Gene name - suppressor of Hairy wing
Cytological map position - 88A12-B2
Function - Transcription factor - Insulator region binding protein
Keywords - Chromatin organization
Symbol - su(Hw)
Genetic map position - 3-54.8
Cellular location - nuclear
|Recent literature||Hsu, S.J., Plata, M.P., Ernest, B., Asgarifar, S. and Labrador, M. (2015). The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila. Dev Biol 403(1):57-68. PubMed ID: 25882370
Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. This study shows that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy revealed that lack of Su(Hw) lead to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there was a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. The study concluded that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.
|Ulianov, S. V., et al. (2015). Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains. Genome Res [Epub ahead of print]. PubMed ID: 26518482
Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, Hi-C and poly(A)+ RNA-seq were performed in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila were found to be only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) was found to be preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, binding of insulator proteins dCTCF and Su(Hw) was found to predict TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, these results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. The mechanism of TAD self-assembly is proposed based on the ability of nucleosomes from inactive chromatin to aggregate, and on the lack of this ability in acetylated nucleosomal arrays. Finally, this hypothesis was tested by polymer simulations and it was found that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.
|Melnikova, L., Shapovalov, I., Kostyuchenko, M., Georgiev, P. and Golovnin, A. (2016). EAST affects the activity of Su(Hw) insulators by two different mechanisms in Drosophila melanogaster. Chromosoma [Epub ahead of print]. PubMed ID: 27136940
Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. This study shows that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.
|Coulthard, A. B., Taylor-Kamall, R. W., Hallson, G., Axentiev, A., Sinclair, D. A., Honda, B. M. and Hilliker, A. J. (2016). Meiotic recombination is suppressed near the histone-defined border of euchromatin and heterochromatin on chromosome 2L of Drosophila melanogaster. Genome 59: 289-294. PubMed ID: 27031007
In Drosophila melanogaster, the borders between pericentric heterochromatin and euchromatin on the major chromosome arms have been defined in various ways, including chromatin-specific histone modifications, the binding patterns of heterochromatin-enriched chromosomal proteins, and various cytogenetic techniques. Elucidation of the genetic properties that independently define the different chromatin states associated with heterochromatin and euchromatin should help refine the boundary. Since meiotic recombination is present in euchromatin, but absent in heterochromatin, it constitutes a key genetic property that can be observed transitioning between chromatin states. Using P element insertion lines marked with a su(Hw) insulated mini-white gene, meiotic recombination was found to transition in a region consistent with the H3K9me2 transition observed in ovaries.
|Kravchuk, O., Kim, M., Klepikov, P., Parshikov, A., Georgiev, P. and Savitsky, M. (2016). Transvection in Drosophila: trans-interaction between yellow enhancers and promoter is strongly suppressed by a cis-promoter only in certain genomic regions. Chromosoma [Epub ahead of print]. PubMed ID: 27300555
Transvection is a phenomenon of interallelic communication whereby enhancers of one allele can activate a promoter located on the homologous chromosome. It has been shown for many independent genes that enhancers preferentially act on the cis-linked promoter, but deletion of this promoter allows the enhancers to act in trans. This study tested whether this cis-preference in the enhancer-promoter interaction could be reconstituted outside of the natural position of a gene. The yellow gene was chosen as a model system. Transgenic flies were generated that carried the yellow gene modified by the inclusion of the strategically placed recognition sites for the Cre and Flp recombinases. To facilitate transvection, an endogenous Su(Hw) insulator (1A2) or gypsy insulator was placed behind the yellow gene. Independent action of the recombinases produced a pair of derivative alleles, one containing the promoter-driven yellow gene, and the other, the enhancers and promoter that failed to produce a functional yellow protein. As a result, strong transvection was observed in many genomic regions, suggesting that a complete cis-preference of the enhancer-promoter interactions is mainly restricted to genes in their natural loci.
