- Email: [email protected]
The third dimension of gene regulation: organization of dynamic chromatin loopscape by SATB1
The third dimension of gene regulation: organization of dynamic chromatin loopscape by SATB1 Sanjeev Galande, Prabhat Kumar Purbey, Dimple Notani and P Pavan Kumar Compartmentalized distribution of functional components is a hallmark of the eukaryotic nucleus. Technological advances in recent years have provided unprecedented insights into the role of chromatin organization and interactions of various structural–functional components toward gene regulation. SATB1, the global chromatin organizer and transcription factor, has emerged as a key factor integrating higher-order chromatin architecture with gene regulation. Studies in recent years have unraveled the role of SATB1 in organization of chromatin ‘loopscape’ and its dynamic nature in response to physiological stimuli. SATB1 organizes the MHC class-I locus into distinct chromatin loops by tethering MARs to nuclear matrix at fixed distances. Silencing of SATB1 mimics the effects of IFNg treatment on chromatin loop architecture of the MHC class-I locus and altered expression of genes within the locus. At genome-wide level, SATB1 seems to play a role in organization of the transcriptionally poised chromatin. Addresses National Centre for Cell Science, Ganeshkhind, Pune 411007, India Corresponding author: Galande, Sanjeev ([email protected])
Current Opinion in Genetics & Development 2007, 17:408–414 This review comes from a themed issue on Differentiation and gene regulation Edited by Denis Duboule and Frank Grosveld
0959-437X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2007.08.003
Introduction Chromatin architecture plays an important role in the regulation of nuclear function [1]. Specialized genomic sequences possessing high affinity for the nuclear matrix are termed matrix attachment regions (MARs) and are utilized in a selective and dynamic manner to tether chromatin loops in vivo [2]. Special AT-rich binding protein 1 (SATB1) is the most well-characterized MARbinding protein (MBP) that participates in the maintenance and compaction of chromatin architecture by organizing it into distinct loops via periodic anchoring of MARs to the nuclear matrix [3]. MARs have been implicated in the regulation of transcription by altering the organization of eukaryotic chromosomes and augmenting the potential of Current Opinion in Genetics & Development 2007, 17:408–414
enhancers to act over large distances. Consistent with this notion, SATB1 was shown to regulate distant genes by selectively tethering MARs to the nuclear matrix resulting in the formation of a characteristic ‘cage-like’ network that circumscribes heterochromatin [3]. Furthermore, SATB1 acts as a ‘docking site’ for several chromatin modifiers including ACF, ISWI, and HDAC1 [4,5] and these chromatin modifiers were suggested to suppress gene expression through histone deacetylation and nucleosome remodeling at SATB1-bound MARs [4]. Despite major advances in defining the precise role for SATB1 in higherorder chromatin organization and global transcriptional regulation, the link between the two is beginning to emerge only recently. Moreover, SATB1 seems to perform two different albeit related roles as it either can act as an architectural component of chromatin and/or can act as a transcription factor by binding to the upstream regulatory elements and recruiting chromatin modifiers. Recently, Kumar et al. identified the promyelocytic leukemia (PML) oncoprotein as a SATB1-interacting protein and showed that SATB1 and PML form unique regulatory complex that governs gene expression in a global manner by establishing distinct chromatin loop architecture [6]. This review focuses on dynamic chromatin looping and transcription mediated by SATB1 using the example of regulation of MHC class-I (MHC-I) chromatin organization and transcription.
Functional interaction between SATB1 and PML at MARs The nucleus of mammalian cells is organized into distinct chromatin territories and specialized subnuclear compartments [1,7]. One of the prominent nuclear substructures is the PML nuclear body (NB). The plethora of colocalizing proteins with PML NBs has led to the proposition that these bodies either function as nuclear depots and/or as organizing centers where numerous nuclear processes are executed and regulated [8]. Recent reports have suggested another potential PML NB function, namely, as a site where the transcriptional activity of a specific chromosomal locus is regulated via higher-order chromatin organization [9]. PML and its associated NBs have been implicated in the regulation of nuclear functions through direct chromatin contacts [8]. As PML lacks DNA-binding activity, it is mandatory for it to ‘piggyback’ onto other DNA-binding protein(s) to regulate transcription. The biology of PML and PML NBs got into an interesting turn when SATB1 was identified as their partner. Recently, Kumar et al. www.sciencedirect.com
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reported that SATB1 is actively recruited to PML NBs in different cell types [6]. Interaction between SATB1 and PML is mediated via their PDZ and coiled-coil domains, respectively, and is dependent on sumoylation of PML [6]. This aspect of their interaction is novel since PDZ domains are known to interact with other proteins via Ctermini or through another PDZ domain [10]. SATB1 and PML were shown to form a trimeric complex with MARs in vitro [6]. Given that both PML as well as SATB1 are known nuclear matrix components [11,12], it is likely that their interaction takes place in matrix-associated NBs. SATB1 and PML recruit large number of chromatin modifiers in vivo [3–5,6,8], and therefore sites at which SATB1 and PML coexist may function as nucleation points for assembly of macromolecular regulatory complexes. Whether such complexes are restricted to the PML NBs and whether they are anchored to the nuclear matrix in vivo require further investigation. The posttranslational modifications of SATB1, PML, and their interaction partners may provide a fine-tuning mechanism for regulation of chromatin architecture and transcription of genes within the loci organized by them.
