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1.  Chromatin Insulators: A Role in Nuclear Organization and Gene Expression 
Advances in cancer research  2011;110:43-76.
Chromatin insulators are DNA-protein complexes with broad functions in nuclear biology. Based on the ability of insulator proteins to interact with each other, it was originally thought that insulators form loops that could constitute functional domains of co-regulated gene expression. Nevertheless, data from genome-wide localization studies indicate that insulator proteins can be present in intergenic regions as well as at the 5′, introns or 3′ of genes, suggesting a broader role in chromosome biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Recent results suggest that insulators mediate intra- and inter-chromosomal interactions to affect transcription, imprinting and recombination. It is possible that these interactions set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation. As a consequence, disruption of insulator activity could result in the development of cancer or other disease states.
doi:10.1016/B978-0-12-386469-7.00003-7
PMCID: PMC3175007  PMID: 21704228
2.  Tissue-Specific Regulation of Chromatin Insulator Function 
PLoS Genetics  2012;8(11):e1003069.
Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.
Author Summary
Mounting evidence in human, mouse, and Drosophila demonstrates a role for the DNA–protein complexes known as chromatin insulators in orchestrating three-dimensional genome organization. Several genes that are only expressed in specific cell types display distinct chromatin configurations correlated with expression status. Recent evidence shows that chromatin insulators play a role in defining tissue-specific chromatin conformation; however, tissue-specific factors that may modulate insulator activity remain unknown. Here we identify a putative RNA–binding protein, Shep, which is expressed most highly in the CNS and interacts directly with insulator complexes. We developed a novel quantitative, tissue-specific insulator assay and found that Shep negatively regulates insulator activity in the CNS. We also find that mutation of shep alters insulator complex nuclear localization in the brain but not other tissues. Finally, we mapped Shep and gypsy insulator protein localization throughout the genome and found that Shep colocalizes with one individual insulator protein but less often than expected with an intact insulator complex. These data suggest that Shep negatively influences insulator activity in a tissue-specific manner.
doi:10.1371/journal.pgen.1003069
PMCID: PMC3510032  PMID: 23209434
3.  A Comprehensive Map of Insulator Elements for the Drosophila Genome 
PLoS Genetics  2010;6(1):e1000814.
Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP–chip the genome-wide binding sites of 6 insulator-associated proteins—dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF—to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.
Author Summary
The spatiotemporal specificity of gene expression is controlled by interactions among regulatory proteins, cis-regulatory elements, chromatin modifications, and genes. These interactions can occur over large distances, and the mechanisms by which they are controlled are poorly understood. Insulators are DNA sequences that can both block the interaction between regulatory elements and genes, as well as block the spread of regions of modified chromatin. To date, relatively few insulators have been identified in developing Drosophila embryos. We here present the genome wide identification of over 14,000 binding sites for 6 insulator-associated proteins. We demonstrate the existence of two broad classes of insulators. Insulators of both classes are enriched at the boundaries of a particular chromatin modification. However, only insulators bound by BEAF-32, CP190, and dCTCF are enriched in regions of open chromatin or demarcate gene boundaries, with a particular enrichment between differentially expressed promoters. Furthermore, insulators of this class are enriched at points of chromosomal rearrangement among the 12 species of sequenced Drosophila, suggesting that insulator defined regulatory boundaries are evolutionarily conserved.
doi:10.1371/journal.pgen.1000814
PMCID: PMC2797089  PMID: 20084099
4.  Effective Blocking of the White Enhancer Requires Cooperation between Two Main Mechanisms Suggested for the Insulator Function 
PLoS Genetics  2013;9(7):e1003606.
Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer–white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer–promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer–promoter pair.
Author Summary
The mechanism underlying enhancer blocking by insulators is unclear. Current models suggest that insulator proteins block enhancers either by formation of chromatin loops or by direct interaction with protein complexes bound to the enhancers and promoters. Here, we tested the role of a chromatin loop in blocking the activity of two Drosophila insulators, gypsy and Fab-7. Both insulators failed to effectively block the interaction between the eye enhancer and the white promoter at most of genomic sites. Insertion of an additional gypsy copy either upstream of the eye enhancer or downstream from the white gene led to complete blocking of the enhancer–promoter communication. In contrast, flanking of the eye enhancer by Fab-7 insulators only weakly improved enhancer blocking. Such a difference in enhancer blocking may be explained by finding that Mod(mdg4)-67.2, a component of gypsy insulator, directly interacts with the Zeste protein, which is critical for enhancer–promoter communication in the white gene.
doi:10.1371/journal.pgen.1003606
PMCID: PMC3701704  PMID: 23861668
5.  Regulation of chromatin organization and inducible gene expression by a Drosophila insulator 
Molecular cell  2011;44(1):29-38.