|Pauli, T., et al. (2016). Transcriptomic data from panarthropods shed new light on the evolution of insulator binding proteins in insects. BMC Genomics 17: 861. PubMed ID: 27809783
Body plan development in multi-cellular organisms is largely determined by homeotic genes. Expression of homeotic genes, in turn, is partially regulated by insulator binding proteins (IBPs). While only a few enhancer blocking IBPs have been identified in vertebrates, the common fruit fly Drosophila melanogaster harbors at least twelve different enhancer blocking IBPs. This study screened ecently compiled insect transcriptomes from the 1KITE project and genomic and transcriptomic data from public databases, aiming to trace the origin of IBPs in insects and other arthropods. The study shows that the last common ancestor of insects (Hexapoda) already possessed a substantial number of IBPs. Specifically, of the known twelve insect IBPs, at least three (i.e., CP190, Su(Hw), and CTCF) already existed prior to the evolution of insects. Furthermore GAF orthologs were found in early branching insect orders, including Zygentoma (silverfish and firebrats) and Diplura (two-pronged bristletails). Mod(mdg4) is most likely a derived feature of Neoptera, while Pita is likely an evolutionary novelty of holometabolous insects. Zw5 appears to be restricted to schizophoran flies, whereas BEAF-32, ZIPIC and the Elba complex, are probably unique to the genus Drosophila. Selection models indicate that insect IBPs evolved under neutral or purifying selection. These results suggest that a substantial number of IBPs either pre-date the evolution of insects or evolved early during insect evolution. This suggests an evolutionary history of insulator binding proteins in insects different to that previously thought. Moreover, this study demonstrates the versatility of the 1KITE transcriptomic data for comparative analyses in insects and other arthropods.
|Melnikova, L., Kostyuchenko, M., Parshikov, A., Georgiev, P. and Golovnin, A. (2018). Role of Su(Hw) zinc finger 10 and interaction with CP190 and Mod(mdg4) proteins in recruiting the Su(Hw) complex to chromatin sites in Drosophila. PLoS One 13(2): e0193497. PubMed ID: 29474480
Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, this study examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Delta10 and CP190. Having studied insulator complex formation in different mutant backgrounds, it is concluded that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.
|Duan, T. and Geyer, P. K. (2018). Spermiogenesis and male fertility require the function of Suppressor of Hairy-Wing in somatic cyst cells of Drosophila. Genetics. PubMed ID: 29739818
Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is an example of a multivalent transcription factor. Although best known for its role in establishing the chromatin insulator of the gypsy retrotransposon, Su(Hw) functions as an activator and repressor at non-gypsy genomic sites. It remains unclear how the different regulatory activities of Su(Hw) are utilized during development. Motivated from observations of spatially restricted expression of Su(Hw) in the testis, this study investigated the role of Su(Hw) in spermatogenesis to advance an understanding of its developmental contributions as an insulator, repressor and activator protein. It was discovered that Su(Hw) is required for sustained male fertility. Although dynamics of Su(Hw) expression coincide with changes in nuclear architecture and activation of co-regulated testis-specific gene clusters, loss of Su(Hw) does not disrupt meiotic chromosome pairing or transcription of testis-specific genes, suggesting that Su(Hw) has minor architectural or insulator functions in the testis. Instead, Su(Hw) has a prominent role as a repressor of neuronal genes, consistent with suggestions that Su(Hw) is a functional homologue of mammalian REST, a repressor of neuronal genes in non-neuronal tissues. Su(Hw) regulates transcription in both germline and somatic cells. Surprisingly, the essential spermatogenesis function of Su(Hw) resides in somatic cyst cells, implying context-specific consequences due to loss of this transcription factor. Together, these studies highlight that Su(Hw) has a major developmental function as a transcriptional repressor, with the impact of its loss dependent upon the cell-specific factors.
|Radion, E., Sokolova, O., Ryazansky, S., Komarov, P. A., Abramov, Y. and Kalmykova, A. (2019). The integrity of piRNA clusters is abolished by insulators in the Drosophila germline. Genes (Basel) 10(3). PubMed ID: 30862119
Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, the effects were studied of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. Transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.
