and recent observations have established the thyroid hormone receptor as a master manipulator of the chromosomal environment in targeting the activation and repression of transcription. Here we review how the thyroid hormone receptor is assembled into chromatin, where in the absence of thyroid hormone the receptor recruits histone deacetylase to silence transcription. On addition of hormone, the receptor undergoes a conformational change that leads to the release of deacetylase, while facilitating the recruitment of transcriptional coactivators that act as histone acetyltransferases. We discuss the biological importance of these observations for gene control by the thyroid hormone receptor and for oncogenic transformation by the mutated thyroid hormone receptor, v-ErbA. The thyroid hormone receptor (TR) regulates gene activity by alternatively silencing or activating transcription, depending on the absence or presence of thyroid hormone (T3)1. The TR plays an important role in development2,3, and mutations in the TR can lead to the clinical syndrome of generalized resistance to T3 in humans4. Interest in TR-regulated developmental pathways is enhanced by the existence of a closely related viral oncoprotein, v-ErbA. The differentiation of erythroblast progenitors in chickens is inhibited by the targeted repression of erythroidspecific genes mediated by v-ErbA, which in turn promotes cell proliferation. In contrast to the TR, v-ErbA does not bind T3 and cannot activate transcription5. An understanding of the molecular mechanisms through which the TR and v-ErbA control transcription is likely to provide insights into regulatory events in oncogenesis and development, while suggesting new avenues for therapeutic intervention in humans. Q. Li, L. Sachs, Y-B. Shi and A.P. Wolffe are at the Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, NIH, Bldg 18T, Rm 106, Bethesda, MD 20892-5431, USA.
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• The Thyroid Hormone Receptor as a Stable Component of Chromatin If a transcriptional regulator is to regulate gene expression through the targeted modification of chromatin, it has to be able to bind to DNA in a chromatin context. Endogenous chromosomes provide the most natural in vivo substrate to test for the binding of a particular transcription factor; however, chromosomal sites are difficult to manipulate. A solution to this problem is to transfect or microinject plasmid DNA into cells under conditions such that a fraction is assembled into chromatin6. However, a common problem with this approach is that large amounts of DNA either do not enter the cells or are degraded in the cytoplasm7. By contrast, plasmid DNA microinjected into the large nuclei of Xenopus oocytes is assembled efficiently into minichromosomes, thereby providing a surrogate and easily manipulated chromatin substrate8. The TR itself is relatively easy to detect within chromatin with radiolabeled T3, antibodies or genomic footprinting. By 1982, Samuels and colleagues had effectively used endogenous chromosomes to establish that the TR could form a stable complex with the
nucleosome9. Their strategy (Fig. 1) used controlled micrococcal nuclease digestion of chromatin isolated from a rat somatotrophic cell line with the presence of the TR in the chromatin digests being assayed by means of radiolabeled T3 (Ref. 10). Micrococcal nuclease preferentially digests the linker DNA between nucleosomes and prolonged digestion leads to the accumulation of a stable kinetic intermediate known as the nucleosome core that normally contains only the core histones and 146 base pairs (bp) of DNA. Samuels et al. discovered that within the chromatin digests released from cell nuclei, a significant fraction of the TR (10%) was stably associated with nucleosome cores as receptor–nucleosome complexes (Fig. 1B and C). The remainder of the TR was associated with linker DNA (Refs 9,10). Assuming one TR per nucleosome, the subset of nucleosomes associated with the receptor was 0.006% of the total or ~2000 per cell nucleus. As there are approximately 33107 nucleosomes in each mammalian cell nucleus, this implies that considerable specificity exists in the recognition of nucleosomal DNA by the TR. These pioneering experiments indicated that the TR can associate with chromatin through interactions with DNA, both within and outside the nucleosome core. More recent studies using model systems have confirmed and extended these observations. • The Chromatin Assembly Pathway Influences Gene Regulation To understand the importance of the capacity of the TR to associate with chromatin it is important to use an experimentally manipulable system. The microinjection of DNA into Xenopus oocyte nuclei leads to the assembly of chromatin that can be alternately transcriptionally repressed or active11. These active or repressed minichromosomes provide a powerful tool to dissect transcriptional regulation in an in vivo context. In vivo chromatin assembly normally occurs during the S phase, when DNA and the histones are synthesized and when the molecular chaperones that direct the deposition of histones onto nascent DNA are maximally