SUMMARY
Insulators are multi-protein-DNA complexes thought to affect gene expression by mediating inter- and intra-chromosomal interactions. Drosophila insulators contain specific DNA binding proteins plus common components, such as CP190, that facilitate these interactions. Here we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to the DNA in order to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli.
doi:10.1016/j.molcel.2011.07.035
PMCID: PMC3190163  PMID: 21981916
Transcription; Insulator; Chromatin; Epigenetics
6.  BEAF Regulates Cell-Cycle Genes through the Controlled Deposition of H3K9 Methylation Marks into Its Conserved Dual-Core Binding Sites 
PLoS Biology  2008;6(12):e327.
Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer–promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element–associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core–associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle.
Author Summary
The genome of eukaryotes is packaged in chromatin, which consists of DNA, histones, and accessory proteins. This leads to a general repression of genes, particularly for those exposed to mostly condensed, heterochromatin regions. DNA sequences called chromatin insulators/boundary elements are able to insulate a gene from its chromosomal context by blocking promiscuous heterochromatin spreading. No common feature has been identified among the insulators/boundary elements known so far. Rather, distinct subfamilies of insulators harbor different DNA consensus sequences targeted by different DNA-binding factors, which confer their insulating activity. Determining whether distinct subfamilies possess distinct cellular functions is important for understanding genome regulation. Here, using Drosophila, we have combined computational and experimental approaches to address the function of the boundary element-associated factor (BEAF) subfamily of insulators. We identify hundreds of BEAF dual-cores that are defined by a particular arrangement of DNA sequence motifs bracketing nucleosome binding sequences, and that mark the genomic BEAF binding sites. BEAF dual-cores are close to hundreds of genes that regulate chromosome organization/segregation and the cell cycle. Since BEAF acts by restricting the deposition of repressing epigenetic histone marks, which affects the accessibility of chromatin, its depletion affects the expression of cell-cycle genes. Our data reveal a new role for BEAF in regulating the cell cycle through its binding to highly conserved chromatin dual-cores.
Chromatin Dual-Cores define new potent nucleosome-associatedcis-regulatory elements that regulate the accessibility of promoters of genes controlling chromosome organization/segregation and the cell cycle.
doi:10.1371/journal.pbio.0060327
PMCID: PMC2605929  PMID: 19108610
7.  CTCF Genomic Binding Sites in Drosophila and the Organisation of the Bithorax Complex 
PLoS Genetics  2007;3(7):e112.
Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C). We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; “Fab-2,” “Fab-3,” and “Fab-4.” With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation.
Author Summary
There is still much to learn about the organisation of regulatory elements that control where, when, and how much individual genes in the genome are transcribed. Several types of regulatory element have been identified; some, such as enhancers, act over large genomic distances. This creates a problem: how do such long-range elements only regulate their appropriate target genes? Insulator elements have been proposed to act as barriers within the genome, confining the effects of long-range regulatory elements. Here we have mapped the locations of one insulator-binding protein, CTCF, in several regions of the Drosophila genome. In particular, we have focussed on the Hox gene cluster in the Bithorax complex; a region whose regulation has been extensively characterised. Previous investigations have identified independent regulatory domains that control the expression of Bithorax complex genes in different segments of the fly, however the molecular nature of the domain boundaries is unclear. Our major result is that we find CTCF binding sites precisely located at the boundaries of these regulatory domains, giving a common molecular basis for these boundaries. This provides a clear example of the link between the positioning of insulators and the organisation of gene regulation in the Drosophila genome.
doi:10.1371/journal.pgen.0030112
PMCID: PMC1904468  PMID: 17616980
8.  The Drosophila eve Insulator Homie Promotes eve Expression and Protects the Adjacent Gene from Repression by Polycomb Spreading 
PLoS Genetics  2013;9(10):e1003883.
Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is “replaced” by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.