Regulatory elements termed enhancers, or locus control regions are capable of exerting their influence over long distances in an orientation-independent manner to orchestrate the complex gene expression patterns required for embryonic development. How are the effects of enhancers confined to the genes they regulate? In recent years the concept of domain boundaries or insulator elements has developed, based on the genetic properties of several eukaryotic genes. These elements serve to isolate chromosomal regions, confining regulatory effects of enhancers to the genes they are meant to serve. For example, specialized chromatin structures (scs and scs') have been found at outside the coding sequence of the Drosophila hsp70 gene. These sequences are associated with chromatin structures and serve as boundaries that can prevent activation by enhancer elements. Similarly the chicken ßglobin insulator can inhibit activation fo the gamma-globin gene promoter by the ß-globin locus control region. The Drosophila gypsy insulator functions similarly, confering position-independent transcription to genes and preventing activation of promoters by enhancers separated from proximal promoters by insulator elements (Gdula, 1996 and references).
The best characterized system is the gypsy chromatin insulator of Drosophila. Gypsy is an infectious retrovirus consisting of two long terminal repeat regions that function in viral replication and integration and three retroviral genes: gag, pol and env, coding for nucleocapsid, reverse transcriptase and envelope proteins respectively (Song, 1994). The gypsy insulator is located in the 5' transcribed untranslated region of the gypsy element. The unlinked gene, suppressor of Hairy-wing plays an essential role in functioning of the gypsy insulator. SU(HW) binds to the insulator element, interacting directly with an octamer motif present in the gypsy insulator. SU(HW) binds to approximately 200 other sites on Drosophila polytene chromosomes, possibly corresponding to endogenous insulators that play a role in the organization of the genome into higher order chromatin domains that allow for the normal regulation of gene expression.
yellow is one of the genes that has been used in studying the insulator properties of gypsy. yellow is a non-essential gene, lending pigmentation to various embryonic, larval and adult cuticular structures. yellow is regulated by a series of tissue-specific transcriptional enhancers (located in the 5' region of the gene). These enhancers regulate expression in the wings, body cuticle, and larval pigmented structures; a second series of intronic enhancers regulate expression in bristles and tarsal claws (Gdula, 1996 and references).
Insertion of gypsy in the 5' region of yellow inhibits the interaction between the upstream wing and body cuticle enhancers but does not affect the larval enhancer located proximal to the promoter. Mutation of su(Hw) results in a non-functional insulator. In flies with mutated su(Hw), upstream enhancers function even in the presence of insulator sequences between the enhancer and the proximal region of the gene. This suggests strongly that su(Hw) is required for the insulating properties of gypsy.
modifier of mdg4 was discovered during an analysis of mutations that alter the ability of su(Hw) to interfere with enhancer-promoter interactions. Mutations in mod(mdg4) cause an enhancement of the yellow phenotype, resulting in flies in which every cuticular structure of the larva and adult is unpigmented. This phenotype can be interpreted as a consequence of the inactivation of the yellow promoter itself, or inactivation of every enhancer controling yellow expression.
Cloning of mod(mdg4) revealed the structure of the protein for which it codes. MOD(MDG4) protein contains a BTB domain in the amino terminal region, a motif found in GAGA protein (known as Trithorax-like), a protein that plays a role in chromatin remodeling. MOD(MDG4) and SU(HW) demonstrate a direct physical interaction. MOD(MDG4) protein lacks a recognizable DNA binding domain and is unable to bind to insulator sequences of gypsy. Nevertheless MOD(MDG)4 is present on polytene chromosomes, in 2-3 times the number of bands containing SU(HW), suggesting that MOD(MDG4) might also interact with other proteins. In fact, mutation of mod(mdg4) is lethal, suggesting thaat MOD(MDG4) has a broad function. In the absence of MOD(MDG4), gypsy loses its uni-directional effects on distal enhancers and instead silences both upstream and downstream enhancers. Thus the function of MOD(MDG4) appears to be establishment of uni-directionality on the gypsy insulator. A mutated mod(mdg4) does not act as simply as a null allele, but instead causes a variegated phenotype similar to those caused by juxtaposing active genes to heterochromatic sequences. The resemblence of mod(mdg4) mutations to position-effect variegation, suggests that the effects of SU(HW) and MOD(MDG4) are mediated by changes in chromatin and not simply by classical transcription factor mediated changes in gene activation (Gdula, 1996).