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Figure 1. The thyroid hormone (T3) receptor (TR–RXR) functions as an integral component of the chromatin infrastructure. (A) Depending upon nucleosome positioning, a thyroid response element (TRE) could be within the linker DNA between two adjacent nucleosome cores (TRE in linker DNA) or it could be positioned within a nucleosome core itself (TRE in nucleosome). In the presence of thyroid hormone, the TR–RXR will recruit transcriptional coactivators that will modify nucleosomal arrays to make them more accessible to nuclease digestion. (B) Controlled micrococcal nuclease digestion of chromatin leads to the accumulation of mononucleosomes and dinucleosomes, together with the TR as a heterodimer with RXR bound either to fragments of linker DNA (receptor–DNA complex) or to nucleosome cores (receptor–nucleosome complex). (C) Isokinetic sucrose gradient centrifugation allows the fractionation of chromatin fragments from the micrococcal nuclease digestion. Free receptor has a sedimentation coefficient of 3.8 S, mononucleosomes of 11.5 S and dinucleosomes of 16 S. Their peaks of distribution in the fractionated sucrose gradient are indicated by the closed triangles. The receptor–DNA complex has a sedimentation coefficient of 6.5 S and the receptor–nucleosome complex of 12.5 S. Their distributions within a representative fractionated sucrose gradient are shown (see Refs 9 and 10 for examples of experimental data). The peaks of distribution for the receptor–DNA complex and the receptor–nucleosome complex are indicated by arrowheads.
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effective. Microinjection of singlestranded DNA into a Xenopus oocyte nucleus leads to second strand synthesis coupled to the rapid and efficient assembly of chromatin, which causes basal transcription to be repressed11. Repression occurs because the molecular mechanisms driving histone deposition outcompete those that direct the association of the basal transcriptional machinery with the promoter. Similar results in which appropriate chromatin assembly occurs on replicating templates are achieved with the use of viral episomes in mammalian cells7. A second pathway of chromatin assembly occurs when double-stranded DNA is microinjected into oocyte nuclei. This assembly pathway is not coupled to replication and resembles the consequence of transiently transfecting or microinjecting plasmid DNA molecules into mammalian tissue culture cells. Under these conditions, chromatin assembly will still occur on the fraction of DNA that is taken up into the nucleus, but does so with much slower kinetics in a process taking at least 4 h in the oocyte. This provides plenty of time for the basal transcriptional machinery to gain access to promoter elements and to assemble an active minichromosome. The T3- and TR-responsive X. laevis TRbA promoter is transcriptionally repressed when assembled into chromatin in a replicative pathway after injection of single-stranded templates into oocyte nuclei, whereas it is active when doublestranded templates are injected. In both instances, the final density of nucleosomes is equivalent, only the pathway of chromatin assembly differs11. This illustrates the potential importance of the chromatin context and assembly pathway on transcriptional control. • Determinants of TR Association with Chromatin The Xenopus oocyte lacks adequate TRs to regulate transcription in a T3-responsive manner. However, microinjection of TR mRNA into the oocyte cytoplasm leads to TR synthesis and the acquisition of T3-responsive transcription. Hormonal responsiveness is greatly enhanced in the presence of the retinoic X receptor RXR, which indicates that TEM Vol. 10, No. 4, 1999