Author Summary
Insulators are specialized DNA elements that can separate the genome into functional units. Most of the current thinking about these elements comes from studies done with model transgenes. Studies of insulators within the specialized Hox gene complexes have suggested that model transgenes can reflect the normal functions of these elements in their native context. However, recent genome-wide studies have called this into question. This work analyzes the native function of an insulator that resides between the Drosophila genes eve and TER94, which are expressed in very different patterns. Also, the eve gene is a Polycomb (Pc) domain, a specialized type of chromatin that is found in many places throughout the genome. We show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. Each of these activities are consistent with those seen with model transgenes, and other known insulators can provide these functions in this context. This work provides a novel and convincing example of the normal role of insulators in regulating the eukaryotic genome, as well as providing insights into their mechanisms of action.
doi:10.1371/journal.pgen.1003883
PMCID: PMC3814318  PMID: 24204298
9.  Coordinated control of dCTCF and gypsy chromatin insulators in Drosophila 
Molecular cell  2007;28(5):761-772.
SUMMARY
CTCF plays a central role in vertebrate insulators and forms part of the Fab-8 insulator in Drosophila. dCTCF is present at hundreds of sites in the Drosophila genome, where it is located at the boundaries between bands and interbands in polytene chromosomes. dCTCF co-localizes with CP190, which is required for proper binding of dCTCF to chromatin, but not with the other gypsy insulator proteins Su(Hw) or Mod(mdg4)2.2. Mutations in the CP190 gene affect Fab-8 insulator activity, suggesting that CP190 is an essential component of both gypsy and dCTCF insulators. dCTCF is present at specific nuclear locations forming large insulator bodies that overlap with those formed by Su(Hw), Mod(mdg4)2.2 and CP190. The results suggest that Su(Hw) and dCTCF may be the DNA-binding components of two different subsets of insulators that share CP190 and cooperate in the formation of insulator bodies to regulate the organization of the chromatin fiber in the nucleus.
doi:10.1016/j.molcel.2007.09.024
PMCID: PMC2579779  PMID: 18082602
CTCF; transcription; insulator; chromatin
10.  Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death 
The Journal of Cell Biology  2013;202(2):261-276.
Insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins.
Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol–p38 mitogen-activated protein kinase–independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization.
doi:10.1083/jcb.201304181
PMCID: PMC3718971  PMID: 23878275
11.  Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila 
Nucleic Acids Research  2013;41(5):2963-2980.
Chromatin insulators are functionally conserved DNA–protein complexes situated throughout the genome that organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the precise mechanisms are not yet understood. Here we identify the exosome, the highly conserved major cellular 3′ to 5′ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. Genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32 and CTCF insulator proteins. Colocalization occurs mainly at promoters but also boundary elements such as Mcp, Fab-8, scs and scs′, which overlaps with a promoter. Surprisingly, exosome associates primarily with promoters but not gene bodies of active genes, arguing against simple cotranscriptional recruitment to RNA substrates. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is required specifically for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel positive relationship between exosome and chromatin insulators throughout the genome.
doi:10.1093/nar/gkt037
PMCID: PMC3597698  PMID: 23358822
12.  The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo 
PLoS Genetics  2013;9(9):e1003847.
Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts.
Author Summary
Mounting evidence in several model organisms collectively demonstrates a role for the DNA-protein complexes known as chromatin insulators in orchestrating the functional domain organization of the eukaryotic genome. Several DNA elements displaying features of insulators, viz barrier and/or directional enhancer-blocking activity, have been identified in yeast, Drosophila, sea urchin, vertebrates and plants; however, proteins that bind these DNA sequences eliciting insulator activities are far less known. Here we identify a novel protein, COMPASS-like (CMPl), which is expressed exclusively by the ambulacrarian group of metazoans and interacts directly with the sea urchin sns5 insulator. Sns5 lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter, where it acts buffering the H1 promoter from the H2A enhancer influence. Intriguingly, we find that CMPl role is absolutely required for the sns5 activity, therefore imposing the different level of accumulation of the linker and nucleosomal transcripts. Overall, our findings add an interesting and novel facet to the chromatin insulator field, highlighting the surprisingly low evolutionary conservation of trans-acting factors binding to chromatin insulators. This opens the possibility that multiple lineage-specific factors modulate chromatin organization in different metazoans.
doi:10.1371/journal.pgen.1003847
PMCID: PMC3784565  PMID: 24086165
13.  Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome 
PLoS Biology  2012;10(11):e1001420.