MHC locus: a paradigm for integrating chromatin organization with gene regulation The mammalian major histocompatibility complex (MHC) locus is a supercluster comprising several clusters of structurally unrelated genes, and has been the preferred model for investigating chromatin-based mechanisms that regulate gene clusters and expression of genes within them. The classical MHC spans a 3.6 Mb region of chromosome 6p21.3 which is one of the most gene-rich regions of the human genome [13,14] and displays open chromatin fiber structure in specific regions [15,16]. From the centromeric to telomeric end, this supercluster is divided into three major regions: classical class-II, class-III, and class-I (Figure 1). PML NBs were shown to be associated specifically with the gene-rich region of the MHC cluster on chromosome 6p21 but not with the gene-poor 6p24 and centromeric regions of chromosome 6 [9]. An interesting caveat here is that PML NBs associate with genomic regions of increased transcription, rather than with individual genes with high transcription levels. Conversely, genes in regions of high activity, although not being highly expressed themselves, have statistically significant associations with PML NBs. Furthermore, within these regions of high local activity, PML NBs may associate with different genes at different times depending upon cell type and stage of cell cycle [9]. Role of PML in the regulation of MHC-I transcription is debated since knockdown of PML expression has differential effect on the transcription of the MHC genes [6,9,17]. However, knockdown of SATB1 has a profound consequence on the expression of majority of MHC-I genes (Figure 1) suggesting that SATB1 is involved in class-I regulation in a manner that mimics IFNg induction [6]. Transcriptional activation of the MHC by IFNg is preceded by massive remodeling of www.sciencedirect.com
the chromatin fiber, which manifests in looping out from the chromosome 6 territory. It has been speculated further that the megabase-sized giant loops formed after IFNg induction of MHC-I expression contain multiple smaller loops that are likely to be attached or adjacent to transcription factories [14]. It is possible that the association of PML bodies with the transcriptionally active regions of the genome including the MHC locus, and the extensive remodeling of this locus upon IFNg treatment are mediated via the SATB1–PML complex. Functional relevance of genome organization, especially towards transcription, has been a matter of intense debate. Two contrasting views have emerged over years, one supporting the notion that genome organization merely reflects nuclear processes including transcription, whereas the other subscribes to the idea that genome organization plays a decisive role in its function. Several independent lines of evidence indicate that the dynamic nuclear organization both reflects and molds genome function [26]. The genome organization-function ‘cause or effect’ riddle is compounded due to lack of a comprehensive knowledge of the various types of interactions between genomic segments in the nucleus, and the proteins that mediate such interactions. The recent emergence of various chromatin conformation capture techniques has provided an opportunity to map inter-chromosomal and intra-chromosomal interactions between multiple loci. Such techniques if combined with a functional screen for genomic segments (such as MARs, promoters, etc.) can provide the missing links toward reconstruction of the functional genomic maps.
MHC class-I loopscape: composition, dynamics, and transcription Kumar et al. probed into the chromatin architecture of a specific, but sufficiently large region of the MHC-I locus by applying a novel methodology that allowed mapping of the positions of individual chromatin loops based on the putative MARs and also the occupancy of proteins at the bases of these loops [6]. The MAR-ligation assay used by Kumar et al. is analogous to the 3C assay [18] except that the crosslinking step is omitted which also mitigates the background noise associated with crosslinking. Instead, the MAR-ligation assay takes advantage of the fact that due to the nature of chromatin looping two anchored MARs become naturally juxtaposed as they are held together by MBPs and consequently have a higher probability of ligation. The ligation products are detected through PCR amplification using specific primers. Such analyses of the 300 kb region of the MHC-I locus spanning HCG-9 and HLA-F genes indicated that regions surrounding base positions 64, 96, 105, 170, 220, and 299 kb are MARs and that intervening regions loop out (Figure 2). SATB1 has been postulated to function as a novel type of gene regulator by acting as a three-dimensional protein Current Opinion in Genetics & Development 2007, 17:408–414
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Figure 1
Organization of the human MHC supercluster. Schematic depiction of human chromosome 6 and gene density covering 183 Mb (www.sanger.ac.uk). Line graph shows an approximation of the chromatin fiber structure across the MHC locus at 6p21.3 spanning MHC classes II, III, and I is depicted in terms of log2 hybridization ratio between purified open chromatin and total input chromatin (log2 open:input chromatin) [15,16] on the y axis. Physical position of the three MHC classes is shown in kb on the x axis. The extended portion depicts selected list of genes across the 300 kb region within MHC class-I in a linear fashion. The transcription status of these genes (up or down) in Jurkat T lymphoblastoid cells upon SATB1 knockdown and IFNg treatment is indicated by arrows below each gene [6].
scaffold to which MAR sequences are tethered [3,6,19]. Indeed, using SATB1 RNAi, Kumar et al. observed that SATB1 is involved in the overall chromatin loop organization of the MHC-I locus, yet is not the sole MBP implicated. Intriguingly, knockdown of SATB1 affects formation of only two loops, suggesting involvement of other yet-to-be identified MBPs in formation of the MHC-I chromatin loopscape [6]. In a manner identical to IFNg treatment, upon SATB1 knockdown the chromatin structure at the MHC-I locus appeared to undergo reorganization leading to the formation of a new loop via anchorage of the region around 256 kb. Although IFNg treatment affected the expression of Current Opinion in Genetics & Development 2007, 17:408–414
majority of the MHC class-I genes in Jurkat T cells, the most profound effect was observed in the form of upregulation of HCG-4 and HCG-4P6 expression. Notably, the HCG-4 locus, which is about 30 kb away from the nearest site of matrix attachment, gets attached to the MAR at 256 kb region in an IFNg-inducible manner, suggesting that the positioning of this gene with respect to the nuclear matrix influences its expression (Figure 2). Strikingly, shRNA-mediated silencing of SATB1 also selectively altered the transcriptional activity of the MHC genes in vivo similar to IFNg treatment (Figure 1). SATB1 seems to be involved specifically in the activation of HCG-9, while it represses most other www.sciencedirect.com
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Figure 2
Dynamic reorganization of the chromatin loop architecture of MHC-I locus upon IFNg treatment. Schematic representation of the chromatin loop structure of the MHC-I locus in control cells and IFNg treated cells as deduced from the ChIP-loop assay [6]. Cartoons on top depict the loopscape in linear fashion while those at bottom indicate the same in circular fashion. In lower left cartoon, the non-random distribution of SATB1 and PML across the MHC-I locus is indicated by depicting the occupancy of the two proteins deduced by chromatin immunoprecipitation assay [6]. The lower right-hand side cartoon depicts only the major changes upon IFNg treatment; namely, attachment of 256 kb region to matrix by SATB1, and replacement of SATB1 by another MBP tethering the region around 220 kb.