the primary functional form of the TR in the oocyte is as a heterodimer with RXR (Ref. 12). In the absence of T3, TR–RXR will repress transcription from the active minichromosome and will further reduce transcription from the already repressed minichromosome assembled during replication. Consistent with these functional studies, in vivo DNase I footprinting indicates that TR–RXR will bind to a thyroid response element (TRE) in the TRbA gene, independent of the prior assembly of this element into chromatin and independent of the presence of T3 (Ref. 11). The phenomenon of nucleosome positioning determines the access of TR–RXR to TREs within chromatin. Nucleosome positioning occurs when a particular DNA sequence is organized in a non-random manner when wrapped around the core histones. Within a translationally positioned nucleosome, histone–DNA contacts begin and end at a particular sequence. In the rotational positioning of DNA in a nucleosome, a particular major or minor groove of the double helix is facing out towards the solution, while another is facing the histones. The translational positioning of nucleosomes and the rotational positioning of DNA on the surface of the histones are important contributory factors in transcriptional regulation of chromatin templates by nuclear receptors13–15. DNA sequences within the first 500 bp of the TRbA gene have the capacity to direct the translational positioning of histone octamers16–18. The type of thyroid receptor recognition element present in the TRbA promoter is a direct repeat separated by 4 bp. The rotational position adopted by the TRE on the surface of the nucleosome assembled on the wild-type TRbA gene is optimal for continued access of the TR–RXR within the chromatin (Fig. 2). The TR–RXR heterodimer is able to bind to the TRE present in the TRbA gene in a nucleosome without significant impediment. The importance of rotational positioning for the continued capacity of the TR–RXR to bind to nucleosomal DNA is shown by a small 3 bp alteration in placement of the TRE, leading to an approximate 102º change in orientation with respect to the histone surface. This TEM Vol. 10, No. 4, 1999
Figure 2. A model for the nucleosome assembled on the thyroid hormone receptor bA (TRbA) gene that contains the thyroid response element (TRE). The TRE is represented by the hatched DNA and the boxed sequences. The dyad position in the nucleosome is indicated, as are integral turns of DNA away from the dyad axis (negative to 25, positive to 12). The histones are shown in the cartoon (see Ref. 42 for details).
rotation leads to a reduction in the capacity of the TR–RXR to bind to nucleosomal DNA in vitro, to a reduction in the capacity of the TR–RXR to bind to polynucleosomal arrays in vivo, and to a reduction in the capacity of the TR–RXR to activate transcription16–18. By contrast, the manipulation of the translational position of nucleosomes over the TRbA promoter is without major consequences for TR–RXR association. Thus, considerable flexibility might exist in terms of exactly where in the nucleosome the TRE is positioned for transcriptional regulation, as long as the TRE is oriented on the histone surface with the appropriate rotational organization. There are several unanticipated consequences of TR–RXR binding to
chromatin. In the absence of T3, the underlying nucleosomal array with which the TR–RXR is associated remains very regular upon binding of the receptors, with no increase in accessibility to micrococcal nuclease16,17. This regularity is surprising because the TR–RXR generates a major site of preferential cleavage by DNase I. We interpret these results to indicate that the unliganded TR–RXR binds to the minichromosome and functions to repress transcription within a regular nucleosomal infrastructure. DNase Ihypersensitive sites are useful markers for chromatin-bound TRs and also potentially for v-ErbA bound in chromatin. Indeed, several transcriptional silencers that contain v-ErbA sites are hypersensitive to DNase I in vivo, 159
Figure 3. A working model for the function of the thyroid hormone (T3) receptor in chromatin. Normal chromatin has a basal level of histone acetylation and transcriptional activity. The binding of the thyroid hormone receptor (TR–RXR) to a thyroid response element (TRE) on a positioned nucleosome in the absence of T3 leads to the recruitment of a corepressor complex (NCoR, Sin3, RPD3) to direct histone deacetylation and transcriptional repression. The binding of the TR–RXR to a TRE in the presence of ligand leads to the recruitment of the coactivator complex (SRC-1, p300/CBP, PCAF) that directs histone acetylation and facilitates transcription.
