The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.
Chromatin; transcription; nucleus; epigenetics
Recent findings provide evidence that tDNAs function as chromatin insulators from yeast to humans. TFIIIC, a transcription factor that interacts with the B-box in tDNAs as well as thousands of ETC sites in the genome, is responsible for insulator function. Though tDNAs are capable of enhancer-blocking and barrier activities for which insulators are defined, new insights into the relationship between insulators and chromatin structure suggest that TFIIIC serves a complex role in genome organization. We review the role of tRNA genes and TFIIIC as chromatin insulators, and highlight recent findings that have broadened our understanding of insulators in genome biology.
chromatin; Epigenetics; CTCF; tRNA genes; ETC loci
Long-range interactions between transcription regulatory elements play an important role in gene activation, epigenetic silencing, and chromatin organization. Transcriptional activation or repression of developmentally regulated genes is often accomplished through tissue specific chromatin architecture and dynamic localization between active transcription factories and repressive Polycomb bodies. However, the mechanisms underlying the structural organization of chromatin and the coordination of physical interactions are not fully understood. Insulators and Polycomb group proteins form highly conserved multi-protein complexes that mediate functional long-range interactions, and have proposed roles in nuclear organization. In this review, we explore recent findings that have broadened our understanding of the function of these proteins and provide an integrative model for the roles of insulators in nuclear organization.
CTCF; Polycomb; TFIIIC; Cohesin; Epigenetics
Myc has been characterized as a transcription factor that activates expression of genes involved in pluripotency and cancer, and as a component of the replication complex. Here we find that Myc is present at promoters and enhancers of D. melanogaster genes during interphase. Myc co-localizes with Orc2, which is part of the pre-replication complex, during G1. As is the case in mammals, Myc associates preferentially with paused genes, suggesting that it may also be involved in the release of RNAPII from promoter proximal pausing in Drosophila. Interestingly, about 40% of Myc sites present in interphase persists during mitosis. None of the Myc mitotic sites correspond to enhancers and only some correspond to promoters. The rest of mitotic Myc sites overlap with binding sites for multiple insulator proteins that are also maintained in mitosis. These results suggest alternative mechanisms to explain the role of Myc in pluripotency and cancer.
Insulators are DNA-protein complexes that can mediate interactions in cis or trans between different regions of the genome. Although originally defined on the basis of their ability to block enhancer–promoter communication or to serve as barriers against the spreading of heterochromatin in reporter systems, recent information suggests that their function is more nuanced and depends on the nature of the sequences brought together by contacts between specific insulator sites. Here we provide an overview of new evidence that has uncovered a wide range of functions for these sequences in addition to their two classical roles.
The functional output of the genome is closely dependent on its organization within the nucleus, which ranges from the 10 nm chromatin fiber to the three-dimensional arrangement of this fiber in the nuclear space. Recent observations suggest that intra-and inter-chromosomal interactions between distant sequences underlie several aspects of transcription regulatory processes. These contacts can bring enhancers close to their target genes, or prevent inappropriate interactions between regulatory sequences via insulators. In addition, intra- and inter-chromosomal interactions can bring co-activated or co-repressed genes to the same nuclear location. Recent technological advances have made it possible to map long-range cis and trans interactions at relatively high resolution. This information is being used to develop three-dimensional maps of the arrangement of the genome in the nucleus and to understand causal relationships between nuclear structure and function.
Chromatin; Epigenetics; Transcription; Nucleus
Chromatin insulators are DNA–protein complexes with broad functions in nuclear biology. Drosophila has at least five different types of insulators; recent results suggest that these different insulators share some components that may allow them to function through common mechanisms. Data from genome-wide localization studies of insulator proteins indicate a possible functional specialization, with different insulators playing distinct roles in nuclear biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Current results suggest that insulators set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation and development.
Drosophila; insulator; transcription; nucleus
Memory deficits in Drosophila nalyot mutants suggest that the Myb family transcription factor Adf-1 is an important regulator of developmental plasticity in the brain. However, the cellular functions for this transcription factor in neurons or molecular mechanisms by which it regulates plasticity remain unknown. Here, we use in vivo 3D reconstruction of identifiable larval motor neuron dendrites to show that Adf-1 is required cell autonomously for dendritic development and activity-dependent plasticity of motor neurons downstream of CaMKII. Adf-1 inhibition reduces dendrite growth and neuronal excitability, and results in motor deficits and altered transcriptional profiles. Surprisingly, analysis by comparative chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of Adf-1, RNA Polymerase II (Pol II), and histone modifications in Kc cells shows that Adf-1 binding correlates positively with high Pol II-pausing indices and negatively with active chromatin marks such as H3K4me3 and H3K27ac. Consistently, the expression of Adf-1 targets Staufen and Fasciclin II (FasII), identified through larval brain ChIP-Seq for Adf-1, is negatively regulated by Adf-1, and manipulations of these genes predictably modify dendrite growth. Our results imply mechanistic interactions between transcriptional and local translational machinery in neurons as well as conserved neuronal growth mechanisms mediated by cell adhesion molecules, and suggest that CaMKII, Adf-1, FasII, and Staufen influence crucial aspects of dendrite development and plasticity with potential implications for memory formation. Further, our experiments reveal molecular details underlying transcriptional regulation by Adf-1, and indicate active interaction between Adf-1 and epigenetic regulators of gene expression during activity-dependent neuronal plasticity.
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.
