InteractiveFly: GeneBrief

CTCF: Biological Overview | References

Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking: Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF (CCCTC-binding factor), with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. This study characterized an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, this study demonstrates that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by CTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans (Moon, 2005).

Expression of the eukaryotic genome is controlled by enhancer and silencer elements, both of which can mediate their function from a distance. Insulator elements with enhancer-blocking activity curb enhancer activity, such that only appropriate promoters are activated. The proteins that mediate insulator function have been identified for only a few Drosophila insulator sequences. These are Zw5, BEAF-32, GAGA factor and Su(Hw) (Moon, 2005).

Another perspective on the requirement of insulators comes from the fact that many genes are controlled by several regulatory elements needed for tissue- and cell-specific expression. For example, the Drosophila gene Abdominal-B (Abd-B) contains an extended 3' regulatory region that is functionally subdivided into distinct enhancer domains. Functional separation of the enhancer sequences is achieved by intervening insulators such as Frontabdominal (Fab)-7 and Fab-8. Although both elements have been shown to mediate enhancer-blocking function, the protein involved in this activity has not been described (Moon, 2005).

In sharp contrast to Drosophila, the genome of vertebrates is much more expanded, due primarily to larger distances between genes. Therefore, the need for insulators to separate genes may not seem as pronounced as it is in Drosophila. Indeed, until now, only a single protein, CTCF, has been identified to mediate enhancer-blocking activity (Ohlsson, 2001). Binding sites for CTCF have been shown to be involved in gene activation (Vostrov, 1997), gene repression (Baniahmad, 1990; Lobanenkov, 1990) and enhancer blocking (Bell, 1999; Hark, 2000; Kanduri, 2000; Szabo, 2000; Filippova, 2001; Lutz, 2003; Tanimoto, 2003). Furthermore, vertebrate- and mammalian-specific functions, such as X-chromosome inactivation and control of the epigenetic DNA methylation state, seem to involve CTCF (Lee, 2003; Moon, 2005 and references therein).

Obviously, the function of enhancer blocking has developed during evolution such that Drosophila uses several proteins and mechanisms for enhancer blocking and insulation (Kuhn, 2003). However, none of the known Drosophila insulator proteins has a counterpart found to be conserved in vertebrates. Rather, vertebrates use CTCF, which has not previously been found in Drosophila. This study characterizes a Drosophila orthologue of CTCF with similarities to many of the features identified for vertebrate CTCF. Furthermore, a previously characterized Drosophila insulator, Fab-8, mediates enhancer blocking by CTCF in Drosophila as well as in vertebrate cells. Thus, the enhancer-blocking protein CTCF and, probably, the mechanisms of CTCF-driven enhancer blocking are both conserved from Drosophila to humans (Moon, 2005).

FlyBase data entries and cDNA sequence analysis revealed an open reading frame (ORF) coding for a protein similar to vertebrate CTCF with respect to the overall structure. dCTCF contains all of the expected 11 zinc fingers (Zn-fingers), separated by both standard and noncanonical inter-finger linkers. Furthermore, most of the crucial DNA base recognition residues at positions −1, 2, 3 and 6 are identical. Variation in position 6 for fingers #6 and #9 generates a change from alanine or serine to methionine; this is of no consequence for the DNA-binding specificity, as the recognition code is not changed (Moon, 2005).

Similarities in Zn-fingers do not necessarily imply similarities in function. Therefore, whether dCTCF can act as a transcriptional repressor, as has been demonstrated previously for vertebrate CTCF (Burcin, 1997), has been examined. The strongest repressive function has been shown to reside within the combined carboxy-terminal plus Zn-finger domains (Lutz, 2000). Equivalent regions of Drosophila and chicken CTCF (chCTCF) were fused to the yeast GAL4 transcription factor DNA-binding domain. Both Drosophila and chicken GAL4-CTCF fusions repressed reporter gene activity to a similar extent in two different cell lines and in a way comparable with the previously characterized strong repressor GAL4-v-erbA362. These results clearly indicate that dCTCF, like its vertebrate counterpart, has transcriptional repressor activity (Moon, 2005).

In vertebrates, CTCF is ubiquitously expressed (Burke, 2002), apparently functioning as a global transcriptional regulator in all cell types (Ohlsson, 2001). In comparison, dCTCF RNA expression levels were monitered at various stages of fly development. Using in situ hybridization, it was found that dCTCF RNA is present in the cytoplasm of the nurse cells within the fly egg chamber, transported into and distributed uniformly in the developing oocyte and in 0-24 h embryos as a maternal. Later stages show expression in all tissues and stages, revealing that dCTCF is a ubiquitous factor as in vertebrates. Location of dCTCF protein is clearly nuclear, exemplified by the nuclear staining of syncytial blastoderm embryos with dCTCF-specific antibodies (Moon, 2005).

To extend the comparison of vertebrate and Drosophila CTCF, in vitro-translated Drosophila and vertebrate CTCF were tested for binding to several previously characterized vertebrate CTCF targets (CTS). The sequences tested included the CTS of the FII insulator element of the β-globin gene, the APP gene, the myc genes and the mouse ARF promoter. With the exception of the two myc FPV and A sites, all the other sequences bound chicken and Drosophila CTCF similarly (Moon, 2005).

A methylation-interference assay was used to determine whether both proteins contact the same guanidine nucleotides on a given target DNA site. Both Drosophila and human CTCF were found to contact the same nucleotides on the β-globin FII insulator fragment. These results indicate that, despite considerable overall sequence divergence, fly and human CTCF show a striking degree of functional conservation with respect to DNA binding (Moon, 2005).

To identify potential Drosophila CTCF regulatory targets, an in vitro screen was performed for CTCF-binding sites, and the Fab-8 element, for which enhancer-blocking and boundary function have been shown, was found. This sequence is situated in the Abd-B locus, separating and insulating enhancer domains infraabdominal-7 (iab-7) from iab-8. Since the protein involved in this mechanism was unknown, and since vertebrate CTCF mediates enhancer-blocking activity (Ohlsson, 2001), whether dCTCF might have a similar role in the context of the Fab-8 element was tested. In vitro binding of dCTCF to Fab-8, as determined by methylation interference, suggested two binding sites for CTCF. Binding site mutations resulting in single-site mutations (mut1 or mut2) and in a double-site mutation (mut1+2) were used for electrophoretic mobility shift assay (EMSA). The wild-type Fab-8 element generates two retarded bands corresponding to a different mobility of the same DNA molecule occupied by CTCF at one of the two closely spaced CTS sequences. These different mobilities are probably caused by a site-specific DNA bending, which has also been observed on other dual binding sites, such as the H19 locus. Excess protein generated a slow mobility complex only resolved after a long run of the gel, reflecting binding of CTCF to both sites (Moon, 2005).

To test in vivo dCTCF binding to this important element, crosslinked chromatin was prepared from Drosophila embryos and CTCF-occupied sites were precipitated with the anti-dCTCF-C antibody. PCR primers for the Fab-8 sequence identified specifically precipitated chromatin, whereas primers against a different non-dCTCF-binding site and mock-precipitated chromatin resulted in no signal (Moon, 2005).

To test the functional similarity between dCTCF and vertebrate CTCF, enhancer blocking of Fab-8 was analyzed in vertebrate K562 cells. In comparison to the known enhancer-blocking effect mediated by the FII sequence, a similar reduction in colony numbers mediated by Fab-8 was seen. More importantly, specific abrogation of CTCF binding by the double mutation, mut1+2, resulted in loss of enhancer blocking (Moon, 2005).

The crucial test for enhancer-blocking activity of dCTCF had to be carried out in flies. Therefore, a vector was used with two regulatory regions containing the iab-5 enhancer from the Abd-B locus and two copies of the minimal twist enhancer, PE, directing an additive pattern of expression when placed between divergently transcribed white and lacZ reporter genes. The iab-5 enhancer directs expression in the posterior one-third of the blastoderm stage embryo, whereas the 2 × PE enhancer activates transcription in the ventral-most region where twist is normally expressed. Enhancer elements are enhancing both the white gene as well as the lacZ gene. Altered patterns of transcription were observed when the 1 kb spacer sequence was replaced by the 680 bp Fab-8 element. On the white promoter, the iab-5 activity was completely abolished (shown as the lack of staining in the iab-5 activity region), while the 2 × PE enhancer was still activating the white gene. The lacZ promoter, conversely, could be activated only by the proximal iab-5 but not by the distal 2 × PE. This result suggests that the Fab-8 fragment blocks the respective distal enhancer for both the white and the lacZ promoters. When the CTCF sites were mutagenized (mut1+2), the iab-5 activity on white was partly restored. Similarly, the 2 × PE element again directed the transcription of the lacZ gene. Chromatin/CTCF immunoprecipitation revealed specific CTCF binding to the Fab-8 element of the enhancer-blocking vector, whereas binding to the Fab-8 mut element was clearly reduced. This correlation between strong CTCF binding and full enhancer-blocking function indicates that the activity of Fab-8 is at least partly mediated by CTCF and that dCTCF, similar to vertebrate CTCF, confers enhancer blocking (Moon, 2005).

Thus, at least one enhancer-blocking protein (CTCF) in Drosophila and vertebrates is conserved with a similar enhancer-blocking function. In addition to enhancer blocking, mammalian CTCF has gained functions involving the control of epigenetic states in the context of imprinted genes and X-chromosome inactivation (Lee, 2003; Lewis, 2004; Moon, 2005 and references therein).

Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). Chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments were performed with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines; evidence was found that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression (Bushey, 2009).

Su(Hw), dCTCF, and BEAF have all been implicated in chromatin loop formation, and the interaction of these different DNA-binding proteins with CP190 could have functional implications for the arrangement of the chromatin fiber within the nucleus. The work presented in this study provides critical insight into the genome-wide distribution of these four insulator proteins and is a first, crucial step toward understanding the role that they play in chromatin organization and the regulation of gene expression (Bushey, 2009).