consistent with stable association of the v-ErbA protein with recognition elements in a chromatin context19. • Transcriptional Coactivators and Corepressors of TR–RXR and v-ErbA that Modify Chromatin and Regulate Transcription Biochemical approaches using the ligand-binding domains of nuclear
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receptors to screen for interacting factors have identified diverse coactivators and corepressors that bind the TR–RXR in a manner that is dependent on the presence or absence of T3 (Ref. 20). In the absence of ligand, the TR has been shown to interact with the corepressors NCoR and SMRT (Refs 20–22). These corepressors also interact with the mammalian proteins
mSin3A and mSin3B (Refs 23,24). The Sin3 proteins associate with histone deacetylase (RPD3)25. These interconnections suggested that the TR–RXR might target transcriptional repression by recruiting histone deacetylase to modify the local chromatin environment. However, it is also possible that components of the recruited corepressor complex such as NCoR, SMRT or Sin3 might exert repressive activities on components of the basal machinery or organize repressive chromatin structures directly without the requirement for histone deacetylase26. Discrimination between these models was possible through the microinjection of doublestranded DNA templates into Xenopus oocyte nuclei. The slow kinetics of chromatin assembly on doublestranded DNA were especially advantageous for following the role of nucleosomes in transcriptional silencing. Expression of histone deacetylase could repress transcription of the Xenopus TRbA gene, depending on the assembly of a nucleosomal template, and this repression could be overcome by hormone-bound TR–RXR. The role of histone deacetylase could also be examined using a highly specific inhibitor trichostatin A (TSA). Incubation of oocytes containing a repressed TRbA gene assembled into nucleosomes with TSA relieved repression of the TRbA promoter to the same extent as the presence of hormone-bound TR–RXR (Ref. 27). This indicates that the activity of histone deacetylase on chromatin has a major role in transcriptional silencing (Fig. 3). A unifying feature of several of the diverse coactivator proteins described earlier is their capacity to function as histone acetyltransferases20,28. Thus, a common theme for transcriptional activation by ligand-bound nuclear receptors is the recruitment of histone acetyltransferase activity. This recruitment might be expected to counteract the repressive influence of histone deacetylase. Mutant forms of the human TR that cannot recruit transcriptional coactivators are responsible for the clinical syndrome of generalized resistance to T3 (Ref. 4). Later we discuss the biological roles of the TEM Vol. 10, No. 4, 1999
oncoprotein v-ErbA, which is also deficient in coactivator recruitment. The dynamics of histone acetylation provide an attractive mechanistic foundation for the reversible activation and repression of transcription. Histone acetylation states provide an example of the dynamic properties of chromatin, the TR itself provides another. Even though the TR–RXR complex can stably associate with chromatin, coactivators and corepressors, the TR is continually being degraded and replaced in vivo. Raaka and Samuels monitored the stability of TRs within the chromatin of mammalian cells through the labeling of TRs with dense amino acids29. Newly synthesized proteins are of higher density than pre-existing proteins and can be separated with gradient centrifugation techniques. The use of radioactive T3 allows the position of normal and dense receptors to be assayed independently in sucrose gradients. By quantifying the abundance of normal receptors at different times after the addition of dense amino acids to the cells, the receptor half-life can be assayed. The accumulation of dense receptors allows the rate of receptor synthesis to be determined. The TR has a half-life of 4.5 h in rat somatotrophic cells cultured in the absence of T3, and of 3 h in the presence of hormone. Importantly, most (95%) of the receptor decays with an identical half-life, indicating that no appreciable nuclear-associated pools exist that have distinct stabilities (Fig. 4). Moreover, the presence or absence of T3 does not influence the rate of TR decay. This suggests that the TR is exchanged from chromatin with equivalent efficiency whether it is engaged in repressing or activating transcription. In addition, more than 1700 molecules of TR were synthesized per hour in each cell to replace those being degraded. These kinetics imply that more than 95% of the receptor will be replaced within chromatin in 24 h. This capacity of a protein to be released from chromatin, degraded and then replaced with newly synthesized molecules is a common feature of both structural and regulatory chromosomal components. This release–replenishment cycle offers conTEM Vol. 10, No. 4, 1999
Figure 4. The thyroid hormone (T3) receptor (TR–RXR) is subject to continual turnover within a single cell cycle. TR is synthesized at a rate of 1700 molecules per cell per hour. The TR is then engaged in repression or activation of transcription as indicated. It is also probable that other pools of TRs exist in the nucleus as sites of storage or degradation. The TR decays with a half-life of 3–5 h independent of gene activity29.