Transcription; Insulator; Chromatin; Epigenetics
CTCF plays diverse roles in nuclear organization and transcriptional regulation. In this issue of Developmental Cell, Essafi et al. (2011) report a mechanism by which the repressive or active state of chromatin in a domain defined by CTCF can be switched by the Wt1 transcription factor to regulate gene expression.
Enhancer function underlies regulatory processes by which cells establish patterns of gene expression. Recent results suggest that enhancers are specified by particular chromatin marks in pluripotent cells, which may be modified later in development to alter patterns of gene expression and cell differentiation choices. These marks may contribute to the repertoire of epigenetic mechanisms responsible for cellular memory and determine the timing of transcription factor accessibility to the enhancer. Mechanistically, cohesin and non-coding RNAs are emerging as critical players responsible for facilitating enhancer-promoter interactions. Surprisingly, these interactions may be required not only to facilitate initiation of transcription but also to activate the release of RNAPII from promoter-proximal pausing.
epigenetics; histones; chromatin; RNA
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.
Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.
Transcription; Chromatin; Epigenetics; Cancer
Although components of the nuclear pore complex have been implicated in gene regulation independent of their role at the nuclear envelope, the evidence so far has been indirect. Capelson et al. (2010) and Kalverda et al. (2010) now reveal that certain nucleoporins are actively involved in transcription inside the nucleoplasm of Drosophila cells.
Nucleoporins; nuclear envelope; transcription; chromatin
CTCF plays diverse roles in the regulation of eukaryotic genes. A new study by Lefevre et al., 2008 reveals a novel mechanism in which non-coding RNA transcription and nucleosome repositioning evicts CTCF from a regulatory element, to facilitate induction of a nearby gene.
CTCF; insulator; transcription; chromatin
Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.
Genetic analysis of the Drosophila leg-arista-wing complex (lawc) gene suggests a role for the Lawc protein in chromatin-related processes based on its classification as a trxG gene but the molecular mechanisms of its function remain elusive. We have found that Lawc is a small, cysteine-rich protein that is present in most of the interbands of polytene chromosomes. In agreement with this observation, Lawc co-localizes with RNA polymerase IIo (Pol IIo) and it is recruited to transcribed loci after elongation by Pol IIo has begun. Lawc interacts with the nuclear proteasome regulator dREGγ in a yeast two-hybrid assay and both proteins co-localize on polytene chromosomes. In addition, a mutation in lawc interacts genetically with a mutation in a component of the proteasome. lawc mutants show decreased expression of some genes, while the levels of Pol IIoSer2 increase. We conclude that Lawc is required for proper transcription by RNA polymerase II in a process that involves the nuclear proteasome.
transcription; Lawc; RNA polymerase II; dREGγ; proteasome
Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function.
Insulators; Epigenetic Inheritance; Chromatin; Nuclear organization
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.
CTCF; transcription; insulator; chromatin
Insulator elements can be classified as enhancer-blocking or barrier insulators depending on whether they interfere with enhancer-promoter interactions or act as barriers against the spreading of heterochromatin. The former class may exert its function at least in part by attaching the chromatin fiber to a nuclear substrate such as the nuclear matrix, resulting in the formation of chromatin loops. The latter class functions by recruiting histone modifying enzymes, although some barrier insulators have also been shown to create chromatin loops. These loops may correspond to functional nuclear domains containing clusters of co-expressed genes. Thus, insulators may determine specific patterns of nuclear organization that are important in establishing specific programs of gene expression during cell differentiation and development.
insulators; chromatin; loops; gene expression; nuclear organization
Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.
insulator; chromatin; transcription; nucleus; retrotransposon
CTCF is a highly conserved zinc finger protein implicated in diverse genomic regulatory functions, including transcriptional activation/repression, insulation, imprinting, and X-chromosome inactivation. Here we re-evaluate data supporting these roles in the context of mechanistic insights provided by recent genome-wide studies and highlight evidence for CTCF-mediated intra- and inter-chromosomal contacts at several developmentally regulated genomic loci. These analyses support a primary role for CTCF in the global organization of chromatin architecture and suggest that CTCF may be a heritable component of an epigenetic system regulating the interplay between DNA methylation, higher-order chromatin structure, and lineage-specific gene expression.
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.
The nuclear factor CTCF has been shown to be necessary for the maintenance of genetic imprinting at the mammalian H19/Igf2 locus. MacDonald and colleagues now report in BMC Biology that the mechanisms responsible for maintaining the imprinted state in Drosophila may be evolutionarily conserved and that CTCF may also play a critical role in this process.
See research article http://www.biomedcentral.com/1741-7007/8/105
Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation.
Activation of gene expression is thought to be regulated mainly at the level of transcription initiation. Nevertheless, many genes in Drosophila and vertebrates contain RNA polymerase that has started transcription but is paused 30–40 bp from the initiation site. Activation of these genes may thus be regulated by releasing the polymerase from the paused state rather than bringing this protein to the promoter. This release requires the recruitment of specialized proteins that modify the polymerase. It appears that the recruitment of these proteins takes place by modification of two chromatin proteins, histones H3 and H4. Here we characterize a process composed of multiple steps required for release of RNA polymerase from the paused state. The process starts with the recruitment of an enzyme that adds a phosphate group to histone H3. This phosphate serves as a signal to recruit a different protein, which in turn recruits a second enzyme capable of adding an acetyl group to the same histone molecule. The multiple steps involved may provide a variety of mechanisms to control the process.