There is a paradox with respect to SU(HW) function that begs for a resolution. On one hand the SU(HW)-MOD(MDG4) interaction suggests a chromatin based mechanism of gene activation and silencing similar to that mediated by Trithorax - and Polycomb-group genes. On the other hand, analysis of enhancers insulated from distal promoters by the gypsy element reveals that these enhancers can still function to regulate genes not protected by the insulator (Cai, 1995 and Scott, 1995). Paradoxically, the enhancer regions themselves are still functional, even if they are insulated form certain promoters by the gypsy element. The continued ability of enhancers to function, suggests that enhancers are not subject to chromatin mediated silencing. The resolution of this paradox will come from a biochemical study of the effects of SU(HW) and MOD(MDG4) chromatin structure. The generality of the role of chromatin in boundary element functioning is illustrated by the characterization of another boundary element associated factor, BEAF-32, which binds and contributes to boundary function at the scs' element of the hsp70 gene (Zhao, 1995).
How does the suHw insulator function to block enhancer-promoter interactions? Using divergently transcribed reporter genes in transgenic Drosophila embryos, it has been shown that an enhancer blocked from the downstream promoter by suHw is fully competent to activate an upstream promoter. To probe the insulator mechanism, the effect of suHw copy number on its insulator strength was tested in Drosophila embryos. The zerknullt enhancer VRE (ventral repression element) has been shown to be partially blocked by suHw. In blastoderm embryos, the V2 transgene containing VRE and E2, an evenskipped stripe 2 enhancer, directs reporter expression in a composite pattern of broad dorsal activation and dominant ventral repression of the E2 stripe. A single 340-bp suHw insulator element in the VS2 transgene partially blocks the upstream VRE enhancer. Two tandem suHw elements (arranged as direct repeats) were inserted between VRE and E2, resulting in VSS2. Instead of enhanced blockage, VSS2 embryos exhibit a loss of suHw insulator activity. This was observed in most VSS2 embryos and in all 10 independent VSS2 lines, indicating that it is unlikely to be caused by chromosomal position effects. Genomic polymerase chain reaction (PCR) analysis of independent VS2 and VSS2 lines further verifies the structural integrity of the transgenes in vivo (Cai, 2001).
To determine whether the loss of insulator function in VSS2 embryos is enhancer-specific, transgenes were constructed using a rhomboid neuroectodermal enhancer (NEE) and a hairy stripe 1 enhancer (H1). The NLH embryos containing NEE and H1 enhancers separated by a 1.4-kb neutral spacer (L) exhibit composite lacZ pattern directed by both enhancers. A single suHw element in the NSH transgene blocks the upstream NEE enhancer, whereas two tandem suHw elements (NSSH) do not block the NEE enhancer. A second group of transgenes uses a twist mesoderm enhancer (PE) and an evenskipped stripe 3 enhancer (E3). Both enhancers are active when separated by the L spacer (PL3). Insertion of a suHw element in the PS3 transgene blocks the upstream PE enhancer, whereas two tandem suHw elements do not block the PE enhancer. Replacing one of the two suHw elements in PSS3 with a spacer of comparable size (A) restores the enhancer-blocking activity of the remaining suHw in PSA3 embryos, indicating that loss of insulator activity with two suHw elements is not due to the spacing change but to the presence of the additional insulator. These results suggest that the loss of insulator activity with tandemly arranged suHw is independent of the enhancer tested (Cai, 2001).