Comparative ChIP-seq data reveal adaptive evolution of insulator protein CTCF binding in multiple Drosophila species.
Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.
Author Summary
A large proportion of the diversity of living organisms results from differential regulation of gene transcription. Transcriptional regulation is thought to differ between species because of evolutionary changes in the physical interactions between regulatory DNA elements and DNA-binding proteins; these can generate variation in the spatial and temporal patterns of gene expression. The mechanisms by which these protein–DNA interactions evolve is therefore an important question in evolutionary biology. Does adaptive evolution play a role, or is the process dominated by neutral genetic drift? Insulator proteins are a special group of DNA-binding proteins—instead of directly serving to activate or repress genes, they can function to coordinate the interactions between other regulatory elements (such as enhancers and promoters). Additionally, insulator proteins can limit the spreading of chromatin condensation and help to demarcate the boundaries of regulatory domains in the genome. In spite of their critical role in genome regulation, little is known about the evolution of interactions between insulator proteins and DNA. Here, we use ChIP-seq to examine the distribution of binding sites for CTCF, a highly conserved insulator protein, in four closely related Drosophila species. We find that genome-wide binding profiles of CTCF are highly dynamic across evolutionary time, with frequent births of new CTCF-DNA interactions, and we demonstrate that this evolutionary process is driven by natural selection. By comparing these with RNA-seq data, we find that gain or loss of CTCF binding impacts the expression levels of nearby genes and correlates with structural evolution of the genome. Together these results suggest a potential mechanism of regulatory re-wiring through adaptive evolution of CTCF binding.
doi:10.1371/journal.pbio.1001420
PMCID: PMC3491045  PMID: 23139640
14.  Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex 
eLife  2012;1:e00171.
Chromatin boundaries subdivide eukaryotic chromosomes into functionally autonomous domains of genetic activity. This subdivision insulates genes and/or regulatory elements within a domain from promiscuous interactions with nearby domains. While it was previously assumed that the chromosomal domain landscape is fixed, there is now growing evidence that the landscape may be subject to tissue and stage specific regulation. Here we report the isolation and characterization of a novel developmentally restricted boundary factor, Elba. We show that Elba is an unusual hetero-tripartite protein complex that requires all three proteins for DNA binding and insulator activity.
DOI: http://dx.doi.org/10.7554/eLife.00171.001
eLife digest
If all of the DNA in a human cell was stretched out, it would be about 2 m long. The nucleus of a human cell, on the other hand, has a diameter of just 6 μm, so the DNA molecules that carry all the genetic information in the cell need to be carefully folded to fit inside the nucleus. Cells meet this challenge by combining their DNA molecules with proteins to form a compact and highly organized structure called chromatin. Packaging DNA into chromatin also reduces damage to it.
But what happens when the cell needs to express the genes carried by the DNA as proteins or other gene products? The answer is that the compact structure of chromatin relaxes and opens up, which allows the DNA to be transcribed into messenger RNA. Indeed, packing DNA into chromatin makes this process more reliable, thus ensuring that the cell only produces proteins and other gene products when it needs them. However, because cross-talk between neighboring genes could potentially disrupt or change gene expression patterns, cells evolved special elements called boundaries or insulators to stop this from happening. These elements subdivide eukaryotic chromosomes into functionally autonomous chromatin domains.
Since the protein factors implicated in boundary function seemed to be active in all tissues and cell types, it was assumed for many years that these boundaries and the resulting chromatin domains were fixed. However, a number of recent studies have shown that boundary activity can be subject to regulation, and thus chromatin domains are dynamic structures that can be defined and redefined during development to alter patterns of gene expression.
Aoki et al. report the isolation and characterization of a new fruit fly boundary factor that, unlike previously characterized factors, is active only during a specific stage of development. The Elba factor is also unusual in that it is made of three different proteins, known as Elba1, Elba2, and Elba3, and all three must be present for it to bind to DNA. While Elba2 is present during most stages of development, the other two Elba proteins are only present during early embryonic development, so the boundary factor is only active in early embryos. In addition to revealing a new mechanism for controlling boundary activity as an organism develops, the studies of Aoki et al. provide further evidence that chromatin domains can be dynamic.
DOI: http://dx.doi.org/10.7554/eLife.00171.002
doi:10.7554/eLife.00171
PMCID: PMC3510454  PMID: 23240086
Boundaries; Insulators; Domains; Chromatin; Bithorax; Development; D. melanogaster
15.  The chromosomal association/dissociation of the chromatin insulator protein Cp190 of Drosophila melanogaster is mediated by the BTB/POZ domain and two acidic regions 
BMC Cell Biology  2010;11:101.