genes within the MHC-I locus. Furthermore, the expression of HCG-9 is enhanced if it is pushed into being part of the giant loop formed by dynamic reorganization in that region [6]. Thus, SATB1 is not only necessary to establish a defined chromatin structure, but more importantly, also for maintaining a conducive transcriptional www.sciencedirect.com
environment tailored for coordinated regulation of the MHC-I locus, including its inducibility by IFNg. The compositional balance of SATB1 and nuclear PML isoforms is crucial for the proper chromatin loop organization and gene regulation of this locus [6]. The transcriptional status of the genes within the locus is dependent on loop Current Opinion in Genetics & Development 2007, 17:408–414
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architecture, their relative distances from the site of attachment of the loops, and occupancy of SATB1 and PML at their upstream regulatory regions.
SATB1 is involved in cell-type-specific organization of MHC class-I loopscape Genome-wide chromatin immunoprecipitation analyses indicated that SATB1 and PML show non-random association across the MHC class-I locus. The ‘clustering’ of these two proteins at MARs and the upstream regulatory sequences of genes indicates a dichotomous role for SATB1 as structural and as regulatory chromatin component (Figure 2). Using MAR-ligation assay, Kumar et al. mapped five chromatin loops within the 300 kb region of the MHC-I locus in Jurkat T cells [6]. Such analysis in CHO cells that do not express SATB1 as abundantly as Jurkat T cells revealed that the MHC-I locus forms six loops. The bases of these loops differ in three positions than that of the Jurkat cells. However, if SATB1 expression is matched with that of Jurkat T cells through transfection of a SATB1 expression construct then it results in detachment of regions surrounding 128 and 256 kb from matrix into chromatin loops and concomitant formation of a new loop at 220 kb region giving rise to a MHC-I loop configuration mirroring the one in Jurkat cells [6]. Thus, the MHC-I loopscape appears to be tissue and cell-type-specific, and that this specificity could be mediated by SATB1 and other MBPs. Moreover, the presence and absence of cell-type-specific MBP(s) as well as their relative abundance both seem to be responsible for the formation and maintenance of cell-type-specific chromatin loop architecture.
Tissue-specific chromatin organization by SATB1: the thymocyte episode SATB1 forms a very distinguished substructure in nuclei, which is seen predominantly in undifferentiated thymocytes and not in differentiated subsets of T cells [3]. SATB1 forms a three-dimensional network assuming shape of a cage surrounding chromatin territories (CTs) (Figure 3). The interchromatin space is occupied by loops containing the gene-dense regions and majority of transcriptionally poised genes. SATB1 binds at the bases of loops and upstream regulatory regions of genes and therefore appears to fill the void between CTs (Figure 3). Whether SATB1 is totally excluded from the CTs or may also play a role in regulation of the transcribed genes within CTs is not yet clear. Since the SATB1 immunostaining signals are not prominent in the CTs, it may be gleaned that SATB1 may not exhibit ‘clustering’ type of occupancy at the various genomic regions embedded within the CTs. The in vivo genomic targets of SATB1 are anchored to the SATB1 network in wild-type thymocytes at the bases of chromatin loops but not in SATB1-deficient thymocytes suggesting role for SATB1 in chromatin organization [3]. Furthermore, binding of SATB1 to its targets elicits site-specific histone modifications that correlate with proper regulation of distant genes [3]. For any gene, SATB1 may act as repressor or activator depending upon the context, physiological need, and the post-translational modification status of SATB1 [20]. SATB1 is required for the formation of the transcriptionally active chromatin loop structure of the 200 kb cytokine locus on chromosome 11 that forms upon T-helper2 (TH2) cell
Figure 3
Global chromatin organization by SATB1. (a) Mouse thymocyte stained with DAPI to reveal heterochromatin blobs or the chromatin territories (CTs). (b) Thymocyte stained with anti-SATB1 to reveal ‘cage-like’ network structure circumscribing the CTs. (c) Schematic representation of an enlarged sector from the thymocyte depicting occupancy of SATB1 at various locations within the decondensed and active chromatin loops that fill the space in between the highly condensed CTs. Current Opinion in Genetics & Development 2007, 17:408–414
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Chromatin loopscape and SATB1 Galande et al. 413
activation [19]. This region has been well characterized with respect to the long-range intrachromosomal interactions [19,21]. A DNaseI hypersensitive site (RHS7) is involved in mediating such interactions among various cis elements within the TH2 cytokine cluster, such as the locus control regions and promoters [22]. Such assembly of cis elements and chromatin looping is crucial for tissuespecific expression of globin genes indicating that clustering of regulatory elements is vital for the creation and maintenance of active chromatin ‘hubs’ and for transcription regulation [23,24]. Interestingly, since SATB1 is required for transcriptional activation of genes within the TH2 cytokine cluster, the molecular events accompanying this activation such as histone acetylation, recruitment of chromatin remodeling factors, and RNA polymerase II could be mediated by SATB1. Thus, SATB1 seems to play a major role in regulating the transcriptionally conducive environment in the interchromatin space.