Although insulator elements containing Su(Hw), dCTCF, and BEAF could, in principle, play similar roles, it was found that they have very different distribution patterns with respect to gene location. Only 20% of Su(Hw) sites are located within 1 kb of the 5' or 3' ends of genes. In contrast, 47% of dCTCF sites and 84% of BEAF sites are found within 1 kb of gene ends, and their distributions are highly skewed toward the 5' end of highly expressed genes. dCTCF and BEAF appear to display further functional compartmentalization in their roles, since BEAF tends to be present at the 5' end of genes involved in metabolic processes and dCTCF is enriched near genes involved in developmental processes. This could indicate that BEAF plays a specific role in the regulation of gene units consisting of metabolic genes, whereas Su(Hw) may play a more general role by setting the foundation for chromatin organization. dCTCF, which shows an intermediate distribution compared with Su(Hw) and BEAF, may sometimes function in large-scale organization and sometimes work at the level of individual developmental gene units. Together, the three CP190 insulator subclasses could create a chromatin web that is part of the framework organizing DNA in the nucleus (Bushey, 2009).

Insulators have been typically characterized as sequences capable of regulating interactions between transcriptional regulatory sequences and/or chromatin states. This function can easily be envisioned in the case of Su(Hw) and dCTCF sites located far from genes, where these sites could flank a group of transcription units that would then represent a domain of coregulated genes. If this is the case, what is the function of the remaining dCTCF and BEAF sites located close to the 5' and 3' ends of genes? This distribution is surprising in the context of what is normally think of as insulator function; however, when CTCF-binding sites were mapped in the human genome, a similar distribution pattern was observed (Kim, 2007; Cuddapah, 2009). This is suggestive of a wider role for insulator proteins than just the establishment of chromatin domains, and, in fact, alternative insulator protein functions have been suggested. For example, CTCF in humans is present in the Igh locus in many of the VH as well as DH and JH exons, suggesting a role in V(D)J recombination (Degner, 2009). Additionally, this study provides evidence that insulator proteins near genes play a role in the regulation of expression of specific genes and suggests that the mechanism behind this regulation differs from classic transcription factors, since the same insulator complexes were seen to both activate and repress transcription. These functions could be a consequence of the ability of these proteins to both interact with each other and mediate intra- and interchromosomal loops. Bringing together various insulator protein-binding sites could facilitate localization to either transcriptionally active or transcriptionally repressed regions of the nucleus depending on the genomic context of the sites (Bushey, 2009).

Comparison of the genome-wide distribution of the three insulator subclasses in two different cell lines has led to insights into possible mechanisms employed during cell differentiation to establish different patterns of gene expression. Overall, the analysis suggests that cells may control insulator function at multiple levels and that these forms of regulation occur throughout the genome. Regulation of insulator function seems to begin at the level of DNA binding, since differential binding was observed at 5%-37% of sites for Su(Hw), dCTCF, and BEAF between two different cell lines even with the most conservative statistical analysis. Similar percentages of cell type-specific binding sites were observed for vertebrate CTCF between different cell lines (Kim, 2007; Cuddapah, 2009). Previous analysis of Su(Hw) binding in various tissues has not revealed any significant tissue-specific binding sites, perhaps because only a small number of sites was analyzed in these studies. Alternatively, the discrepancy could be due to the use of whole tissues in previous studies that contain multiple cell types, making it difficult to detect cell type-specific sites (Bushey, 2009).

After Su(Hw), dCTCF, and BEAF bind DNA, they are thought to recruit other proteins such as CP190. Regulation at this level was observed throughout the genome, where a subset of the Su(Hw), dCTCF, and BEAF sites recruit CP190 in a cell type-specific manner. The additional Su(Hw), dCTCF, and BEAF sites that do not recruit CP190 in either Kc or Mbn2 cells may do so in other cell types or other growth conditions not tested in this study. This idea is supported by the two dCTCF sites in the bithorax region that were found to contain CP190 in third instar larvae brains (Mohan, 2007) but not in the data sets collected in Kc cells or Mbn2 cells. Although further study is needed to determine which sites of insulator protein localization participate in chromatin organization, it is expected that sites lacking CP190 do not, since mutations in CP190 are known to disrupt insulator body formation and only those sites that recruit CP190 seem to affect gene expression. Therefore, these sites may represent insulators that are poised for incorporation into chromatin loops upon recruitment of CP190. On the other hand, these sites could function through the recruitment of an alternative cofactor and in this way represent a functionally distinct subset of Su(Hw)-, dCTCF-, and BEAF-binding sites (Bushey, 2009).

An additional layer of regulation may then occur at the level of protein-protein interactions mediated by CP190. This type of regulation cannot be gleaned from ChIP-chip data, but other experiments have shown that sumoylation of insulator proteins is able to inhibit protein-protein interactions affecting Su(Hw) insulator body formation but not association of insulator proteins with DNA. Similarly, vertebrate CTCF insulator function has been linked to poly(ADP-ribosyl)ation (PARlation), and it has been suggested that PARlation facilitates CTCF self-interaction. Furthermore, the presence of RNA and RNA-binding proteins may also contribute to the formation or maintenance of insulator bodies required to create chromatin loops. Finally, insulator bypass that results in the inactivation of insulator activity through pairing of nearby insulator elements, and specialized sequences such as the promoter targeting sequences (PTS), can allow an enhancer to bypass an insulator. These forms of regulation may alter the ability of insulator proteins to interact with one another to regulate insulator loop formation (Bushey, 2009).

It is expected that these various forms of regulation including DNA binding, CP190 recruitment, and loop formation result in changes in gene expression between different cell lines. However, transcription analysis with insulator proteins is difficult since insulator elements are thought to control regulatory elements such as enhancers and silencers that can be found far away from their target promoters. Therefore, determining which genes are controlled by an insulator site is not a trivial process. In the transcription analysis, genes were considered with a cell type-specific insulator site only within the gene or the 1 kb surrounding region. Despite this limitation, a significant enrichment was still seen for genes that change expression between cell types when they have a cell type-specific insulator site nearby, supporting the idea that insulator proteins are involved in the regulation of gene expression. Genes that did not change expression despite being located near a cell type-specific insulator protein-binding site may not be the actual target genes of the insulator sites; therefore, this analysis probably greatly underestimates the effect of insulator proteins on gene expression. Additionally, it was found that insulator protein-binding sites that localize to genes are enriched at genes with certain expression signals, high expression for dCTCF and BEAF, and low expression for Su(Hw). However, comparison between the two cell lines revealed that expression can be either positively or negatively regulated by sites with each DNA-binding protein. Therefore, although an insulator protein associates with a highly expressed gene, it may lead to either an increase or decrease in transcription of this gene. The observed level of expression may be an additive effect of many different regulatory elements, including multiple insulator sites. Different mechanisms may be used to regulate a highly transcribed gene versus a gene with low levels of transcription, and therefore the different insulator subclasses may target these different mechanisms (Bushey, 2009).

The transcription analysis in this study suggests that insulator proteins play a role in the regulation of gene expression, but has just begun to explore the depth of their effect. Numerous steps at which insulator activation can be subject to regulation allow for a vast amount of variation between different cell types and could play a major role in establishing the diverse patterns of chromatin organization necessary for cell type-specific gene expression. The different CP190 insulator subclasses might have distinct roles in this cell type-specific nuclear organization. In vertebrates, CTCF is the only insulator known thus far, and an important question to address in the future is the apparent disparity between genome complexity and insulator diversification between flies and vertebrates. It is possible that vertebrates have insulator subclasses represented by DNA-binding proteins other than CTCF that have not yet been identified. Alternatively, it is possible that vertebrate CTCF has acquired all the functions of the various Drosophila insulator subclasses. The distribution pattern of dCTCF suggests that it can play a role in both global organization and in the regulation of individual genes, making it the most likely candidate of the three Drosophila subclasses to play this overarching organizational role in vertebrates. Therefore, vertebrates may use methods other than variant DNA-binding proteins to distinguish insulator subclasses, such as recruitment of different CTCF interaction partners at different insulator sites. This would make it difficult to distinguish between the various layers of insulator control in the vertebrate genome. If this is the case, Drosophila could provide a powerful model system to dissect the various functions and levels of regulation of chromatin insulators (Bushey, 2009).

A comprehensive map of insulator elements for the Drosophila Genome

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. The genome-wide binding sites of 6 insulator associated proteins (dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF) was studied to obtain the first comprehensive map of insulator elements in Drosophila embryos. Over 14,000 putative insulators, including all classically defined insulators, were identified. Two major classes of insulators were 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 (Négre, 2010).

This study the embryonic binding profile of six factors previously known to be associated with insulator function in Drosophila. The analysis of insulator binding site distributions and protein composition suggest there exist 2 principal categories of insulator elements (Class I and Class II). In particular, it was shown that Class I insulators, identified by the binding of CTCF, CP190 or BEAF-32, segregate differentially expressed genes and delimit the boundaries of chromatin silencing, while they are depleted between known CRMs and their target genes. No evidence was found supporting a significant distinction between CP190/BEAF and CP190/CTCF or CTCF/BEAF. In contrast, the analyses suggest that BEAF-32, CP190, and CTCF are distributed and function quite similarly, while Su(Hw) appears distinct. The Class II insulators, bound by Su(Hw), are often exceptional in this analyses. It is noted that the analysis of genome-wide mapping data, expression data, and genome annotation provides an endogenous boundary assay that demonstrates that, while Su(Hw) has been described as an insulator before, it is not systematically associated with the boundaries of the gene units (Négre, 2010).

By helping to delimit the regulatory boundaries of genes, the Class I insulator map presented in this study will aid in the identification of transcription factor target genes and the construction of transcriptional regulatory networks. As an example of this concept, the distribution of known regulatory elements and insulators across the Antennapedia Complex (ANT-C) of homeotic genes is presented. This region quite strikingly demonstrates the potential utility of insulator binding data for cis-regulatory annotation. Across approximately 500 kb, cis-regulatory elements and their target promoters are found between insulator pairs. For example, a single insulator separates the lab and Edg84A genes, with their respective cis-regulatory elements narrowly partitioned on either side. The adjacent regulatory elements and promoters of zen and bcd are similarly insulator segregated (Négre, 2010).