The enhancer-blocking activity of suHw may require its interaction with other sites (or insulators) within the nucleus. A second suHw nearby may compete dominantly for the existing suHw and affect the neighboring enhancer-promoter interactions, depending on the cis arrangement of these elements. To test this hypothesis, the SVS2 transgene was constructed in which the VRE enhancer is flanked by two suHw elements. In contrast to the loss of insulator function seen in VSS2 embryos, the VRE enhancer is more effectively blocked in SVS2 embryos than in VS2 embryos. Thus, it is the tandem arrangement rather than physical proximity that causes the loss of insulator activity. VRE-mediated dorsal activation of the divergently transcribed miniwhite is also diminished in SVS2 embryos, indicating that VRE is blocked from promoters on either side. suHw-mediated blockage of VRE is significantly reduced in SVS2/mod(mdg4)u1 embryos, indicating that a MOD(MDG4)-mediated complex is required for the enhanced insulator activity. VSS2, NSSH, and PSS3 transgenes were also examined in a mod(mdg4)u1 background, and no change in the staining patterns was seen (Cai, 2001).
Insulators block enhancer-promoter interactions only when positioned between them. How can this occur without inactivating the enhancer or promoter? It has been hypothesized that insulators may interact with each other to form chromatin loop domains, restricting interactions among neighboring regulatory elements. The enhancer-blocking activity mediated by suHw is abolished when insulators are in tandem, and is enhanced when they flank the enhancer. Thus, suHw does not seem to block distal enhancers by locally capturing the enhancer complex or its associated proteins, because two tandem elements abolish rather than enhance the insulator function. Instead, a single intervening suHw insulator may interact with other insulators or chromosomal/nuclear sites, separating the enhancer and the promoter into topologically distinct chromatin domains. Two tandem suHw elements may preferentially interact with each other, excluding other interactions necessary to sequester the enhancer from the promoter, and may even augment the enhancer-promoter interaction by 'looping out' the intervening DNA. In contrast, suHw elements flanking an enhancer may readily interact as a result of their proximity, leading to better blockage of the enhancer. Loss of insulator function was seen when the distance between the two tandem suHw elements is 50, 150, and 170 bp (VSS2, PSS3, and NSSH, respectively). It has also been observed with spacers ranging from 200 bp to 5 kb in length. Therefore, it is unlikely to be caused by nonspecific steric hindrance due to the close juxtaposition of the insulators. DNA looping has been observed between interacting regulatory elements as close as 100 bp apart. Insulator assembly may induce alternative chromatin structure, resulting in DNA bending or nuclease-hypersensitive sites, which often indicate nucleosome-free DNA, to facilitate loop formation. Insulators or chromatin boundaries are frequently found in multiple copies, flanking enhancers or the genetic locus they regulate, such as the scs and scs' elements, the Mcp-1 and Fab boundaries, and the chicken ß-globin 5' and 3' boundaries. Selective interactions between neighboring insulators may regulate the access of tissue-specific enhancers to target promoters by forming alternative chromatin loop domains. It is conceivable that these domains not only block inappropriate enhancers but also facilitate interaction between distant enhancers and the target promoter (Cai, 2001).
Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into Topologically Associating Domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, Hi-C and PolyA+ RNA-seq was performed in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, this study found that regions between TADs (i.e. the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, it was found that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted interbands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, these results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. The mechanism of TAD self-assembly is proposed based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability is found in acetylated nucleosomal arrays. Finally, this hypothesis is tested by polymer simulations, and it was found that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin (Ulianov, 2015).