isoforms originated from Dr Oliver Bischof. We regret that due to space restrictions the interesting work of several colleagues could not be cited. Work in SG Laboratory is supported by grants from the Department of Biotechnology, Government of India, and the Wellcome Trust, UK. PKP, DN, and PK are supported by fellowships from the Council of Scientific and Industrial Research, India. SG is an international senior research fellow of the Wellcome Trust, UK.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Spector DL: The dynamics of chromosome organization and gene regulation. Annu Rev Biochem 2003, 72:573-608.
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Heng HH, Goetze S, Ye CJ, Liu G, Stevens JB, Bremer SW, Wykes SM, Bode J, Krawetz SA: Chromatin loops are selectively anchored using scaffold/matrix-attachment regions. J Cell Sci 2004, 117:999-1008.
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Cai S, Han HJ, Kohwi-Shigematsu T: Tissue-specific nuclear architecture and gene expression regulated by SATB1. Nat Genet 2003, 34:42-51.
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Yasui D, Miyano M, Cai S, Varga-Weisz P, Kohwi-Shigematsu T: SATB1 targets chromatin remodelling to regulate genes over long distances. Nature 2002, 419:641-645.
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Kumar PP, Purbey PK, Ravi DS, Mitra D, Galande S: Displacement of SATB1-bound histone deacetylase 1 corepressor by the human immunodeficiency virus type 1 transactivator induces expression of interleukin-2 and its receptor in T cells. Mol Cell Biol 2005, 25:1620-1633.
Conclusions and prospects The MHC class-I locus is organized into distinct chromatin loops by SATB1-mediated tethering of MARs to nuclear matrix at defined positions. IFNg treatment or SATB1 knockdown induces alterations both in chromatin loop architecture of the MHC-I locus and the expression profile of distinct set of MHC-I genes. Moreover, an exquisite collaboration between individual PML isoforms and SATB1 determines dynamics of the chromatin organization of the MHC-I locus and its expression. Post-translational modifications of SATB1 and PML may add further complexity in regulation by providing additional layers of regulation. CTs are interspersed with a variety of non-chromatin domains such as Cajal bodies, PML NBs, and transcription factories [1,7,25,26]. A central question regarding this organization is how the activities of the genome and non-chromatin domains are coordinated to regulate nuclear processes. Recent studies have suggested a role for SATB1 at the interface of the CTs and interchromatin compartments. Understanding the molecular constitution and organization of the SATB1 superstructure in thymocyte nuclei may help in resolving mysteries of the enigmatic interchromatin compartment which appears to be decorated by SATB1. Such studies will also benefit from genome-wide analysis of SATB1 targets and defining a consensus for SATB1 binding. Comprehensive knowledge of the repertoire of genome-wide SATB1 targets and various factors recruited by SATB1 will further enhance our understanding of the intricate relationship between chromatin architecture and genome function.
Acknowledgements We thank Amita Limaye for microscopy and members of SG laboratory for discussion. The idea for studying contributions of different PML www.sciencedirect.com
6.
Kumar PP, Bischof O, Purbey PK, Notani D, Urlaub H, Dejean A, Galande S: Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus. Nat Cell Biol 2007, 9:45-56. This article reports for the first time that SATB1 bridges PML onto chromatin. Using MAR-ligation assay the involvement of SATB1 and PML in the organization of MHC class I locus in form of a dynamic ‘loopscape’ and for proper gene expression was demonstrated. 7.
Cremer T, Cremer M, Dietzel S, Muller S, Solovei I, Fakan S: Chromosome territories — a functional nuclear landscape. Curr Opin Cell Biol 2006, 18:307-316.
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Ching RW, Dellaire G, Eskiw CH, Bazett-Jones DP: PML bodies: a meeting place for genomic loci? J Cell Sci 2005, 118:847-854.
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Wang J, Shiels C, Sasieni P, Wu PJ, Islam SA, Freemont PS, Sheer D: Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions. J Cell Biol 2004, 164:515-526.
10. Harris BZ, Lim WA: Mechanism and role of PDZ domains in signaling complex assembly. J Cell Sci 2001, 114:3219-3231. 11. Chang KS, Fan YH, Andreeff M, Liu J, Mu ZM: The PML gene encodes a phosphoprotein associated with the nuclear matrix. Blood 1995, 85:3646-3653. 12. Seo J, Lozano MM, Dudley JP: Nuclear matrix binding regulates SATB1-mediated transcriptional repression. J Biol Chem 2005, 280:24600-24609. 13. Kumanovics A, Takada T, Lindahl KF: Genomic organization of the mammalian MHC. Annu Rev Immunol 2003, 21:629-657. 14. Volpi EV, Chevret E, Jones T, Vatcheva R, Williamson J, Beck S, Campbell RD, Goldsworthy M, Powis SH, Ragoussis J et al.: Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. J Cell Sci 2000, 113:1565-1576. 15. Gilbert N, Boyle S, Fiegler H, Woodfine K, Carter NP, Bickmore WA: Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers. Cell 2004, 118:555-566. Current Opinion in Genetics & Development 2007, 17:408–414
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16. Sproul D, Gilbert N, Bickmore WA: The role of chromatin structure in regulating the expression of clustered genes. Nat Rev Genet 2005, 6:775-781.