Consistent with their observed regulatory boundary functions, Class I insulators are embedded within local regions of active chromatin and are frequently associated with syntenic breakpoints between species. Previous work has demonstrated that active promoters in yeast and Drosophila are associated with reduced nucleosome occupancy and low-salt soluble and high-salt insoluble chromatin. Therefore, surprisingly, dynamic chromatin is a shared feature between promoters and most classes of insulators. It is notable however that some studies have revealed functional similarities between insulators and promoters in transgenic assays. These results have been described as paradoxical, as insulators can negatively affect promoters by blocking communication between enhancers and promoters. One proposed model for insulator function is that they act as promoter 'decoys' by recruiting away factors necessary for transcriptional initiation. Alternatively, insulators and promoters might require common chromatin features to function by mechanisms that are still unknown. One potential interpretation is that the dynamic chromatin at insulators forms a flexible chromatin joint that would affect the probability of productive contact between separated regulatory elements. In this way, the similarity between promoters and insulators would be a consequence of their common requirement for dynamic chromatin, although with very different consequences. This model may explain why promoters are so frequently scored as insulators in the classical insulator assay, when an element is placed between an enhancer and a promoter (Négre, 2010).

Analysis of chromatin boundary activity in Drosophila cells

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 limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. This paper reports the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. The activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are shown to be supported in these cells. It was further shown 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 this cell-based assay for probing insulator function. It is concluded that 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 (Li, 2009).

Despite their diverse genomic origins and distinct cis- and trans- components, the Drosophila suHw, SF1, Fab7 and Fab8 elements function as potent enhancer-blockers in the Drosophila cells. This finding suggests that chromatin boundary represents a basic cell function that is shared by diverse tissues. The cell-based insulator assay was combined with RNAi-mediated gene knockdown to systematically test the requirement of SuHw and dCTCF in the function of several Drosophila insulators. RNAi-mediated knockdown of SuHw and dCTCF specifically disrupted the function of the suHw and Fab8 boundaries, respectively, thereby validating the functional specificity of the assay. The results suggest that multiple independent pathways in Drosophila mediate insulator function. This is in contrast with the pivotal role the CTCF protein plays in the enhancer-blocking activities in vertebrates (Li, 2009).

Cell culture assays have several important advantages that complement studies using in vivo system. The homogeneous cell populations in these assays can be used in biochemical and cell biological analyses. They allow more efficient and quantitative assessment of reporter readout from a large number of individual cells. Insulator activity has previously been demonstrated in Drosophila cells; this system has improved the assay with several novel features. First is the use of P-element-based transgene vector, which is known to mediate single to low copy number, non-tandem genomic integration of the assay transgenes. This would provide more native genomic and regulatory environment for studying chromatin boundary function. Large numbers of stably transfected cells with randomly integrated transgenes also provide a broader sampling of the genomic environment, a feature that can be exploited to examine boundary activity in blocking chromosomal position effect. The second improvement is the use of divergently transcribed dual reporters, which provides a linked readout to control for the 'off-targets' effects on the non-insulator components in the assay system, such as enhancers, promoters, reporters, the state of general transcription or other cellular functions that impact the reporter readout. It should also provide an important control for the chromosomal position effect near the transgene integration site in stably transfected cells. The use of fluorescent protein reporters further allows rapid and quantitative FACS assessment of the enhancer-blocking activity, a feature particular important in high-throughput applications. The activity of multiple Drosophila insulators has been established, along with the efficiency of RNAi-mediated gene knockdown; this should facilitate biochemical dissection of insulator function and genome-wide high throughput RNAi screens for novel boundary components (Li, 2009).

As most cell-based systems, the enhancer-blocking assay is limited in its application by potential tissue or developmental stage incompatibilities of the insulator and the cell. Studies have suggested that certain chromatin boundaries, such as Fab7 and SF1, are composed of distinct insulator activities that function in different tissues and/or developmental stages. Although this study has documented the functionality of several Drosophila insulators in S2 and Kc cells, both derived from embryonic cell lineages, other insulators may not function in these two cell lines. In addition, cultured cells may have, over the course of many passages, lost the physiological stoichiometry of relevant DNA or protein components, resulting in impaired function of certain insulators. Furthermore, the dynamic regulation of insulator activity in response to developmental and physiological cues would depend on the context of the whole animal. Therefore, the cell-based insulator assay presented in this study provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements (Li, 2009).

CTCF genomic binding sites in Drosophila and the organisation of the Bithorax Complex

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). This study 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 construction of a robust CTCF binding-site consensus sequence (AGGNGGC, the same ase mammalian CTCF). 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 localization of three additional presumptive insulator elements; 'Fab-2', 'Fab-3', and 'Fab-4'. With the exception of Fab-7, these 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 (Holohan, 2007).

The multiple zinc-finger DNA-binding protein CTCF is known to be required for the enhancer blocking action of vertebrate insulators, and a clear role for CTCF in the regulation of endogenous gene expression has been demonstrated at the imprinted Igf2. The mode of action of CTCF is, however, still unclear, although several studies have implicated CTCF in the formation of higher-order chromatin structure. CTCF molecules can interact to form clusters and thereby may mediate the formation of chromatin loop domains (Kurukuti, 2006; Splinter; 2006; Yusufzai, 2004). Partitioning of regulatory elements into independent chromatin loop domains is postulated to play a key role in the interactions between enhancers and promoters. The CTCF homolog of Drosophila is required for the insulator function of the Fab-8 element in the BX-C. This observation has opened up the prospect of utilising the wealth of genetic and molecular characterisation of BX-C transcriptional regulation for the analysis of CTCF function. This study used ChIP-array to investigate CTCF binding sites in regions of the Drosophila genome with a particular focus on the BX-C. CTCF not only associates with the Fab-8 insulator, but also with other mapped boundary elements, Fab-6 and Mcp. In addition, CTCF sites are located at other postulated boundaries within the BX-C; 'Fab-2', 'Fab-3', and 'Fab-4'. This provides a precise mapping of regulatory domain boundaries and a specific molecular foundation for the domain model of BX-C regulation (Holohan, 2007).

It is noted that the Fab-7 boundary may differ from the other characterised boundaries in the BX-C since no strong Patser match was found to the CTCF consensus in the functionally mapped Fab-7 boundary element. Although Fab-7 was not demonstrably enriched in the ChIP-array, significant CTCF association with Fab-7 was found in the more sensitive PCR-base ChIP assay. Given the lack of a strong Patser match (ChIP enrichment) this may suggest an indirect association. No CTCF site was seen between the abx/bx and the bxd/pbx regulatory elements. However, these elements are separated by a long distance, and it is not clear whether they require insulation (Holohan, 2007).

According to the domain model, the parasegment-specific regulatory domains that control the expression patterns of the Ubx, abd-A, and Abd-B genes of the BX-C are initially activated in appropriate parasegments by the early pattern-forming genes acting on initiator elements. Each regulatory domain is predicted to contain a particular initiator element, tuned to respond to a specific combination of gap and pair-rule gene products, thus activating the regulatory domain in the appropriate set of parasegments. This activation would be read by maintenance elements consisting of PREs that thereafter autonomously maintain each regulatory domain in either the OFF (silenced) or ON (active) state. Within a domain in the ON state, enhancers present in that domain would be able to engage with the relevant gene promoter and regulate expression of the gene. Boundary elements that flank each domain are proposed to restrict the effects of the initiator and maintenance elements to a single domain (Holohan, 2007).

Although boundary elements are postulated to have the common property of insulating the regulatory domains, no sequence similarity between the mapped boundary elements has been reported until now. This study shows that a set of these boundary elements contain CTCF binding sites and bind CTCF in vivo. CTCF has been shown to be required for the insulator activity of Fab-8, and it seems likely that CTCF will also be a required component at the other boundary elements. In support of this suggestion, it was found that the CTCF sites are well conserved within the sequenced insect genomes. The observation that CTCF sites flank a set of regulatory domains in the BX-C, together with the vertebrate studies that suggest that CTCF can mediate the formation of chromatin loops (Splinter, 2006; Yusufzai, 2004) supports the idea that interaction between CTCF sites may organise these domains into chromatin loops. However, how such a looping mechanism enables the autonomy of the individual regulatory domains and facilitates appropriate enhancer/promoter interactions is still unclear (Holohan, 2007).

A key feature of the domain model is the relationship between the boundary and maintenance elements. For the domains to be capable of independently being set to the ON or OFF state, the range of influence of PREs needs to be restricted by the domain boundaries. Each domain would require at least one PRE. Precise mapping of in vivo CTCF binding sites has enabled examination of their relationship with Polycomb target sites. In strong support of the domain model, it was found that the domains demarcated by CTCF sites contain Polycomb target sites. Indeed, an intimate relationship was found between CTCF and Polycomb binding sites for 'Fab-4', Mcp, Fab-6, and CTCF site 'C'. This fits with previous functional mapping indicating that boundary elements and PREs are closely associated at Fab-7, Fab-8, and Mcp. This arrangement would impose a polarity on the spread of chromatin modification from the PRE, such that modification may start at the PRE abutting one boundary and spread across the domain in one direction towards the next boundary. At the boundaries, CTCF may play many possible roles. It could participate in boundary element function allowing the independence of chromatin domains by acting as a chromatin insulator blocking the spread of chromatin modification. However, at the chicken ß-globin locus, the chromatin boundary appears to be separable from the CTCF binding site . Another possibility is suggested by that fact that CTCF has been demonstrated to block the progression of RNA polymerase (Zhao, 2004). This could potentially play an important role at boundaries in the BX-C to enable the independent function of PREs in neighbouring domains. There is considerable evidence that transcription through PREs may control their state, and many noncoding RNAs have been detected in the regulatory regions of the BX-C. One role for CTCF could be to act as a barrier to such noncoding transcription, preventing transcripts arising in one regulatory domain from crossing into the neighbouring domain and affecting the PRE state. Such a role would be consistent with the observed location of CTCF sites in this region, as a CTCF site closely abuts one side of each PRE (Holohan, 2007).