Recently developed 3C-based methods coupled with high-throughput sequencing have enabled genome-wide investigation of chromatin organization. Studies performed in human, mouse, Drosophila, yeasts, Arabidopsis and several other species have unraveled general principles of genome folding. Chromosomes in mammals and Drosophila are organized hierarchically. At the megabase scale, mammalian chromosomes are partitioned into active and inactive compartments. At the sub-megabase scale, these compartments are subdivided into a set of self-interacting domains called Topologically Associating Domains (TADs); TADs themselves are often hierarchical and are split into smaller domains. Similar to mammals, Drosophila chromosomes are partitioned into TADs that are interspaced with short boundaries or longer inter-TAD regions (inter-TADs) (Ulianov, 2015).
Partitioning of mammalian genomes into TADs appears to be largely cell-lineage independent and evolutionary conserved. Disruption of certain TAD boundaries leads to developmental defects in humans and mice. TADs correlate with units of replication timing regulation in mammals and colocalize with epigenetic domains (either active or repressed) in Drosophila. The internal structure of TADs was reported to change in response to environmental stress, during cell differentiation, and embryonic development. In addition, comparative Hi-C analysis has demonstrated that genomic rearrangements between related mammalian species occur predominantly at TAD boundaries. Consequently, TADs appear to evolve primarily as constant and unsplit units. Previous studies in Drosophila embryonic nuclei and embryo-derived Kc167 cells detected TADs of various sizes roughly corresponding to epigenetic domains. Additionally, long-range genomic contacts and clustering of pericentromeric regions were revealed, and TAD boundaries were found to be enriched with active chromatin marks and insulator proteins. Both active and inactive TADs were identified, and their spatial segregation was observed (Ulianov, 2015).
Despite extensive studies, mechanisms underlying TAD formation remain obscure. Architectural proteins, including cohesin and CTCF, are often found at TAD boundaries; thus, they have been proposed to play a key role in the demarcation of TADs. However, several studies suggest that other mechanisms may be responsible for partitioning and formation of TADs. Firstly, depletion of various insulator proteins did not affect the profile of chromosome partitioning into TADs, but rather decreased intra-TAD interactions. Secondly, CTCF may mediate loops that occur between the start and the end of the so-called 'loop domains'. However, domains of similar sizes but without a loop were observed as well (so-called 'ordinary domains'. Thirdly, polymer simulations of a permanent chromatin loop yield a noticeable interaction between the loop bases on a simulated Hi-C map, but without a characteristic square shape of a TAD. Loops of this kind are thought to occur between insulator proteins such as Su(Hw) in the 'topological insulation' model. Finally, chromosomal domains similar to TADs in the bacterium Caulobacter crescentus are demarcated by actively transcribed genes, and are not affected by the knockout of SMC, a homolog of cohesin subunits (Ulianov, 2015).
This study presents evidences that question the role of insulators in the organization of TAD boundaries in Drosophila . The results suggest that TADs are self-organized and potentially highly dynamic structures formed by numerous transient interactions between nucleosomes of inactive chromatin, while inter-TADs and TAD boundaries contain highly acetylated nucleosomes that are less prone to interactions. Finally, a polymer model of TAD formation is developed based on the two types of nucleosomes, and it was found that a polymer composed of active and inactive chromatin blocks forms TADs on a simulated Hi-C map (Ulianov, 2015).
This study and others (Hou, 2012; Sexton, 2012) revealed that boundaries and inter-TADs in Drosophila, as opposed to TADs, are strongly enriched with active chromatin and its individual marks, as well as with active transcription and with constitutively transcribed housekeeping genes. Consequently, active chromatin marks, in the simplest case only total transcription and H3K4me3 (a mark of active promoters), can relatively well predict a TAD/inter-TAD profile. The existence of long inter-TADs composed of active chromatin is per se an argument for the ability of this type of chromatin to separate TADs. Furthermore, the current observations demonstrate that the presence of active chromatin and transcribed regions within TAD undermines the TAD integrity making TAD less compact and generating weak boundaries inside TAD. Consequently, a bona fide TAD is inactive; TADs containing active chromatin become less dense, acquire weak internal boundaries and eventually split into smaller TADs that are composed of inactive chromatin. The observation that the majority of housekeeping genes are located within inter- TADs and TAD boundaries suggests that evolutionary conservation and cell-type independence of TAD/inter-TAD profiles may be explained by conservation of positions of housekeeping genes along the chromosomes (Ulianov, 2015).