21. Spilianakis CG, Flavell RA: Long-range intrachromosomal interactions in the T helper type 2 cytokine locus. Nat Immunol 2004, 5:1017-1027.
17. Bruno S, Ghiotto F, Fais F, Fagioli M, Luzi L, Pelicci PG, Grossi CE, Ciccone E: The PML gene is not involved in the regulation of MHC class I expression in human cell lines. Blood 2003, 101:3514-3519.
22. Lee GR, Spilianakis CG, Flavell RA: Hypersensitive site 7 of the TH2 locus control region is essential for expressing TH2 cytokine genes and for long-range intrachromosomal interactions. Nat Immunol 2005, 6:42-48.
18. Splinter E, Grosveld F, de Laat W: 3C technology: analyzing the spatial organization of genomic loci in vivo. Methods Enzymol 2004, 375:493-507.
23. Tolhuis B, Palstra R-J, Splinter E, Grosveld F, de Laat W: Looping and interaction between hypersensitive sites in the active bglobin locus. Mol Cell 2002, 10:1453-1465.
19. Cai S, Lee CC, Kohwi-Shigematsu T: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes. Nat Genet 2006, 38:1278-1288. This is the first report showing involvement of a T lineage enriched MARbinding protein SATB1 in regulation of chromatin organization and transcription of clustered genes. SATB1 was shown to be essential for the formation of transcriptionally active chromatin structure of the TH2 cytokine locus on mouse chromosome 11.
24. de Laat W, Grosveld F: Spatial organization of gene expression: the active chromatin hub. Chromosome Res 2003, 11:447-459.
20. Pavan Kumar P, Purbey PK, Sinha CK, Notani D, Limaye A, Jayani RS, Galande S: Phosphorylation of SATB1, a global gene regulator, acts as a molecular switch regulating its transcriptional activity in vivo. Mol Cell 2006, 22:231-243.
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25. Albiez H, Cremer M, Tiberi C, Vecchio L, Schermelleh L, Dittrich S, Ku¨pper K, Joffe B, Thormeyer T, von Hase J: Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks. Chromosome Res 2006, 14:707-733. 26. Fraser P, Bickmore W: Nuclear organization of the genome and the potential for gene regulation. Nature 2007, 447:413-417. An excellent review article summarizing the current knowledge and concepts regarding spatial relationships and interactions of genes and regulatory elements in the nuclear space and their effects on gene expression.
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Current Opinion in Genetics & Development 2007, 17:408–414 This review comes from a themed issue on Differentiation and gene regulation Edited by Denis Duboule and Frank Grosveld
0959-437X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2007.08.003
Introduction Chromatin architecture plays an important role in the regulation of nuclear function [1]. Specialized genomic sequences possessing high affinity for the nuclear matrix are termed matrix attachment regions (MARs) and are utilized in a selective and dynamic manner to tether chromatin loops in vivo [2]. Special AT-rich binding protein 1 (SATB1) is the most well-characterized MARbinding protein (MBP) that participates in the maintenance and compaction of chromatin architecture by organizing it into distinct loops via periodic anchoring of MARs to the nuclear matrix [3]. MARs have been implicated in the regulation of transcription by altering the organization of eukaryotic chromosomes and augmenting the potential of Current Opinion in Genetics & Development 2007, 17:408–414
enhancers to act over large distances. Consistent with this notion, SATB1 was shown to regulate distant genes by selectively tethering MARs to the nuclear matrix resulting in the formation of a characteristic ‘cage-like’ network that circumscribes heterochromatin [3]. Furthermore, SATB1 acts as a ‘docking site’ for several chromatin modifiers including ACF, ISWI, and HDAC1 [4,5] and these chromatin modifiers were suggested to suppress gene expression through histone deacetylation and nucleosome remodeling at SATB1-bound MARs [4]. Despite major advances in defining the precise role for SATB1 in higherorder chromatin organization and global transcriptional regulation, the link between the two is beginning to emerge only recently. Moreover, SATB1 seems to perform two different albeit related roles as it either can act as an architectural component of chromatin and/or can act as a transcription factor by binding to the upstream regulatory elements and recruiting chromatin modifiers. Recently, Kumar et al. identified the promyelocytic leukemia (PML) oncoprotein as a SATB1-interacting protein and showed that SATB1 and PML form unique regulatory complex that governs gene expression in a global manner by establishing distinct chromatin loop architecture [6]. This review focuses on dynamic chromatin looping and transcription mediated by SATB1 using the example of regulation of MHC class-I (MHC-I) chromatin organization and transcription.