The individual regulatory domains must not only be able to act autonomously to set and maintain their activity state, but they must also be able to interact appropriately with the relevant gene promoters. Boundaries may play a role in this, and recently a long-range interaction has been demonstrated between Fab-7 and the Abd-B-RB promoter. This interaction was associated with lack of Abd-B expression, but similar interactions, bringing in appropriate enhancers, may also activate expression. The ability of CTCF to form clusters may facilitate such interactions, and it is intriguing that there are CTCF sites not only at the boundaries but also close to Abd-B promoters; the CTCF site 'B' is 300 bp upstream of the Adb-B-RB promoter. Clustering of boundaries together with Abd-B promoter sequences may enable interaction between the promoter and enhancers in domains in the ON state. The clustering may also be more selective; in S2 cells, which specifically express Abd-B-RB, several boundaries are embedded in chromatin bearing the repressive H3K27me3 modification, whereas Fab-8, CTCF site 'B', and the Abd-B-RB promoter are in the unmodified, presumably 'open', chromatin domain. It could be speculated that the expression of Abd-B-RB in these cells might be facilitated by interaction of the CTCF sites in the 'open' domain, Fab-8 and site 'B', enabling Fab-8 to bring appropriate enhancers to the Abd-B-RB promoter (Holohan, 2007).

ChIP-array analysis of CTCF genomic sites can be compared with ChIP-array analysis of binding sites for another Drosophila insulator-binding protein, Su(Hw). CTCF and Su(Hw) are both multi-zinc- finger DNA-binding proteins, and in both cases relatively long (~20 bp) consensus binding sites have been identified. In contrast to most DNA-binding proteins, it was found that strength of match to the consensus binding sites is a good predictor of in vivo occupancy. It was also investigated whether the data indicate any collaboration between CTCF and Su(Hw). This seemed an attractive possibility since removing Su(Hw) function in vivo has little effect; su(Hw) null mutant flies are female-sterile but viable. Also, the insulating activity of Fab-8 was significantly reduced when the CTCF sites were mutated but not completely abolished. However this study found no evidence for general colocalisation between CTCF and Su(Hw). A total of 60 Su(Hw) sites were identified in the Adh region, and only one of the fragments covering this region contained both CTCF and Su(Hw) sites. The single CTCF site identified in the achaete-scute complex was also some distance from the two Su(Hw) sites found. Subsequent ChIP-array analysis in the BX-C led to the identification of only one Su(Hw) site within the entire BX-C region, in a location devoid of CTCF binding sites. Indeed while the BX-C appears relatively enriched in CTCF sites compared to the Adh region, the converse is true for Su(Hw). For CTCF there are 4.7 sites/100 kb in the BX-C and 1.7 sites/100 kb in the Adh region, whereas for Su(Hw) the BX-C is depleted in sites with only 0.29/100 kb in comparison to 2.7/100 kb in the Adh region. Clearly, although CTCF and Su(Hw) both possess insulating ability, their sites of action do not correlate and there is no evidence from this analysis, covering approximately 3% of the Drosophila genome, for cooperative activity (Holohan, 2007).

By comparing the sequences of ChIP-enriched fragments a strong Drosophila consensus CTCF binding site was identified. Analysis of vertebrate CTCF target sequences leads to a proposal that vertebrate CTCF also binds to a similar consensus sequence. These findings do not support the current view that CTCF binds to divergent DNA sequences by engaging different subsets of the zinc fingers. Indeed, the binding site revealed here has been previously noted. Bell (1999) identified a CTCF binding site in the chicken β-globin insulator, and sequence comparisons between this site and other known CTCF sites identified a conserved 3' region, the mutation of which completely abolished CTCF binding and enhancer blocking. Filippova (2001) extended this comparison to include the Dm1 sites, mouse H19 DMD4 and DMD7 and human MYC A, and again identified a conserved region within the larger approximately 50-bp DNase footprint for each site. It is this conserved region that corresponds to the vertebrate CTCF site found here. Very recently, an analysis of CTCF binding in the human genome has generated a vertebrate CTCF consensus site (Kim, 2007), and a CTCF consensus has also been derived from analysis of conserved regions in the human genome (Xie, 2007). Both these sites are very similar to the consensus identified in this study; in particular they share the strong features of the CC at positions 1 and 2, the AG at positions 6 and 7, and the GGC at positions 10, 11, and 12. Overall, these findings indicate that CTCF in both Drosophila and vertebrates binds to a single core consensus sequence (Holohan, 2007).

In summary, ChIP-array analysis has enabled construction of a CTCF binding site consensus. Mapping of genomic binding sites leads to a proposal that all known or predicted insulators in the BX-C (with the possible exception of Fab-7) function in a CTCF dependent manner (Holohan, 2007).

The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning

Insulator sequences guide the function of distantly located enhancer elements to the appropriate target genes by blocking inappropriate interactions. In Drosophila, five different insulator binding proteins have been identified, Zw5, BEAF-32, GAGA factor, Su(Hw) and dCTCF. Only dCTCF has a known conserved counterpart in vertebrates. This study found that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlaps with dCTCF binding sites and interacts with dCTCF. Binding of dCTCF to targets requires CP190 in many cases, whereas others are independent of CP190. Analysis of the bithorax complex revealed that six of the borders between the parasegment specific regulatory domains are bound by dCTCF and by CP190 in vivo. dCTCF null mutations affect expression of Abdominal-B, cause pharate lethality and a homeotic phenotype. A short pulse of dCTCF expression during larval development rescues the dCTCF loss of function phenotype. Overall, this study demonstrates the importance of dCTCF in fly development and in the regulation of abdominal segmentation (Mohan, 2007).

The CP190 protein contains three classical C2H2 zinc-finger motifs and an N-terminal BTB/POZ domain. Both domains could potentially be involved in chromatin binding. In contrast, chromatin binding might be achieved by interaction with other factors, such as dCTCF. A possible interaction of dCTCF with CP190 was tested using co-immunoprecipitation. Precipitation of CP190 from Schneider cell extracts resulted in the detection of dCTCF. To confirm the interaction a FLAG-dCTCF fusion protein was expressed in Schneider cells and precipitated with either an antibody against CP190 or an antibody against FLAG. The CP190 precipitate contained endogenous dCTCF as well as FLAG-dCTCF in the same ratio as the input, suggesting that both dCTCF proteins are similarly associated with CP190. Furthermore, the reverse experiment using FLAG precipitation demonstrated that dCTCF and CP190 interact in vivo (Mohan, 2007).

Because CP190 and dCTCF colocalize on polytene chromosomes and interact in vivo, it was asked whether the overall amount of dCTCF protein might be changed in CP190-deficient third instar larvae. A Western blot analysis of both Cp190¹ homozygotes (deficient in CP190) and wild-type larval extracts showed that the amount of dCTCF is reduced in Cp190¹ homozygotes (Mohan, 2007).

Next it was of interest to know whether the reduced amount of dCTCF caused by the loss of CP190 affects dCTCF binding on the polytene chromosomes. It was found that the total number of dCTCF labeled sites is reduced in the Cp190¹ mutant, whereas the number of CP190 sites was not affected by dCTCF mutants. The analysis of dCTCF binding in the two hypomorphic mutants CTCF^EY15833/CTCF^EY15833 and GE24185/GE24185 revealed that that the number of bound sites is reduced to about 50% and 25%, respectively. By close inspection of the chromosomes it was found that the set of dCTCF sites missing in the CP190 or in the dCTCF mutants overlap but are not identical. Thus, different sites vary in their requirement for CP190/dCTCF cooperation (Mohan, 2007).

Insulator elements with enhancer blocking activity establish independent regulatory domains. An analysis of binding sites (CTS) for the enhancer blocking factor dCTCF on salivary gland polytene chromosomes resulted in the identification of several hundred sites bound by dCTCF. All of these sites are found in interbands, and when inspected more precisely are often at the borders of interbands next to bands. Interbands harbor active housekeeping genes or regulatory regions of inactive genes, whereas bands contain the bodies of inactive genes. Interbands and bands differ in chromatin composition and modification. Thus, there is a clear border between interbands and bands. Any factors generating functional chromatin boundaries would be expected to be localized to the interband/band transition. This is not only the case for dCTCF, as a similar location has been found for Su(Hw). Also, BEAF-32 and Zw5 are located in interbands at hundreds of binding sites throughout the genome (Mohan, 2007).

The obvious question was whether dCTCF has a redundant function and therefore similar targets as the other Drosophila enhancer blocking factors. No significant colocalization of dCTCF with either BEAF-32 or with Su(Hw) on polytene chromosomes was detected. This may provide an explanation of how an organism with a small genome, such as Drosophila, can prevent promiscuous enhancer interaction with any nearby gene. Apparently, an elaborate system of different enhancer blockers and barrier factors fulfills the insulation of regulatory units (Mohan, 2007).