siderable regulatory opportunities and would benefit from further investigation in easily manipulated model systems such as the Xenopus oocyte. Thus, just as histone acetylation states are dynamic, it would also appear that the TR, which targets chromatin modification, will be exchanged from corepressor and coactivator complexes within a given cell cycle. Together, these dynamic qualities should provide ample opportunity to reset patterns of gene expression rapidly in response to physiological demands. • Chromatin Disruption by the Ligand-bound TR–RXR We have discussed how the TR–RXR can bind to nucleosomal DNA to target both transcriptional repression and
activation. The TR–RXR silences transcription effectively in the context of a positioned nucleosomal array11,16. On the addition of ligand this regular nucleosomal array is disrupted. The requirements for this disruption offer additional insight into the potential roles for histone acetylation and chromatin modification in gene control. The TR–RXR alone and its recognition element are sufficient to initiate a process of chromatin disruption over an extended segment of DNA sequence, as assayed by micrococcal nuclease cleavage16. Each TRE can target the loss of topological constraint found in three to four nucleosomes, provided the TREs are separated by at least 400 bp of DNA. Transcription is not required for chromatin disruption, and additional 161
proximal promoter elements are necessary to facilitate hormone-dependent activation of transcription by the TR–RXR. The T3-dependent chromatin disruption depends on the transcription activation domain at the C-terminus of the TR. An important, yet unresolved, question concerns the role of histone acetyltransferases in this disruption process. At this time, the roles of transcriptional coactivators such as PCAF, p300 and SRC-1 in chromatin disruption have not been assayed; however, the structural and functional consequences of histone acetylation have been investigated intensively. Chromatin disruption targeted by ligand-bound TR–RXR leads to major topological changes in minichromosomes consistent with a loss of wrapping of DNA around the histones. Although histone acetyltransferases are recruited to the ligand-bound TR–RXR, it is difficult to account for the large topological change observed through acetylation alone30. In fact, transcription can be activated without significant topological change from a TRbA promoter complexed in chromatin with unliganded TR–RXR simply by the addition of the deacetylase inhibitor TSA (Ref. 27). Thus, histone hyperacetylation in isolation seems unlikely to account for chromatin disruption. More importantly, the chromatin disruption phenomenon in a minichromosomal context, as assayed by topological change and increases in micrococcal nuclease digestion, is not necessary for transcriptional activation. Chromatin disruption, as assayed by topological change and increases in micrococcal nuclease cleavage, has generally been interpreted as essential for transcriptional activation. However, transcriptional activation can occur with minimal changes in DNA topology31,32. This would be anticipated if histone acetylation were the only alteration to chromatin structure necessary for transcriptional activation. It is possible that the pathways directing chromatin disruption in the oocyte nucleus in response to ligand-bound TR–RXR do not involve histone acetylation, and that alternative means of disrupting histone–DNA contacts are employed,
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leading to similar transcriptional consequences. Candidate mechanisms include recruitment of molecular machines such as vertebrate chromatin remodeling complexes33,34. • Biological Implications of TR and v-ErbA Function in Chromatin Investigation of the requirements for the TR in animal development reveal relatively minor determinative roles, but more extensive requirements for the TR in adaptive responses and differentiation2,3,35. These observations might be explained by the TR exerting gene control predominantly by binding in chromatin and repressing gene activity. Thus, ablation of the TR will have the same consequence as adding hormone, in that repression will be relieved and transcription will return to high basal levels. If