Functional interaction between SATB1 and PML at MARs The nucleus of mammalian cells is organized into distinct chromatin territories and specialized subnuclear compartments [1,7]. One of the prominent nuclear substructures is the PML nuclear body (NB). The plethora of colocalizing proteins with PML NBs has led to the proposition that these bodies either function as nuclear depots and/or as organizing centers where numerous nuclear processes are executed and regulated [8]. Recent reports have suggested another potential PML NB function, namely, as a site where the transcriptional activity of a specific chromosomal locus is regulated via higher-order chromatin organization [9]. PML and its associated NBs have been implicated in the regulation of nuclear functions through direct chromatin contacts [8]. As PML lacks DNA-binding activity, it is mandatory for it to ‘piggyback’ onto other DNA-binding protein(s) to regulate transcription. The biology of PML and PML NBs got into an interesting turn when SATB1 was identified as their partner. Recently, Kumar et al. www.sciencedirect.com
Chromatin loopscape and SATB1 Galande et al. 409
reported that SATB1 is actively recruited to PML NBs in different cell types [6]. Interaction between SATB1 and PML is mediated via their PDZ and coiled-coil domains, respectively, and is dependent on sumoylation of PML [6]. This aspect of their interaction is novel since PDZ domains are known to interact with other proteins via Ctermini or through another PDZ domain [10]. SATB1 and PML were shown to form a trimeric complex with MARs in vitro [6]. Given that both PML as well as SATB1 are known nuclear matrix components [11,12], it is likely that their interaction takes place in matrix-associated NBs. SATB1 and PML recruit large number of chromatin modifiers in vivo [3–5,6,8], and therefore sites at which SATB1 and PML coexist may function as nucleation points for assembly of macromolecular regulatory complexes. Whether such complexes are restricted to the PML NBs and whether they are anchored to the nuclear matrix in vivo require further investigation. The posttranslational modifications of SATB1, PML, and their interaction partners may provide a fine-tuning mechanism for regulation of chromatin architecture and transcription of genes within the loci organized by them.
MHC locus: a paradigm for integrating chromatin organization with gene regulation The mammalian major histocompatibility complex (MHC) locus is a supercluster comprising several clusters of structurally unrelated genes, and has been the preferred model for investigating chromatin-based mechanisms that regulate gene clusters and expression of genes within them. The classical MHC spans a 3.6 Mb region of chromosome 6p21.3 which is one of the most gene-rich regions of the human genome [13,14] and displays open chromatin fiber structure in specific regions [15,16]. From the centromeric to telomeric end, this supercluster is divided into three major regions: classical class-II, class-III, and class-I (Figure 1). PML NBs were shown to be associated specifically with the gene-rich region of the MHC cluster on chromosome 6p21 but not with the gene-poor 6p24 and centromeric regions of chromosome 6 [9]. An interesting caveat here is that PML NBs associate with genomic regions of increased transcription, rather than with individual genes with high transcription levels. Conversely, genes in regions of high activity, although not being highly expressed themselves, have statistically significant associations with PML NBs. Furthermore, within these regions of high local activity, PML NBs may associate with different genes at different times depending upon cell type and stage of cell cycle [9]. Role of PML in the regulation of MHC-I transcription is debated since knockdown of PML expression has differential effect on the transcription of the MHC genes [6,9,17]. However, knockdown of SATB1 has a profound consequence on the expression of majority of MHC-I genes (Figure 1) suggesting that SATB1 is involved in class-I regulation in a manner that mimics IFNg induction [6]. Transcriptional activation of the MHC by IFNg is preceded by massive remodeling of www.sciencedirect.com
the chromatin fiber, which manifests in looping out from the chromosome 6 territory. It has been speculated further that the megabase-sized giant loops formed after IFNg induction of MHC-I expression contain multiple smaller loops that are likely to be attached or adjacent to transcription factories [14]. It is possible that the association of PML bodies with the transcriptionally active regions of the genome including the MHC locus, and the extensive remodeling of this locus upon IFNg treatment are mediated via the SATB1–PML complex. Functional relevance of genome organization, especially towards transcription, has been a matter of intense debate. Two contrasting views have emerged over years, one supporting the notion that genome organization merely reflects nuclear processes including transcription, whereas the other subscribes to the idea that genome organization plays a decisive role in its function. Several independent lines of evidence indicate that the dynamic nuclear organization both reflects and molds genome function [26]. The genome organization-function ‘cause or effect’ riddle is compounded due to lack of a comprehensive knowledge of the various types of interactions between genomic segments in the nucleus, and the proteins that mediate such interactions. The recent emergence of various chromatin conformation capture techniques has provided an opportunity to map inter-chromosomal and intra-chromosomal interactions between multiple loci. Such techniques if combined with a functional screen for genomic segments (such as MARs, promoters, etc.) can provide the missing links toward reconstruction of the functional genomic maps.
MHC class-I loopscape: composition, dynamics, and transcription Kumar et al. probed into the chromatin architecture of a specific, but sufficiently large region of the MHC-I locus by applying a novel methodology that allowed mapping of the positions of individual chromatin loops based on the putative MARs and also the occupancy of proteins at the bases of these loops [6]. The MAR-ligation assay used by Kumar et al. is analogous to the 3C assay [18] except that the crosslinking step is omitted which also mitigates the background noise associated with crosslinking. Instead, the MAR-ligation assay takes advantage of the fact that due to the nature of chromatin looping two anchored MARs become naturally juxtaposed as they are held together by MBPs and consequently have a higher probability of ligation. The ligation products are detected through PCR amplification using specific primers. Such analyses of the 300 kb region of the MHC-I locus spanning HCG-9 and HLA-F genes indicated that regions surrounding base positions 64, 96, 105, 170, 220, and 299 kb are MARs and that intervening regions loop out (Figure 2). SATB1 has been postulated to function as a novel type of gene regulator by acting as a three-dimensional protein Current Opinion in Genetics & Development 2007, 17:408–414
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Figure 1
Organization of the human MHC supercluster. Schematic depiction of human chromosome 6 and gene density covering 183 Mb (www.sanger.ac.uk). Line graph shows an approximation of the chromatin fiber structure across the MHC locus at 6p21.3 spanning MHC classes II, III, and I is depicted in terms of log2 hybridization ratio between purified open chromatin and total input chromatin (log2 open:input chromatin) [15,16] on the y axis. Physical position of the three MHC classes is shown in kb on the x axis. The extended portion depicts selected list of genes across the 300 kb region within MHC class-I in a linear fashion. The transcription status of these genes (up or down) in Jurkat T lymphoblastoid cells upon SATB1 knockdown and IFNg treatment is indicated by arrows below each gene [6].