The biochemical composition and function of insulator complexes involving Su(Hw) have been studied in detail. The best studied binding site is the gypsy transposon with a 350-bp sequence containing 12 binding sites for Su(Hw). A functional complex of Su(Hw), Mod(mdg4)67.2, CP190, and possibly other factors has been documented (Capelson, 2005; Lei, 2006). Although there is no colocalization of Su(Hw) with dCTCF on polytene chromosomes, and only partial colocalization with Mod(mdg4), it was of interest to examine whether CP190 plays a role in dCTCF function. Vertebrate CTCF is a centrosomal factor during mitosis and a nuclear protein during interphase (Zhang, 2004), and that CP190 (centrosome binding protein) is associated with centrosomes as well. CP190 is essential for viability, but is not required for cell division (Butcher, 2004). CP190 knockdown in Schneider cells has no effect, whereas a null mutation in flies leads to pharate lethality. A similar phenotype is seen after dCTCF depletion in Schneider cells and in the pharate lethality in flies. The centrosomal function of CP190 is not required for the insulator activity in the context of Su(Hw) bound to gypsy (Pai, 2004). The localization of CP190 on polytene chromosomes overlaps with sites bound by Su(Hw) or by Mod(mdg4)67.2. In addition, CP190 is found at loci devoid of Su(Hw) or Mod(mdg4)67.2, suggesting that other factors might recruit CP190 to these sites (Pai, 2004). There is a significant overlap in dCTCF with CP190 binding sites. A functional dependence is seen, because at many sites binding of dCTCF depends on CP190. Although there is an overall reduction in the dCTCF amount observed in the CP190 mutant, differences in dCTCF occupancy in dCTCF and CP190 mutants indicate a discrimination between CP190-independent and -dependent sites. Furthermore, the previously characterized insulator Fab-8 is impaired in the absence of dCTCF (Moon, 2005) and by the reduction of CP190 (Mohan, 2007).

Another perspective on the requirement of insulators comes from the fact that many genes are controlled by several regulatory elements that are required for tissue and cell-specific expression. A prominent example is the Drosophila BX-C. This is one of two Hox gene clusters, which contain regulator genes controlling development. The BX-C is responsible for the correct specification of the posterior thorax segment (T3) and all of the abdominal segments. Within BX-C, only three protein coding genes, Ubx, abd-A and Abd-B, are responsible for the segment-specific development of organs and tissues. On the other hand, nine separate groups of many mutations are affecting segment-specific functions. The borders of some of these domains are genetically defined by elements Fab-6, Fab-7, Fab-8 and by Mcp. Proteins involved in such a functional separation are the GAGA factor in case of the Fab-7 element, and dCTCF for the Fab-8 sequence. Recently, it has been demonstrated that six of the BX-C domain junctions are bound by dCTCF (Holohan, 2007). Consequently, if these sites contribute to boundary function, gene activity within this locus should be changed. Indeed, a homeotic phenotype and a reduced expression of Abd-B was found in larval nerve cord. If dCTCF plays a central role in separating the different regulator domains in the BX-C and elsewhere in the genome, it is difficult to predict the dCTCF phenotype. The situation could be complicated as the three BX-C genes are controlling realizator genes as well as other regulators. Furthermore, individual BX-C genes repress others, for example Abd-B as well as the miRNA iab-4 and bxd expression repress Ubx. In addition, other factors, such as CP190 and perhaps additional unknown factors may contribute to the enhancer blocking function of dCTCF. For all of the CTS in the BX-C, dCTCF and CP190 binding was found. Although both factors clearly interact as seen by co-immunoprecipitation, CP190 may contact other DNA-bound factors as well, or may be directly targeted to chromatin (Mohan, 2007).

Thus, dCTCF shares several biochemical and functional features with Su(Hw), but is clearly targeted to dCTCF-specific sites. Overall, this study has shown that dCTCF is important for fly development, and has important functions in the regulation of abdominal segmentation (Mohan, 2007).

CTCF is required for Fab-8 enhancer blocking activity in S2 cells

CTCF is a conserved transcriptional regulator with binding sites in DNA insulators identified in vertebrates and invertebrates. The Drosophila Abdominal-B locus contains CTCF binding sites in the Fab-8 DNA insulator. Previous reports have shown that Fab-8 has enhancer blocking activity in Drosophila transgenic assays. This study now confirms the enhancer blocking capability of the Fab-8 insulator in stably transfected Drosophila S2 cells and shows this activity depends on the Fab-8 CTCF binding sites. Furthermore, knockdown of Drosophila CTCF by RNAi in these stable cell lines demonstrates that CTCF itself is critical for Fab-8 enhancer blocking (Ciavatta, 2007).

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq was performed in two different embryonic cell lines; AGO2 was found to localize to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. It is proposed that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome (Moshkovich, 2011).

This study provides the first evidence for an Argonaute protein functioning directly on euchromatin to effect changes in gene expression. The genome-wide binding profile of AGO2 displays striking overlap with insulator proteins. Genetic analysis revealed that AGO2, independent of its catalytic activity, promotes Fab-8 insulator activity. Like known insulator proteins, AGO2 also associates with promoters and can oppose PcG function. Genome-wide AGO2 recruitment to chromatin is dependent on CTCF and CP190 binding and may be partially achieved via looping interactions among cis-regulatory regions and promoters. It is proposed that AGO2 may act to facilitate or stabilize looping that is needed to partition the genome into independent transcriptional domains (Moshkovich, 2011).

These results suggest that the main function of AGO2 on chromatin resides in euchromatin and not in heterochromatin. Immunofluorescence localization of AGO2 on polytene chromosomes and cell lines indicates exclusion from heterochromatic and HP1-enriched regions. Furthermore, the majority of chromatin-associated AGO2 resides in nonrepetitive euchromatic but not repeat-rich regions, as determined by genome-wide ChIP-seq. It is suggested that the role of AGO2 in RNAi-dependent silencing of TEs occurs primarily at the post-transcriptional level and that AGO2 harbors a second RNAi-independent activity to promote chromatin insulator function (Moshkovich, 2011).

Several observations suggest that AGO2 chromatin association is mainly, if not exclusively, independent of the RNAi pathway. First, AGO2 chromatin association does not correspond to regions of the genome that produce high levels of endo-siRNAs, which are dependent on Dcr-2 and AGO2. Second, AGO2, but not Dcr-2, is required for Fab-8 insulator function. Finally, a catalytically inactive AGO2 protein, which is defective for RNAi, retains the ability to associate with chromatin and is functional with respect to both TrxG function and Fab-8 insulator activity (Moshkovich, 2011).

An intriguing question raised by these findings is whether or not the functions of AGO2 in RNAi and chromatin insulator activity are completely distinct. CP190 mutants were found to remain competent for silencing, suggesting that AGO2 chromatin association is not required for RNAi. Nevertheless, it remains possible that chromatin-associated AGO2 is loaded with siRNA. Future work will address how AGO2 subcellular localization and seemingly disparate functions in RNAi and chromatin insulator activities are regulated (Moshkovich, 2011).

A unique positive role for AGO2 but not other RNA silencing factors was identified in Fab-8 insulator function. Importantly, a catalytically inactive mutant form of AGO2 expressed at wild-type levels retains insulator activity, further suggesting that the RNAi pathway is dispensable for Fab-8 insulator function. A significant fraction of AGO2 resides in the nucleus, and physical interaction is observed between AGO2 and CP190. This interaction is insensitive to RNaseA, suggesting that RNA does not mediate the interaction between AGO2 and CP190. It remains possible that AGO2 can interact with siRNA or other RNA while associated with the insulator complex, although there is no evidence to support this hypothesis (Moshkovich, 2011).

This study shows that chromosomal looping in the Abd-B locus is dependent on CTCF, CP190, and AGO2. Confirming and extending previous studies, it was found that the Abd-B RB promoter interacts frequently with Fab-7, Fab-8, and the iab-8 enhancer and, moreover, that the Fab-8 region also contacts Fab-7 as well as multiple Abd-B promoters. Currently, the significance of insulator protein promoter association is unclear, but insulators may be thus situated to control looping interactions between promoters and cis-regulatory elements. Depletion of CP190 or CTCF reduces these high-frequency looping interactions, and loss of this specialized chromatin configuration could result in disassociation of AGO2. Given this possibility, AGO2 may act to detect the insulator-dependent conformation of this locus (Moshkovich, 2011).

AGO2 is recruited to chromatin insulator sites as well as noninsulator sites in a CTCF/CP190-dependent manner. It is speculated that AGO2 chromatin association with insulator sites could result from physical interactions with CP190 complexes, while AGO2 recruitment to other sites may be achieved at least in part by chromatin looping mediated by CP190 and CTCF. In fact, it was recently shown that PcG proteins can be transferred from a PRE to a promoter as a result of intervening insulator-insulator interactions. Once recruited to chromatin, AGO2 could perform a primarily structural function to promote or stabilize the frequency of CTCF/CP190-dependent looping interactions (Moshkovich, 2011).

AGO2 appears to promote Fab-8 insulator activity independently of an effect on gypsy insulator body localization. Previous work showed that both the gypsy class and CTCF/CP190 insulators colocalize to insulator bodies, suggesting that these subnuclear structures may be important for both gypsy and Fab-8 activities. However, since Fab-8 activity is not affected by RNA silencing components that disrupt gypsy insulator body localization, this subnuclear structure appears to be dispensable for Fab-8 function. Recent work indicates that the BX-C harbors multiple redundant cis-regulatory elements that can maintain looping interactions of this locus, suggesting that the configuration of the BX-C may not require a nuclear scaffold such as the gypsy insulator body (Moshkovich, 2011).

AGO2 mutations suppress the Polycomb phenotype, indicating that AGO2 behaves similarly to trxG genes and opposes PcG function. A previous study proposed that RNA silencing factors promote long-range PRE-dependent chromosomal pairing as well as PcG body formation but did not examine AGO2. This study found that the AGO2^51B-null mutation has no effect on Fab-X PRE pairing-dependent silencing on sd as assayed in that study, and genetic results suggest that AGO2 is unlikely to promote PRE-dependent interactions or PcG body formation, which are both positively correlated with PcG function. Interestingly, it has recently been shown in the case of AGO2-associated Fab-7 and Mcp boundary elements that long-range interactions are dependent on insulator sequences and not PREs. Future studies will elucidate the complex interplay between PcG and insulator organization as well as the role of AGO2 in the regulation of these structures (Moshkovich, 2011).