It is noted that chromosomal interaction domains similar to TADs have been observed in the bacterium Caulobacter crescentus, where they are demarcated by sites of active transcription. Although the basic level of chromosomal folding is different in bacteria and eukaryotes, the model proposed in (Le, 2013) and the model stem from common principles. In Caulobacter, active transcription is thought to disrupt the fiber of supercoils (plectonemes) by creating a stretch of non-packaged DNA, free of plectonemes, which spatially separates chromosomal regions flanking it. In the model, transcription disrupts chromatin organization by introducing a 'non-sticky' region of chromatin, which is less compact and more unfolded in space, and thus spatially separates two flanking regions. Computer modeling shows that stickiness of non-acetylated (inactive) nucleosomes and the absence of stickiness for acetylated (active) nucleosomes are sufficient for chromatin partitioning into TADs and inter-TADs. Self-association of nucleosomes may be explained by the interaction of positively charged histone tails (in particular, the tail of histone H4) of one nucleosome with the acidic patch of histones H2A/H2B at an adjacent nucleosome. Acetylation of histone tails, which is typical of active chromatin, may interfere with inter-nucleosomal associations. In addition to a high level of histone acetylation, other features of active chromatin including lower nucleosome density in inter-TADs, manifested as the decreased histone H3 occupancy, might contribute to the generation of TAD profiles (Ulianov, 2015).
It should be mentioned that a significant difference between the polymer simulations and models previously suggested by the Cavalli and Vaillant groups (Jost, 2014) is the use of saturating interactions between inactive nucleosomes. In the case of volume interactions, all nucleosomes of the same type adjacent in 3D space will attract each other; in the case of saturating interactions, each molecule may attract only one neighbor. Using volume interactions leads to the formation of a single dense blob, and does not produce TADs in a simulated Hi-C map.It is noted that the saturating nature of interactions between nucleosomes is based on the known properties of nucleosomal particles. Previous studies considered a variety of mechanisms that may lead to the formation of TADs. In particular, Barbieri (2012) studied segregation of two TADs using cubic lattice simulations of a short 152-monomer chain consisting of two TADs, assuming that inter- monomer interactions could only form between monomers belonging to the same TAD. In the current model, this study shows that TADs emerge without requiring such specific interactions; any two regions of sticky monomers separated by a non-sticky linker would form TADs. Another study proposed that transcription-induced supercoiling may be responsible for the formation of TADs (Benedetti, 2014). Although this model is consistent with the current observation that sites of active transcription demarcate TAD boundaries, there is limited evidence that supercoiling of chromatinized DNA exists in Drosophila and other organisms. On the contrary, the current model is based on known biochemical properties of nucleosomes (Ulianov, 2015).
The fact that a minor fraction of TADs is built mostly from active chromatin apparently contradicts the current model, suggesting that additional ways of chromatin self-organization could exist. One possibility is the establishment of long-range contacts between enhancers and their cognate promoters, as well as loops between pairs of insulators. Such loops formed inside active unstructured chromatin linkers (i.e., inter-TADs) could probably be sufficient to compact them and thus to fold into TADs (Ulianov, 2015).
TAD profiles of X chromosomes are almost identical in the male and female cell lines, that is in agreement with recently published observations (Ramírez, 2015). Thus, it seems that hyperacetylation of male X-chromosomes due to dosage compensation does not generate new TAD boundaries. However, it should be noted that MOF histone acetyltransferase of the MSL complex introduces only the H4K16ac mark. Although this modification is important to prevent inter-nucleosomal interactions, acetylation at other histone positions and H2B ubiquitylation contribute as well. Additionally, H4K16 acetylation generated by the dosage compensation system occurs preferentially at regions enriched with transcribed genes and hence within inter-TADs (Ulianov, 2015).