Background
Chromatin insulators or boundary elements are a class of functional elements in the eukaryotic genome. They regulate gene transcription by interfering with promoter-enhancer communication. The Cp190 protein of Drosophila melanogaster is essential to the function of at least three-types of chromatin insulator complexes organized by Su(Hw), CTCF and BEAF32.
Results
We mapped functional regions of Cp190 in vivo and identified three domains that are essential for the insulator function and for the viability of flies: the BTB/POZ domain, an aspartic acid-rich (D-rich) region and a C-terminal glutamic acid-rich (E-rich) region. Other domains including the centrosomal targeting domain and the zinc fingers are dispensable. The N-terminal CP190BTB-D fragment containing the BTB/POZ domain and the D-rich region is sufficient to mediate association with all three types of insulator complexes. The fragment however is not sufficient for insulator activity or viability. The Cp190 and CP190BTB-D are regulated differently in cells treated with heat-shock. The Cp190 dissociated from chromosomes during heat-shock, indicating that dissociation of Cp190 with chromosomes can be regulated. In contrast, the CP190BTB-D fragment didn't dissociate from chromosomes in the same heat-shocked condition, suggesting that the deleted C-terminal regions have a role in regulating the dissociation of Cp190 with chromosomes.
Conclusions
The N-terminal fragment of Cp190 containing the BTB/POZ domain and the D-rich region mediates association of Cp190 with all three types of insulator complexes and that the E-rich region of Cp190 is required for dissociation of Cp190 from chromosomes during heat-shock. The heat-shock-induced dissociation is strong evidence indicating that dissociation of the essential insulator protein Cp190 from chromosomes is regulated. Our results provide a mechanism through which activities of an insulator can be modulated by internal and external cues.
doi:10.1186/1471-2121-11-101
PMCID: PMC3022720  PMID: 21194420
16.  Functional sub-division of the Drosophila genome via chromatin looping 
Nucleus  2013;4(2):115-122.
Insulators help in organizing the eukaryotic genomes into physically and functionally autonomous regions through the formation of chromatin loops. Recent findings in Drosophila and vertebrates suggest that insulators anchor multiple loci through long-distance interactions which may be mechanistically linked to insulator function. Important to such processes in Drosophila is CP190, a common co-factor of insulator complexes. CP190 is also known to associate with the nuclear matrix, components of the RNAi machinery, active promoters and borders of the repressive chromatin domains. Although CP190 plays a pivotal role in insulator function in Drosophila, vertebrates lack a probable functional equivalent of CP190 and employ CTCF as the major factor to carry out insulator function/chromatin looping. In this review, we discuss the emerging role of CP190 in tethering genome, specifically in the perspective of insulator function in Drosophila. Future studies aiming genome-wide role of CP190 in chromatin looping is likely to give important insights into the mechanism of genome organization.
doi:10.4161/nucl.23389
PMCID: PMC3621743  PMID: 23333867
Insulators; CP190; long-range interactions; chromatin organization
17.  An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster 
BMC Cell Biology  2009;10:8.
Background
Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells.
Results
The Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei.
Conclusion
We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription.
doi:10.1186/1471-2121-10-8
PMCID: PMC2654426  PMID: 19178707
18.  DNA Topoisomerase II Modulates Insulator Function in Drosophila 
PLoS ONE  2011;6(1):e16562.
Insulators are DNA sequences thought to be important for the establishment and maintenance of cell-type specific nuclear architecture. In Drosophila there are several classes of insulators that appear to have unique roles in gene expression. The mechanisms involved in determining and regulating the specific roles of these insulator classes are not understood. Here we report that DNA Topoisomerase II modulates the activity of the Su(Hw) insulator. Downregulation of Topo II by RNAi or mutations in the Top2 gene result in disruption of Su(Hw) insulator function. This effect is mediated by the Mod(mdg4)2.2 protein, which is a unique component of the Su(Hw) insulator complex. Co-immunoprecipitation and yeast two-hybrid experiments show that Topo II and Mod(mdg4)2.2 proteins directly interact. In addition, mutations in Top2 cause a slight decrease of Mod(mdg4)2.2 transcript but have a dramatic effect on Mod(mdg4)2.2 protein levels. In the presence of proteasome inhibitors, normal levels of Mod(mdg4)2.2 protein and its binding to polytene chromosomes are restored. Thus, Topo II is required to prevent Mod(mdg4)2.2 degradation and, consequently, to stabilize Su(Hw) insulator-mediated chromatin organization.
doi:10.1371/journal.pone.0016562
PMCID: PMC3029388  PMID: 21304601
19.  Analysis of chromatin boundary activity in Drosophila cells 
BMC Molecular Biology  2008;9:109.