scaffold to which MAR sequences are tethered [3,6,19]. Indeed, using SATB1 RNAi, Kumar et al. observed that SATB1 is involved in the overall chromatin loop organization of the MHC-I locus, yet is not the sole MBP implicated. Intriguingly, knockdown of SATB1 affects formation of only two loops, suggesting involvement of other yet-to-be identified MBPs in formation of the MHC-I chromatin loopscape [6]. In a manner identical to IFNg treatment, upon SATB1 knockdown the chromatin structure at the MHC-I locus appeared to undergo reorganization leading to the formation of a new loop via anchorage of the region around 256 kb. Although IFNg treatment affected the expression of Current Opinion in Genetics & Development 2007, 17:408–414
majority of the MHC class-I genes in Jurkat T cells, the most profound effect was observed in the form of upregulation of HCG-4 and HCG-4P6 expression. Notably, the HCG-4 locus, which is about 30 kb away from the nearest site of matrix attachment, gets attached to the MAR at 256 kb region in an IFNg-inducible manner, suggesting that the positioning of this gene with respect to the nuclear matrix influences its expression (Figure 2). Strikingly, shRNA-mediated silencing of SATB1 also selectively altered the transcriptional activity of the MHC genes in vivo similar to IFNg treatment (Figure 1). SATB1 seems to be involved specifically in the activation of HCG-9, while it represses most other www.sciencedirect.com
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Figure 2
Dynamic reorganization of the chromatin loop architecture of MHC-I locus upon IFNg treatment. Schematic representation of the chromatin loop structure of the MHC-I locus in control cells and IFNg treated cells as deduced from the ChIP-loop assay [6]. Cartoons on top depict the loopscape in linear fashion while those at bottom indicate the same in circular fashion. In lower left cartoon, the non-random distribution of SATB1 and PML across the MHC-I locus is indicated by depicting the occupancy of the two proteins deduced by chromatin immunoprecipitation assay [6]. The lower right-hand side cartoon depicts only the major changes upon IFNg treatment; namely, attachment of 256 kb region to matrix by SATB1, and replacement of SATB1 by another MBP tethering the region around 220 kb.
genes within the MHC-I locus. Furthermore, the expression of HCG-9 is enhanced if it is pushed into being part of the giant loop formed by dynamic reorganization in that region [6]. Thus, SATB1 is not only necessary to establish a defined chromatin structure, but more importantly, also for maintaining a conducive transcriptional www.sciencedirect.com
environment tailored for coordinated regulation of the MHC-I locus, including its inducibility by IFNg. The compositional balance of SATB1 and nuclear PML isoforms is crucial for the proper chromatin loop organization and gene regulation of this locus [6]. The transcriptional status of the genes within the locus is dependent on loop Current Opinion in Genetics & Development 2007, 17:408–414
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architecture, their relative distances from the site of attachment of the loops, and occupancy of SATB1 and PML at their upstream regulatory regions.
SATB1 is involved in cell-type-specific organization of MHC class-I loopscape Genome-wide chromatin immunoprecipitation analyses indicated that SATB1 and PML show non-random association across the MHC class-I locus. The ‘clustering’ of these two proteins at MARs and the upstream regulatory sequences of genes indicates a dichotomous role for SATB1 as structural and as regulatory chromatin component (Figure 2). Using MAR-ligation assay, Kumar et al. mapped five chromatin loops within the 300 kb region of the MHC-I locus in Jurkat T cells [6]. Such analysis in CHO cells that do not express SATB1 as abundantly as Jurkat T cells revealed that the MHC-I locus forms six loops. The bases of these loops differ in three positions than that of the Jurkat cells. However, if SATB1 expression is matched with that of Jurkat T cells through transfection of a SATB1 expression construct then it results in detachment of regions surrounding 128 and 256 kb from matrix into chromatin loops and concomitant formation of a new loop at 220 kb region giving rise to a MHC-I loop configuration mirroring the one in Jurkat cells [6]. Thus, the MHC-I loopscape appears to be tissue and cell-type-specific, and that this specificity could be mediated by SATB1 and other MBPs. Moreover, the presence and absence of cell-type-specific MBP(s) as well as their relative abundance both seem to be responsible for the formation and maintenance of cell-type-specific chromatin loop architecture.