It remains to be seen whether Drosophila AGO2 euchromatin association and function may be conserved in other organisms. In Caenorhabditis elegans, the nuclear NRDE RNAi pathway can block transcriptional elongation of Pol II on a target transcript when treated with exogenous complementary dsRNA. Interestingly, this negative transcriptional effect is contemporaneous with an increase in H3K9me3. Whether the Argonaute protein NRDE-3/WAGO-12, which lacks Slicer activity, associates with euchromatin to effect this repression is not yet known. Furthermore, the C. elegans Argonaute Csr-1, loaded with 22G endo-siRNAs antisense to mRNAs of holocentric chromosomes, may serve as chromosomal attachment points to promote efficient chromosome segregation. Recently, it has been shown that Schizosaccharomyces pombe Ago1 participates in surveillance mechanisms to prevent readthrough transcription of mRNA. However, the majority of Ago1 associates with heterochromatic regions, and it is not clear thus far whether Ago1 directly associates with euchromatin or acts post-transcriptionally. An emerging theme from studies of RNAi in various model systems is that genome integrity and control of gene expression may be achieved by multiple yet overlapping mechanisms (Moshkovich, 2011).

Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene

Boundary elements have been found in the regulatory region of the Drosophila Abdominal-B gene, which is subdivided into a series of iab domains. The best-studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF (dCTCF) were identified in the Fab-8 boundary and upstream of Abd-B promoter A, with no binding of CTCF to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when its binding sites are separated by a 5-kb yellow gene, a study was performed of the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing a dCTCF binding site. It was found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggests that additional, as yet unidentified proteins are involved in long-distance interactions between them. Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter (Kyrchanova, 2008).

Previously it was found that the relative orientation of Mcp elements defines the mode of loop formation that either allows or blocks stimulation of the white promoter by the GAL4 activator. This study has demonstrated that two PTS/F8 boundaries or Fab-8 insulators alone are also capable of orientation-dependent interaction. When these elements are located in opposite orientations, the loop configuration is favorable for communication between regulatory elements located beyond the loop. The loop formed by two insulators located in the same orientation juxtaposes two elements located within and beyond the loop, which leads to partial isolation of the GAL4 binding sites and the white promoter placed on the opposite sides of the insulators (Kyrchanova, 2008).

The orientation-dependent interaction may be accounted for by at least two proteins bound to the insulator that are involved in specific protein-protein interactions. In the case of a Fab-8 insulator, dCTCF is likely to be directly involved in pairing between two insulators. Since mutated Fab-8 insulators devoid of dCTCF binding sites proved to be incapable of interacting with each other, it is hypothesized that dCTCF facilitates the binding of a certain as yet unidentified protein (or proteins) that, in combination with dCTCF, accounts for orientation-dependent interaction between the Fab-8 insulators. Functional interactions between the Fab-7 boundary devoid of dCTCF binding sites and PTS/F8 or the upstream Abd-B A promoter region are also evidence for the existence of unidentified proteins that support organization of distance interactions in the Abd-B locus (Kyrchanova, 2008).

Recently it was shown that in the repressed state of the bithorax complex, all of its major regulatory elements binding PcG proteins, including PREs with adjacent boundaries and core promoters, interact at a distance, giving rise to a topologically complex structure (Lanzuolo, 2007). The question arises as to what proteins are important for such interactions. All PREs tested (Lanzuolo, 2007) were flanked by boundaries, suggesting that all these regulatory elements may be involved in long-distance interactions. As shown previously, the Fab-7 or Mcp boundaries including PREs can support physical association between even transposons located on different chromosomes. One of relevant models proposes that PcG proteins are capable of supporting highly specific long-distance interactions between transposons (Lanzuolo, 2007). However, it is known that many PcG complexes with similar properties can bind to Drosophila chromosomes, which leaves open the question as to how such protein complexes can ensure a high specificity of interactions between distantly located transposons. Moreover, there is no experimental evidence that PREs without additional regulatory elements can support long-distance interactions. In contrast, there are many proven cases showing that insulator proteins are involved in physical association between distant chromosomal regions. For example, the interaction between gypsy insulators can support activation of the yellow promoter by enhancers separated by many megabases. The Mod(mdg4)-67.2 and Su(Hw) proteins bound to the gypsy insulator are essential for such long-distance interactions. In mammals, the interaction of the imprinting control region on chromosome 7 with the Wsb1/Nf1 locus on chromosome 11 depends on the presence of the CTCF protein. In vivo interaction between Fab-7 and the Abd-B promoter is absolutely dependent on the presence of the Fab-7 insulator. Finally, this study has demonstrated the functional interaction between the Fab-7 and Fab-8 boundaries and the Abd-B promoter. These results support the model that transcriptional factors bound to boundaries can facilitate enhancer-promoter interactions in the bithorax complex. Further studies are necessary for identifying new proteins involved in long-distance interactions and for elucidating the mechanisms that allow interactions either between proper active enhancers and promoters or between only silenced enhancers and promoters (Lanzuolo, 2008).

CTCF is expressed during the transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila

In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. This study focused on post-meiotic stages and shows that also after meiosis, histone H3 shows a high overall methylation of K9 and K27; it was hypothesised that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, histones are lost during the early canoe stage, and just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. It is believed that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, the first transition protein-like chromosomal protein to be found in Drosophila, Tpl94D, is reported. It is proposed that Tpl94D (an HMG box protein) and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, it is showm that histone modifications and removal are independent of protamine synthesis (Rathke, 2007).

The switch between a nucleosome-based chromatin configuration and a protamine-based structure is a specialised form of chromatin remodelling in the male germline. The mammalian zinc finger protein CTCF is involved in many epigenetic processes. Furthermore, paralogous variant of CTCF which is testis-specifically expressed, called BORIS, is exclusively expressed in the mammalian male germline. The function of BORIS in this context is still not clear (Loukinov, 2002). Drosophila, in contrast to mammals, contains only one CTCF gene (Moon, 2005). It was therefore asked whether Drosophila CTCF is also expressed in the testes, and immunostaining and anti-histone staining was performed on testes of transgenic flies expressing protamine-eGFP. CTCF expression was observed during pre-meiotic and meiotic stages at the chromosomes as has been shown for mitotic cell division in mammalian cell culture. Shortly after meiosis, CTCF is visible in young elongating nuclei, where it co-localises with the chromatin as indicated by the histone distribution. CTCF is also present in the early and late canoe stage spermatid heads. At the early canoe stage, CTCF is very diffusely distributed in comparison to histones. CTCF does not co-localise with the chromatin which starts to condense at one side of the nucleus. This diffuse distribution is still visible at the late canoe stage when protamine-eGFP starts to be deposited to the chromatin. CTCF is no longer detectable after the canoe stage. The earlier chromatin-associated CTCF localisation might indicate a very early role in chromatin reorganisation at the switch between the nebenkern and canoe stage. Furthermore, CTCF might be associated primarily with the chromatin, which is not yet condensing during these stages. The late canoe stage is the only post-meiotic stage where distinct regions of RNA polymerase II are found with an antibody directed against a phosphorylated subunit of active polymerase, indicative of transcription. At this precise stage, only a very small set of genes is thought to be transcribed. Also CTCF expression during chromatin reorganisation in the nucleus was detected in D. hydei (Rathke, 2007).

Sperm morphogenesis is characterised by an impressive degree of changes in cell architecture based on stored, translationally repressed mRNAs that are recruited at the appropriate time to the polysomes. Among these are mRNAs that encode Tpl^94D and protamines. A dramatic switch in structure from the nucleosomal- to the protamine-based structure of chromatin takes place, and this remarkable chromatin reorganisation of the complete genome is a typical feature depending on stored mRNAs, e.g. for protamine synthesis. This process ultimately leads to an extremely condensed state of the haploid genome in the sperm, which is essential for male fertility in mammals. This study focused on the switch between a nucleosomal- and a protamine-based chromatin reorganisation. The major steps in chromatin organisation take place in the canoe stage of spermatid development. A candidate for a transition protein in Drosophila was identified. The corresponding gene tpl^94D (CG31281) encodes a predicted basic high mobility group (HMG) protein of 18.8 kDa. In transgenic flies, Tpl^94D-eGFP fusion proteins are expressed solely during the switch between histones and protamines, as is typical for mammalian transition proteins. Since a highly similar chain of events to those reported in mammals is observed, the Drosophila system is considered an excellent choice to study the mechanism of chromatin remodelling during male germ cell development (Rathke, 2007).

Generally, the bulk of histones, including their diverse modifications in the N-terminal tail, appear to be removed during the canoe stage. Furthermore, the nucleus accumulates ubiquitin at the early canoe stage, when mono-ubiquitylation of histone H2A is no longer detectable. Therefore, taking into account the known presence of proteasomes in the nucleus at this stage of chromatin reorganisation and the overlap of expression shown in this study, it is hypothesised that this ubiquitylation is targeting histones for degradation. This study investigated several mutants having mutations in ubiquitin-conjugating enzymes or ubiquitin ligases, exhibiting arrested spermiogenesis during spermatid development and that are male sterile. However, in all investigated mutants, histone removal is indistinguishable from that of wild-type flies (Rathke, 2007).

Many histone modifications were found after meiosis and were categorised into three classes (Rathke, 2007).

1. Histone modifications that persist from pre-meiotic stages and keep the genome silent.
   
   The vast majority of the genome is transcriptionally silent in post-meiotic stages. This is accompanied by multiple histone modifications that persist from pre-meiotic stages and indicate silencing such as H3K9 and H3K27 methylation. These modifications do not change significantly during post-meiotic stages, which is in agreement with the hypothesis that these modifications predominantly play a role in maintaining transcriptional silencing. Previously, phosphorylation of histones have been analysed during spermatogenesis. Phosphorylated histone H4S1 and H3S10 are present during meiotic divisions. H3S10 phosphorylation is hardly detectable after meiosis, whereas phosphorylation of H4S1 persists until chromatin compaction starts.
   