Background
Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared.
Results
We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function.
Conclusion
The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements.
doi:10.1186/1471-2199-9-109
PMCID: PMC2621236  PMID: 19077248
20.  Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle 
Cell Cycle  2013;12(10):1605-1615.
DREF was first characterized for its role in the regulation of transcription of genes encoding proteins involved in DNA replication and found to interact with sequences similar to the DNA recognition motif of the BEAF-32 insulator protein. Insulators are DNA-protein complexes that mediate intra- and inter-chromosome interactions. Several DNA-binding insulator proteins have been described in Drosophila, including BEAF-32, dCTCF and Su(Hw). Here we find that DREF and BEAF-32 co-localize at the same genomic sites, but their enrichment shows an inverse correlation. Furthermore, DREF co-localizes in the genome with other insulator proteins, suggesting that the function of this protein may require components of Drosophila insulators. This is supported by the finding that mutations in insulator proteins modulate DREF-induced cell proliferation. DREF persists bound to chromatin during mitosis at a subset of sites where it also co-localizes with dCTCF, BEAF-32 and CP190. These sites are highly enriched for sites where Orc2 and Mcm2 are present during interphase and at the borders of topological domains of chromosomes defined by Hi-C. The results suggest that DREF and insulator proteins may help maintain chromosome organization during the cell cycle and mark a subset of genomic sites for the assembly of pre-replication complexes and gene bookmarking during the M/G1 transition.
doi:10.4161/cc.24742
PMCID: PMC3680540  PMID: 23624840
transcription; chromatin; epigenetics; replication; cell cycle; mitosis
21.  Identification and Characterization of Proteins Involved in Nuclear Organization Using Drosophila GFP Protein Trap Lines 
PLoS ONE  2013;8(1):e53091.
Background
Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.
Methodology/Principal Findings
We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.
Conclusions/Significance
These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.
doi:10.1371/journal.pone.0053091
PMCID: PMC3547006  PMID: 23341925
22.  Insulators Target Active Genes to Transcription Factories and Polycomb-Repressed Genes to Polycomb Bodies 
PLoS Genetics  2013;9(4):e1003436.
Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs), mediate associations among Polycomb Group (PcG) targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.
Author Summary
We have studied the nuclear localization of genes that are regulated by Polycomb mechanisms. The genomes of higher eukaryotes contain hundreds of genes that are regulated by Polycomb mechanisms. Once repressed by Polycomb complexes, they tend to stay repressed; but, when activated, they bind Trithorax protein and tend to maintain the active state epigenetically. Polycomb repression has been reported to make these genes associate in the nucleus to form “Polycomb bodies.” We find that this association is not caused by Polycomb complexes but by insulator elements accompanying the genes. We show that, when these genes are in the active state, the binding of Trithorax targets them to other nuclear regions where transcription occurs, so-called transcription factories. In these nuclear re-positionings the insulator provides the associative power while the state of activity determines whether a gene goes to a Polycomb body or to a transcription factory. The strong effect of Trithorax suggests the possibility that the stable association with a transcription factory it produces may account for the epigenetic memory of the active state.
doi:10.1371/journal.pgen.1003436
PMCID: PMC3630138  PMID: 23637616
23.  Genome-Wide Identification of Chromatin Transitional Regions Reveals Diverse Mechanisms Defining the Boundary of Facultative Heterochromatin 
PLoS ONE  2013;8(6):e67156.