TR function is not to activate transcription much above basal levels in chromatin then removing the receptor will provide a phenotype similar to that of an animal constitutively exposed to T3. v-ErbA lacks the capacity to activate transcription and to interact with thyroid hormone as a consequence of small deletions in the ligand-binding domain. Neoplastic transformation by v-ErbA appears to be a result, in part, of the inhibition of genes regulated by the TR and the retinoic acid receptor (RAR)5. v-ErbA interacts with corepressors constitutively21 and requires histone deacetylase activity to silence transcription36. Transformation-defective variants of v-ErbA that fail to repress transcription also fail to interact with corepressors21. The capacity of v-ErbA to repress specific transcription, presumably via the recruitment of histone deacetylase, inhibits cell differentiation and allows cell proliferation to proceed. This mode of action of nuclear receptors appears to be co-opted by diverse oncoproteins containing nuclear receptor corepressor-binding domains37,38. Recent results demonstrate that in media depleted of T3 and retinoids, overexpressed TRa functions exactly like v-ErbA (Ref. 36). These experiments establish that unliganded TRa could impede differentiation, associate with histone deacetylase and repress
carbonic anhydrase and c-myb. These phenomena were reversed on the addition of hormone. These authors further suggest that TRa can function as a ligand-operated molecular switch, regulating the balance between erythroblast self-renewal and differentiation. It is attractive to speculate that TRa in Xenopus might have a similar function during tadpole development before the differentiation of a thyroid gland and the synthesis of thyroid hormone. Unliganded Xenopus TRa accumulates early in larval development, when it may serve to repress the expression of genes that are not required until metamorphosis. Thus, the TR might facilitate the proliferation of adult stem cell lineages in the absence of differentiation. Once thyroid hormone is synthesized, the same TRa bound within chromatin will serve to activate gene expression, including that of the TRb gene. The surge in TRb gene expression may be important in facilitating metamorphic climax by activating genes. In this scenario, TRb is the immediate-early gene whose transcription factor product initiates the cascade of gene activity associated with metamorphosis39. • Summary and Conclusions The TR and the related oncoprotein vErbA provide useful tools to investigate the functional capabilities of chromatin architecture and modification. The TR has the capacity to undergo regulated interactions with a rich variety of transcriptional coactivators and corepressors. A unifying feature of these interactions is the capacity of coactivators to acetylate histones and of corepressors to deacetylate histones. The TR also facilitates other structural modifications to chromatin, with as yet unknown functional consequences. For the vast majority of genes regulated by the TR, acetylation of proteins in the vicinity of the TRE appears to activate transcription, while deacetylation represses. Exactly how this comes about is unknown at present. However, it is clear that chromatin structure is destabilized as a consequence of acetylating the N-termini of the core histones, and that this modification facilitates the accessibility of DNA in TEM Vol. 10, No. 4, 1999
chromatin to the transcriptional machinery. Evidence also exists for direct contacts between the TR and components of the basal transcriptional machinery40,41. An understanding of nucleoprotein architecture within which the basal transcriptional machinery functions is undoubtedly essential to interpret the regulatory roles of the TR. However, the key point is that the activation or repression of a particular promoter will depend on the recruitment of a molecular machinery that modifies or disrupts the chromatin infrastructure. Large gaps in our knowledge as to how this occurs exist at both the structural and functional levels. Filling these gaps will contribute greatly to our understanding of the molecular mechanisms during cell transformation and also in generating targeted therapeutic strategies for intervention in human clinical disorders.