   
2. Histone modifications that persist from pre-meiotic stages and characterise transcriptionally active chromatin.
   
   The primary spermatocyte phase is characterised by a high level of transcriptional activity of housekeeping genes. In addition, genes are transcribed that are needed for the subsequent steps in spermatogenesis, as the majority of transcription ceases once meiotic division starts. H4 acetylation and H3K4 and H4R3 methylation of histones were investigated. These histone modifications, which are indicative of transcriptional activity, persist until histone degradation.
   
   
3. Increasing or de novo appearance of histone modifications that decrease the affinity between histones and DNA as a prelude to histone removal.
   
   It might be that H4 hyper-acetylation, as postulated for mammals and/or other secondary modifications of histones are the first step towards histone removal. The fact that these modifications are conserved between mammals and flies adds support to this hypothesis. Indeed, histone H4 acetylation is very pronounced at the canoe stage and de novo mono-ubiquitylation of histone H2A is seen in round spermatids. Both types of histone modifications are proposed to be necessary for opening the chromatin and decreasing the contact between DNA and histones. The fact that histone H2A mono-ubiquitylation vanishes before the early canoe stage, thus before the hyper-acetylation of histone H4, leads to thinking about a stepwise remodelling of the chromatin. This study proposes that these histone modifications open the chromatin, so that enzymes and regulators have access to histone-based chromatin and can induce and prepare the reorganisation of the genome in the male germline.
   

It remains to be clarified whether and how these histone modifications influence the topology of the chromatin as a prelude to histone removal as well as for Tpl^94D, Mst77F and protamine deposition. A functional approach based on analysis of mutants of histone-modifying enzymes is difficult, as all characterised histone-modifying enzymes are already active during Drosophila development or at least in spermatogonia and spermatocytes. Therefore a tissue-specific knock-out mutant would most probably exhibit arrest of spermatogenesis before meiosis, rendering it useless for experimental purposes (Rathke, 2007).

At the first glance, it might seem surprising that histones and all their modifications are removed. Instead of specifically reverting the differentially modified histones to their unmodified state, they are removed together with all histones. This might allow the paternal genome to form nucleosomes with unmodified histones after fertilisation and before zygote formation. Thus, the paternal genome starts embryogenesis with a nucleosomal chromatin lacking histone modifications (Rathke, 2007).

The data show that most of the histones are removed between the early and late canoe stage; such a process requires a loosening of contact between the histones and DNA, which in turn requires an unwinding of the chromatin structure. It is proposed that this unwinding process is facilitated by DNA nicks as they were widespread at this stage of chromatin reorganisation. Finally, Tpl^94D, UbcD6 and SUMO were also observed to accumulate in the chromatin during this process. DNA breaks, Tpl^94D, UbcD6 and SUMO were no longer detectable when protamines were fully expressed. Thus, it is proposed that all these proteins and the DNA breaks act together in an unknown manner to allow chromatin remodelling (Rathke, 2007).

The CTCF protein is present during pre-meiotic stages in the nucleus and stays associated with the chromosomes during meiosis. After meiosis, however, strong localisation to the nucleus is detected during the transition from round spermatid nuclei to the early canoe stage of spermiogenesis. It is speculated that CTCF might set borders in the chromatin for the histone modifications, which are characteristic of the canoe stage, such as acetylation and ubiquitylation. CTCF is visible for longer than histones and disappears together with active RNA polymerase II. CTCF might maintain chromatin accessibility to RNA polymerase II since a few genes are known to be transcribed at this time. In addition, transient occurrence of RNA polymerase II at the late canoe stage might require CTCF to insulate active genes from inactive ones. This idea needs to be tested in tissue-specific CTCF loss-of-function mutants; such mutants are, however, currently unavailable (Rathke, 2007).

The question of whether histone removal is dependent on a signal that monitors the start of protamine and Mst77F mRNA translation was addressed. Both histone modification and degradation are indistinguishable from the wild-type in loss-of-function mutants of Mst35Ba and Mst35Bb, the genes encoding protamine A and B, respectively. Also in nc3 mutants of Mst77F, histone removal is not disturbed. It is concluded that N-terminal tail modification of histones and histone degradation, on the one hand, and protamine deposition, on the other, are controlled by different pathways in the cell (Rathke, 2007).

In mammals, it is well known that after meiosis the nucleosomal conformation is lost. This is accompanied by the appearance of testis-specific linker histones. So far, no linker histone variants have been identified in Drosophila, but variants of H2A (H2AvD) and H3 (H3.3) are known. In mammals, histones are hyper-acetylated before being displaced from the DNA, and phosphorylation and ubiquitylation have also been proposed to occur. For Drosophila, H2A mono-ubiquitylation and a strong increase in H4 acetylation occur shortly before histone removal and degradation. In mammals, histones are replaced first by transition proteins (major types: TP1 and TP2). This study identified the high mobility group protein Tpl^94D, a first probable candidate for a functional homologue of mammalian transition proteins. In mammals, transition proteins are subsequently replaced by protamines leading to chromatin with a doughnut structure. In Drosophila, it has recently been shown that the sperm nucleus also contains protamines. Protamines A and B are encoded by two closely related protamine genes, Mst35Ba and Mst35Bb. In addition, the identification of Mst77F shows that sperm nuclei contain at least one further abundant chromatin component. Moreover, in human sperm several new putative protamines have been identified by 2D gel electrophoresis and protein sequencing. In mammals, this chromatin reorganisation is essential for male fertility. Male flies carrying the deletion protDelta38.1, where both protamines as well as three additional ORFs are removed, show severely reduced fertility (Rathke, 2007).

In summary, a step-by-step scheme is proposed for chromatin reorganisation: (1) histone modifications lead to subsequent histone removal and degradation; (2) the exposed chromatin becomes nicked, resulting in DNA breaks; (3) Tpl^94D deposition constitutes an intermediate stage that triggers subsequent protamine-based chromatin organisation (Rathke, 2007).

Since many features concerning spermiogenesis are conserved between Drosophila and mammals, it is proposed that Drosophila is an ideal system to gain further insight into the mechanism of chromatin reorganisation in spermatid nuclei, a process that is crucial for male fertility (Rathke, 2007).

The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome

Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. An integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays revealed an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and they were estimated to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106-164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from this analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which genome-wide binding data was available. It is suggested that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function (Fu, 2008).

Gene-specific repression of the p53 target gene PUMA via intragenic CTCF-Cohesin binding

The p53 transcriptional program orchestrates alternative responses to stress, including cell cycle arrest and apoptosis, but the mechanism of cell fate choice upon p53 activation is not fully understood. PUMA (p53 up-regulated modulator of apoptosis), a key mediator of p53-dependent cell death, is regulated by a noncanonical, gene-specific mechanism. Using chromatin immunoprecipitation assays, it was found that the first half of the PUMA locus (approximately 6 kb) is constitutively occupied by RNA polymerase II and general transcription factors regardless of p53 activity. Using various RNA analyses, it was found that this region is constitutively transcribed to generate a long unprocessed RNA with no known coding capacity. This permissive intragenic domain is constrained by sharp chromatin boundaries, as illustrated by histone marks of active transcription (histone H3 Lys9 trimethylation [H3K4me3] and H3K9 acetylation [H3K9Ac]) that precipitously transition into repressive marks (H3K9me3). Interestingly, the insulator protein CTCF (CCCTC-binding factor) and the Cohesin complex occupy these intragenic chromatin boundaries. CTCF knockdown leads to increased basal expression of PUMA concomitant with a reduction in chromatin boundary signatures. Importantly, derepression of PUMA upon CTCF depletion occurs without p53 activation or activation of other p53 target genes. Therefore, CTCF plays a pivotal role in dampening the p53 apoptotic response by acting as a gene-specific repressor (Gomes, 2010).

Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. This study showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function (Yao, 2010).

A number of investigations have examined the role of factors that interact with CTCF and affect its function as an insulator protein. The chromodomain helicase protein CHD8 has been shown to be important for insulation, although its mode of action is not known. Also, it has been shown that CTCF recruits the cohesin complex to its binding sites, and that the presence of cohesin is essential to insulator activity, probably because it stabilizes long-range intranuclear interactions between CTCF sites (Li, 2008; Parelho, 2008; Wendt, 2008). This study showed that the DEAD-box RNA-binding protein p68 (DDX5) interacts with CTCF both in vivo and in vitro, and that the noncoding RNA SRA, a functionally important RNA known to associate with p68, immunoprecipitates with CTCF. It was also shown that both p68 protein and SRA are necessary for the activity of CTCF as an insulator element in vivo (Yao, 2010).

The DEAD-box RNA helicase p68 and the partially homologous protein p72 are RNA-binding proteins that are involved in a wide variety of regulatory and biosynthetic functions. p68 is required for ribosome biogenesis, and its ATPase/helicase activities are important for pre-mRNA splicing and microRNA processing. Notably, p68 is an essential component of the Drosha complex. p68 also functions as a cofactor for a variety of transcriptional regulatory proteins, including ERα, the p53 tumor suppressor, and MyoD. At least some of these do not require the helicase activity of p68, and probably involve a distinct independent mechanism. In many of these cases, the active form of p68 may involve a complex with p72. Although this study focused on the role of p68, data suggest that p72 also plays a role in CTCF function. The p68/p72 protein specifically bind SRA, a functionally important RNA, and it has been shown that the coactivation of MyoD by p68 depends on the presence of SRA. SRA has also been shown to bind other proteins and modulate their activity. Since some splice variants of SRA code for protein, it is necessary to distinguish the activity of the RNA from that of the protein (Yao, 2010).