The current analysis does not support the previously reported (Hou, 2012; Sexton, 2012) strong enrichments of insulator proteins Su(Hw) and dCTCF at TAD boundaries in Drosophila. To assess the possible reasons of this divergence, the dCTCF distribution was re-analyzed with respect to TAD positions in the current dataset using the raw ChIP-seq data. No strong difference was observed in the dCTCF coverage in TADs and inter-TADs. Interestingly, this study obtained the same result while analyzing dCTCF and Su(Hw) binding within TAD boundaries identified by Hou (2012). However, a strong enrichment of dCTCF at TAD boundaries was observed when the peak distribution was analyzed instead of read coverage. Additionally, the effect was much weaker when modENCODE peaks were used. Hence, the discrepancy may be caused by a different peak calling procedure in modENCODE and in Hou. (2012). The biological significance of these observations remains to be determined. It is noted that disruption of the cohesin/CTCF complex in mammals, as well as depletion of the Vtd (also known as Rad21) cohesin subunit in Drosophila, did not lead to disappearance of TAD boundaries, but rather only slightly decreased interactions inside TADs (in mammals) and reduced TAD boundary strength in the Drosophila genome. These observations favor a role for the cohesin/CTCF complex, which is known to form loops, in chromatin compaction inside the TADs (Ulianov, 2015).
Binding of insulator proteins might contribute to establishing TAD boundaries through introducing active chromatin marks. Indeed, when inserted into an ectopic position, a classical insulator triggers hyperacetylation of the local chromatin domain and recruits chromatin-remodeling complexes. However, absence of strong enrichment of dCTCF at TAD boundaries and preferential location of Su(Hw) inside TADs mean that at least dCTCF- and Su(Hw)-dependent insulators are not the major determinants of TAD boundaries and inter-TADs (Ulianov, 2015).
TADs are predicted based on the analysis of averaged data from a cell population. Although they are usually represented as large chromatin globules, direct experimental evidence for the existence of such globules in individual cells is controversial. Using confocal and 3D-SIM microscopy, ~1-Mb globular domains have been observed within chromosomal territories. However, using STORM microscopy, chromatin in individual mammalian cells has been found to be organized into 'clutches' composed of several nucleosomes, and that increased histone acetylation dramatically reduces size of these clutches. It is thus possible that sub-megabase TADs revealed by Hi-C represent a set of nucleosome clutches separated by relatively short spacers of various sizes. These short clutches may occupy various positions within TADs in different cells and stochastically assemble to form short-living aggregates. The stochastic nature of TADs is supported by computer simulations (Ulianov, 2015).
Exons - 7
SU(HW) contains 12 repeats of the C2H2 zinc finger motif. These zinc finger domains are located in the central portion of the protein. In addition, a highly acidic region, containing 48% Asp and Glu, is present between amino acids 154 and 202, immediately proceeding the first Zn finger motif (Parkhurst, 1988). A leucine zipper domain is found between the zinc finger domains and the C-terminal end of the protein (Harrison, 1993).
It is thought that su(Hw) forms discrete domains of gene activity by segregating promoters from enhancer elements through a change in chromatin organization. Functional domains of the su(Hw) protein were characterized that mediate the silencing effect of mod(mdg4) mutations. Two of three regions of su(Hw), regions B and C, that are located between the leucine zipper motif and the C-terminal acidic domain are conserved across Drosophila species and are necessary for both the unidirectional and bidirectional repression of transcription by su(Hw). These domains are implicated in an interaction with mod(mdg4) which is thought to mediate the unidirectional repression due to insulator function. In contrast, two acidic domains, the N-terminal acidic domain and the C-terminal acidic domain, dispensable for the unidirectional repression of enhancer elements, are critical for the bidirectional silencing of enhancer activity observed in mutants lacking functional mod(mdg4) protein. Bidirectional repression is thought to be due to changes in large blocks of chromatin structure (Gdula, 1997).
date revised: 10 May 98
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