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Role of Pax Genes in Endoderm-derived Organs Ahmed Mansouri, Luc St-Onge and Peter Gruss
Pax genes, which encode a family of transcription factors, are essentially required for the formation of several tissues from all germ layers in the mammalian embryo. Specifically, in organogenesis, they are involved in triggering early events of cell differentiation. The differentiation of endodermderived endocrine pancreas is mediated through Pax4 and Pax6. In the thyroid gland, Pax8 is essential for the formation of thyroxine-producing follicular cells, also of endodermal origin. The analysis of loss-of-function mutants revealed a common function of Pax genes in organogenesis. The Pax gene family of transcription factors has been isolated on the basis of their sequence homology with Drosophila segmentation genes1–3. They share a common conserved DNA-binding domain of 128 amino acids (reviewed in Ref. 4). During embryogenesis, Pax genes exhibit highly restricted temporal and spatial expression patterns4. Functional studies of mouse mutants revealed that these genes are required for early events to generate a variety of tissues of all germ layers4. Specifically, in the endoderm, Pax genes are essential for the differentiation of endocrine cells in the pancreas and follicular cells in the mature thyroid gland. In this review, we will emphasize the role of Pax4 and Pax6 in the endocrine pancreas and Pax8 in the generation of thyroid hormone-producing follicular cells. The three genes are necessary to establish the differentiation of endoderm progenitors in A. Mansouri, L. St-Onge and P. Gruss are at the Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg, D-37077 Göttingen, Germany.
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specific cell types and indicate a common mechanism in the role of Pax genes in organogenesis. • Pax4 and Pax6 in the Developing Pancreas The mammalian pancreas is derived from the endoderm5,6. In the mouse, the first morphological appearance of the developing pancreas is evident at embryonic day (e) 9.5 as a dorsal, and later as a ventral, bud between the foreand midgut, in the region of the future duodenum. During further differentiation, a ductal branching system is formed, giving rise to the adult pancreas7–9. The major part consists of exocrine cells (acini) that produce the digestive enzymes. The second population (endocrine cells) is organized in units called islets of Langerhans6,9. Four endocrine cell types are found in the adult pancreas, a, b, d and PP, which produce glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. The genes encoding several transcription factors are expressed in the pancreas9–15, including Pax4 and Pax6.
mone receptor coactivator complex. Proc. Natl. Acad. Sci. U. S. A. 93, 8329–8333 42 Pruss, D., Hayes, J.J. and Wolffe, A.P. (1995) Nucleosomal anatomy – where are the histones? BioEssays 17, 161–170
Expression of Pax4 and Pax6 in the Developing Pancreas Pax4 and Pax6 expression occurs as early as e9.0 (15 somites) in the foregut of the mouse embryo16–18. At e10.5, Pax4 gene expression is observed in the dorsal and, by e11.0, in the ventral pancreatic bud. Already, at these early stages, Pax4 expression is restricted to a few cells, which also express the insulin gene. This pattern is maintained during further development and, in newborns, Pax4 is only detected in insulin-producing b cells16. Thus, Pax4 is expressed selectively in b cells. Pax6, on the other hand, is also first localized in a few cells, including cells coexpressing glucagon, but from e15.5 on its expression expands to all pancreatic endocrine cells. In newborn animals, the Pax6 protein is detected in all cells of the islets of Langerhans17,18. Taken together, these expression data show that at early stages of pancreatic development Pax4 and Pax6 gene expression is confined to endocrine precursor cells. Later, Pax4 gene expression continues to be restricted to insulinproducing b cells, but Pax6 expression is found in all pancreatic endocrine cells16–18. However, expression is never seen in the exocrine tissue. This suggests that Pax4 and Pax6 are required during early steps to generate specific endocrine cells of the pancreas. Role of Pax4 and Pax6 in the Pancreas An analysis of Pax4 function in pancreas development has been performed in mice generated by replacing the DNA-binding domain of genes with the bacterial β-galactosidase (lacZ) gene via homologous recombination in embryonic stem cells. In these embryos, Pax4expressing cells can be imaged by staining for LacZ (Ref. 16). Newborn mice homozygous for the mutated Pax4 gene develop to term but die soon after birth. Histological analysis
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