Tissue-specific chromatin organization by SATB1: the thymocyte episode SATB1 forms a very distinguished substructure in nuclei, which is seen predominantly in undifferentiated thymocytes and not in differentiated subsets of T cells [3]. SATB1 forms a three-dimensional network assuming shape of a cage surrounding chromatin territories (CTs) (Figure 3). The interchromatin space is occupied by loops containing the gene-dense regions and majority of transcriptionally poised genes. SATB1 binds at the bases of loops and upstream regulatory regions of genes and therefore appears to fill the void between CTs (Figure 3). Whether SATB1 is totally excluded from the CTs or may also play a role in regulation of the transcribed genes within CTs is not yet clear. Since the SATB1 immunostaining signals are not prominent in the CTs, it may be gleaned that SATB1 may not exhibit ‘clustering’ type of occupancy at the various genomic regions embedded within the CTs. The in vivo genomic targets of SATB1 are anchored to the SATB1 network in wild-type thymocytes at the bases of chromatin loops but not in SATB1-deficient thymocytes suggesting role for SATB1 in chromatin organization [3]. Furthermore, binding of SATB1 to its targets elicits site-specific histone modifications that correlate with proper regulation of distant genes [3]. For any gene, SATB1 may act as repressor or activator depending upon the context, physiological need, and the post-translational modification status of SATB1 [20]. SATB1 is required for the formation of the transcriptionally active chromatin loop structure of the 200 kb cytokine locus on chromosome 11 that forms upon T-helper2 (TH2) cell
Figure 3
Global chromatin organization by SATB1. (a) Mouse thymocyte stained with DAPI to reveal heterochromatin blobs or the chromatin territories (CTs). (b) Thymocyte stained with anti-SATB1 to reveal ‘cage-like’ network structure circumscribing the CTs. (c) Schematic representation of an enlarged sector from the thymocyte depicting occupancy of SATB1 at various locations within the decondensed and active chromatin loops that fill the space in between the highly condensed CTs. Current Opinion in Genetics & Development 2007, 17:408–414
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Chromatin loopscape and SATB1 Galande et al. 413
activation [19]. This region has been well characterized with respect to the long-range intrachromosomal interactions [19,21]. A DNaseI hypersensitive site (RHS7) is involved in mediating such interactions among various cis elements within the TH2 cytokine cluster, such as the locus control regions and promoters [22]. Such assembly of cis elements and chromatin looping is crucial for tissuespecific expression of globin genes indicating that clustering of regulatory elements is vital for the creation and maintenance of active chromatin ‘hubs’ and for transcription regulation [23,24]. Interestingly, since SATB1 is required for transcriptional activation of genes within the TH2 cytokine cluster, the molecular events accompanying this activation such as histone acetylation, recruitment of chromatin remodeling factors, and RNA polymerase II could be mediated by SATB1. Thus, SATB1 seems to play a major role in regulating the transcriptionally conducive environment in the interchromatin space.
isoforms originated from Dr Oliver Bischof. We regret that due to space restrictions the interesting work of several colleagues could not be cited. Work in SG Laboratory is supported by grants from the Department of Biotechnology, Government of India, and the Wellcome Trust, UK. PKP, DN, and PK are supported by fellowships from the Council of Scientific and Industrial Research, India. SG is an international senior research fellow of the Wellcome Trust, UK.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Conclusions and prospects The MHC class-I locus is organized into distinct chromatin loops by SATB1-mediated tethering of MARs to nuclear matrix at defined positions. IFNg treatment or SATB1 knockdown induces alterations both in chromatin loop architecture of the MHC-I locus and the expression profile of distinct set of MHC-I genes. Moreover, an exquisite collaboration between individual PML isoforms and SATB1 determines dynamics of the chromatin organization of the MHC-I locus and its expression. Post-translational modifications of SATB1 and PML may add further complexity in regulation by providing additional layers of regulation. CTs are interspersed with a variety of non-chromatin domains such as Cajal bodies, PML NBs, and transcription factories [1,7,25,26]. A central question regarding this organization is how the activities of the genome and non-chromatin domains are coordinated to regulate nuclear processes. Recent studies have suggested a role for SATB1 at the interface of the CTs and interchromatin compartments. Understanding the molecular constitution and organization of the SATB1 superstructure in thymocyte nuclei may help in resolving mysteries of the enigmatic interchromatin compartment which appears to be decorated by SATB1. Such studies will also benefit from genome-wide analysis of SATB1 targets and defining a consensus for SATB1 binding. Comprehensive knowledge of the repertoire of genome-wide SATB1 targets and various factors recruited by SATB1 will further enhance our understanding of the intricate relationship between chromatin architecture and genome function.
Acknowledgements We thank Amita Limaye for microscopy and members of SG laboratory for discussion. The idea for studying contributions of different PML www.sciencedirect.com
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Kumar PP, Bischof O, Purbey PK, Notani D, Urlaub H, Dejean A, Galande S: Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus. Nat Cell Biol 2007, 9:45-56. This article reports for the first time that SATB1 bridges PML onto chromatin. Using MAR-ligation assay the involvement of SATB1 and PML in the organization of MHC class I locus in form of a dynamic ‘loopscape’ and for proper gene expression was demonstrated. 7.
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19. Cai S, Lee CC, Kohwi-Shigematsu T: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes. Nat Genet 2006, 38:1278-1288. This is the first report showing involvement of a T lineage enriched MARbinding protein SATB1 in regulation of chromatin organization and transcription of clustered genes. SATB1 was shown to be essential for the formation of transcriptionally active chromatin structure of the TH2 cytokine locus on mouse chromosome 11.
24. de Laat W, Grosveld F: Spatial organization of gene expression: the active chromatin hub. Chromosome Res 2003, 11:447-459.
20. Pavan Kumar P, Purbey PK, Sinha CK, Notani D, Limaye A, Jayani RS, Galande S: Phosphorylation of SATB1, a global gene regulator, acts as a molecular switch regulating its transcriptional activity in vivo. Mol Cell 2006, 22:231-243.
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25. Albiez H, Cremer M, Tiberi C, Vecchio L, Schermelleh L, Dittrich S, Ku¨pper K, Joffe B, Thormeyer T, von Hase J: Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks. Chromosome Res 2006, 14:707-733. 26. Fraser P, Bickmore W: Nuclear organization of the genome and the potential for gene regulation. Nature 2007, 447:413-417. An excellent review article summarizing the current knowledge and concepts regarding spatial relationships and interactions of genes and regulatory elements in the nuclear space and their effects on gene expression.
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