Due to the self-propagating nature of the heterochromatic modification H3K27me3, chromatin barrier activities are required to demarcate the boundary and prevent it from encroaching into euchromatic regions. Studies in Drosophila and vertebrate systems have revealed several important chromatin barrier elements and their respective binding factors. However, epigenomic data indicate that the binding of these factors are not exclusive to chromatin boundaries. To gain a comprehensive understanding of facultative heterochromatin boundaries, we developed a two-tiered method to identify the Chromatin Transitional Region (CTR), i.e. the nucleosomal region that shows the greatest transition rate of the H3K27me3 modification as revealed by ChIP-Seq. This approach was applied to identify CTRs in Drosophila S2 cells and human HeLa cells. Although many insulator proteins have been characterized in Drosophila, less than half of the CTRs in S2 cells are associated with known insulator proteins, indicating unknown mechanisms remain to be characterized. Our analysis also revealed that the peak binding of insulator proteins are usually 1–2 nucleosomes away from the CTR. Comparison of CTR-associated insulator protein binding sites vs. those in heterochromatic region revealed that boundary-associated binding sites are distinctively flanked by nucleosome destabilizing sequences, which correlates with significant decreased nucleosome density and increased binding intensities of co-factors. Interestingly, several subgroups of boundaries have enhanced H3.3 incorporation but reduced nucleosome turnover rate. Our genome-wide study reveals that diverse mechanisms are employed to define the boundaries of facultative heterochromatin. In both Drosophila and mammalian systems, only a small fraction of insulator protein binding sites co-localize with H3K27me3 boundaries. However, boundary-associated insulator binding sites are distinctively flanked by nucleosome destabilizing sequences, which correlates with significantly decreased nucleosome density and increased binding of co-factors.
doi:10.1371/journal.pone.0067156
PMCID: PMC3696093  PMID: 23840609
24.  The gypsy Insulator Can Act as a Promoter-Specific Transcriptional Stimulator 
Molecular and Cellular Biology  2001;21(22):7714-7720.
Insulators define chromosomal domains such that an enhancer in one domain cannot activate a promoter in a different domain. We show that the Drosophila gypsy insulator behaves as a cis-stimulatory element in the larval fat body. Transcriptional stimulation by the insulator is distance dependent, as expected for a promoter element as opposed to an enhancer. Stimulation of a test alcohol dehydrogenase promoter requires a binding site for a GATA transcription factor, suggesting that the insulator may be facilitating access of this DNA binding protein to the promoter. Short-range stimulation requires both the Suppressor of Hairy-wing protein and the Mod(mdg4)-62.7 protein encoded by the trithorax group gene mod(mdg4). In the absence of interaction with Mod(mdg4)-62.7, the insulator is converted into a short-range transcriptional repressor but retains some cis-stimulatory activity over longer distances. These results indicate that insulator and promoter sequences share important characteristics and are not entirely distinct. We propose that the gypsy insulator can function as a promoter element and may be analogous to promoter-proximal regulatory modules that integrate input from multiple distal enhancer sequences.
doi:10.1128/MCB.21.22.7714-7720.2001
PMCID: PMC99942  PMID: 11604507
25.  Loss of the insulator protein CTCF during nematode evolution 
BMC Molecular Biology  2009;10:84.
Background
The zinc finger (ZF) protein CTCF (CCCTC-binding factor) is highly conserved in Drosophila and vertebrates where it has been shown to mediate chromatin insulation at a genomewide level. A mode of genetic regulation that involves insulators and insulator binding proteins to establish independent transcriptional units is currently not known in nematodes including Caenorhabditis elegans. We therefore searched in nematodes for orthologs of proteins that are involved in chromatin insulation.
Results
While orthologs for other insulator proteins were absent in all 35 analysed nematode species, we find orthologs of CTCF in a subset of nematodes. As an example for these we cloned the Trichinella spiralis CTCF-like gene and revealed a genomic structure very similar to the Drosophila counterpart. To investigate the pattern of CTCF occurrence in nematodes, we performed phylogenetic analysis with the ZF protein sets of completely sequenced nematodes. We show that three ZF proteins from three basal nematodes cluster together with known CTCF proteins whereas no zinc finger protein of C. elegans and other derived nematodes does so.
Conclusion
Our findings show that CTCF and possibly chromatin insulation are present in basal nematodes. We suggest that the insulator protein CTCF has been secondarily lost in derived nematodes like C. elegans. We propose a switch in the regulation of gene expression during nematode evolution, from the common vertebrate and insect type involving distantly acting regulatory elements and chromatin insulation to a so far poorly characterised mode present in more derived nematodes. Here, all or some of these components are missing. Instead operons, polycistronic transcriptional units common in derived nematodes, seemingly adopted their function.
doi:10.1186/1471-2199-10-84
PMCID: PMC2749850  PMID: 19712444

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