The CTCF-p68 interaction is critical to CTCF function as an enhancer-blocking insulator, as demonstrated by transient expression experiments with a reporter carrying CTCF-binding sites in which p68 is depleted by shRNA. Additionally, ChIP experiments show that p68 is present at the ICR of the human IGF2/H19 locus on chromosome 11 in HeLa cells, as well as the equivalent site on mouse chromosome 7 in MEF cells. Depletion of p68 results in an increase in IGF2 expression and a decrease in H19 expression, similar to that observed in HeLa cells upon depletion of cohesin components (Wendt, 2008). Loss of p68 also results in an increase in genomic contacts, as measured by 3C, between the endodermal enhancer and sites upstream of IGF2, consistent with loss of insulator function. The loss of p68 is not accompanied by a decrease in CTCF binding. It was also found that the binding of p68 to CTCF is RNA-dependent: Depletion of ssRNA by RNase A or down-regulation of SRA inhibited the CTCF-p68 interaction. This is similar to the behavior of the interaction between p68 and p53. It is therefore not surprising that the ability of CTCF to act as an insulator also depends on SRA. The protein SRAP did not coprecipitate with CTCF either in vitro or in vivo, suggesting that it is not involved in the CTCF-p68 interaction (Yao, 2010).

It has been reported that the Drosophila DEAD-box putative RNA helicase protein Rm62, which is homologous to p68, interacts physically with the DNA-binding insulator protein CP190 in an ssRNA-dependent manner and negatively regulates gypsy insulator function (Lei, 2006). It is striking that, in the case of Drosophila, the interacting factors are different from (CP190 rather than CTCF) and the effects are the opposite of (inhibitory rather than activating) those in vertebrates. These results hint at a common mechanism of action that has diverged (Yao, 2010).

What is the role of p68 in CTCF-dependent insulator function? In addition to its interaction with CTCF, p68 also bound to a component of the cohesin complex in vitro. Cohesin interacts with CTCF and is essential to insulator function. Previous studies have shown that loss of cohesin does not affect CTCF binding at most sites (Parelho, 2008). Similarly, depletion of p68 or its associated RNA, SRA, did not affect CTCF binding to the IGF2/H19 ICR in vivo; however, depletion of either did result in loss of cohesin from those sites, showing that the interactions observed in vitro are important in vivo. These data support a model in which cohesin binding to CTCF at the IGF2/H19 locus is further stabilized by cohesin interaction with p68/SRA. It is suggested that the effects on insulator function that were observed when p68/SRA is depleted reflect, at least in part, the loss of cohesin from the site. p68 is also found at sites occupied by ERα and cohesin. It will be interesting to determine whether, at such sites, p68 plays a role in stabilizing cohesin localization (Yao, 2010).

These results show that CTCF sites, the majority of which recruit cohesin, may require additional components to establish long-range interactions and maintain an active insulator complex. It remains to be determined whether p68/SRA, known to have multiple regulatory activities, contributes in other ways to CTCF function (Yao, 2010).

Nonallelic transcriptional roles of CTCF and cohesins at imprinted loci

The cohesin complex holds sister chromatids together and is essential for chromosome segregation. Recently, cohesins have been implicated in transcriptional regulation and insulation through genome-wide colocalization with the insulator protein CTCF, including involvement at the imprinted H19/Igf2 locus. CTCF binds to multiple imprinted loci and is required for proper imprinted expression at the H19/Igf2 locus. This study reports that cohesins colocalize with CTCF at two additional imprinted loci, the Dlk1-Dio3 and the Kcnq1/Kcnq1ot1 loci. Similar to the H19/Igf2 locus, CTCF and cohesins preferentially bind to the Gtl2 differentially methylated region (DMR) on the unmethylated maternal allele. To determine the functional importance of the binding of CTCF and cohesins at the three imprinted loci, CTCF and cohesins were depleted in mouse embryonic fibroblast cells. The monoallelic expression of imprinted genes at these three loci was maintained. However, mRNA levels for these genes were typically increased; for H19 and Igf2 the increased level of expression was independent of the CTCF-binding sites in the imprinting control region. Results of these experiments demonstrate an unappreciated role for CTCF and cohesins in the repression of imprinted genes in somatic cells (Lin, 2011).

Search PubMed for articles about Drosophila CTCF

Baniahmad, A., Steiner, C., Kohne, A. C. and Renkawitz, R. (1990). Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site. Cell 61: 505-514. Medline abstract: 2159385

Bell, A. C., West, A. G. and Felsenfeld, G. (1999). The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98: 387-396. Medline abstract: 10458613

Burcin, M., et al. (1997). Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF. Mol Cell Biol 17: 1281-1288. Medline abstract: 9032255

Burke, L. J., Hollemann, T., Pieler, T. and Renkawitz, R. (2002). Molecular cloning and expression of the chromatin insulator protein CTCF in Xenopus laevis. Mech Dev 113: 95-98. Medline abstract: 11900981

Bushey, A. M., Ramos, E. and Corces, V. G. (2009). Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions. Genes Dev. 23(11): 1338-50. PubMed Citation: 19443682

Butcher, R. D., et al. (2004). The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division. J. Cell Sci. 117: 1191-1199. Medline abstract: 14996941

Capelson, M. and Corces, V. G. (2005). The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator. Mol Cell 20: 105-116. Medline abstract: 16209949

Ciavatta, D., Rogers, S. and Magnuson, T. (2007). Drosophila CTCF is required for Fab-8 enhancer blocking activity in S2 cells. J. Mol. Biol. 373(2): 233-9. Medline abstract: 17825318

Cuddapah, S., et al. (2009). Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains. Genome Res. 19: 24-32. PubMed Citation: 19056695

Degner, S. C., et al. (2009). Cutting edge: Developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development. J. Immunol. 182: 44-48. PubMed Citation: 19109133

Filippova, G. N., et al. (2001). CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus. Nat Genet 28: 335-343. Medline abstract: 11479593

Fu, Y., Sinha, M., Peterson, C. L. and Weng, Z. (2008). The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome. PLoS Genet. 4(7): e1000138. PubMed Citation: 18654629

Gomes, N. P. and Espinosa, J. M. (2010). Gene-specific repression of the p53 target gene PUMA via intragenic CTCF-Cohesin binding. Genes Dev. 24(10): 1022-34. PubMed Citation: 20478995

Hark, A. T., Schoenherr, C. J., Katz, D. J., Ingram, R. S., Levorse, J. M. and Tilghman, S. M. (2000). CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405: 486-489. Medline abstract: 10839547

Holohan, E. E., et al.. (2007). CTCF genomic binding sites in Drosophila and the Organisation of the Bithorax Complex. PLoS Genet. 3(7): e112. Medline abstract: 17616980

Kanduri, C., et al. (2000). Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive. Curr. Biol. 10: 853-856. Medline abstract: 10899010

Kim, T. H., et al. (2007). Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128: 1231-1245. Medline abstract: 17382889

Kuhn, E. J., Viering, M. M., Rhodes, K. M. and Geyer, P. K. (2003). A test of insulator interactions in Drosophila. EMBO J 22: 2463-2471. Medline abstract: 12743040

Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, et al. (2006) CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc. Natl. Acad. Sci. 103: 10684-10689. Medline abstract: 16815976

Kyrchanova, O., Toshchakov, S., Podstreshnaya, Y., Parshikov, A. and Georgiev, P. (2008). Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene. Mol. Cell. Biol. 28(12): 4188-95. PubMed Citation: 18426914

Lanzuolo, C., et al. (2007). Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex. Nat. Cell Biol. 9: 1167-1174. PubMed Citation: 17828248

Lee, J. T. (2003). Molecular links between X-inactivation and autosomal imprinting: X-inactivation as a driving force for the evolution of imprinting? Curr. Biol. 13: R242-R254. Medline abstract: 12646153

Lei, E. P. and Corces, V. G (2006). RNA interference machinery influences the nuclear organization of a chromatin insulator. Nat. Genet. 38: 936-941. Medline abstract: 16862159

Lei, E. P. and Corces, V. G. (2006). RNA interference machinery influences the nuclear organization of a chromatin insulator. Nat. Genet. 38: 936-941. PubMed Citation: 16862159

Lewis, A. and Murrell, A. (2004). Genomic imprinting: CTCF protects the boundaries. Curr. Biol. 14: R284-R286. Medline abstract: 15062124

Li, M., Belozerov, V. E. and Cai, H. N. (2009). Analysis of chromatin boundary activity in Drosophila cells. BMC Mol. Biol. 9: 109. PubMed Citation: 19077248

Li, T., et al. (2008). CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop. Mol Cell Biol 28: 6473-6482. PubMed Citation: 18662993

Lin, S., et al. (2011). Nonallelic transcriptional roles of CTCF and cohesins at imprinted loci. Mol. Cell Biol. 31(15): 3094-104. PubMed Citation: 21628529

Lobanenkov VV, Nicolas RH, Adler VV, Paterson H, Klenova EM, Polotskaja AV, Goodwin GH. (1990). A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene. Oncogene 5: 1743-1753. Medline abstract: 2284094

Loukinov, D. I., et al. (2002). BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc. Natl. Acad. Sci. 99: 6806-6811. Medline abstract: 12011441

Lutz M et al. (2000). Transcriptional repression by the insulator protein CTCF involves histone deacetylases. Nucleic Acids Res 28: 1707-1713. Medline abstract: 10734189

Mohan, M., et al. (2007). The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. EMBO J. 26(19): 4203-14. Medline abstract: 17805343

Moon, H., et al. (2005). CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator. EMBO Rep. 6(2): 165-70. Medline abstract: 15678159

Moshkovich, N., et al. (2011). RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function. Genes Dev. 25(16): 1686-701. PubMed Citation: 21852534

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Ohlsson R, Renkawitz R, Lobanenkov V. (2001). CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet 17: 520-527. Medline abstract: 11525835

Pai, C. Y., Lei, E. P., Ghosh, D. and Corces, V. G. (2004). The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol. Cell 16: 737-748. Medline abstract: 15574329

Parelho, V., et al. (2008). Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132: 422-433. PubMed Citation: 18237772

Rathke, C., et al. (2007). Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila. J. Cell Sci. 120(Pt 9): 1689-700. Medline abstract: 17452629

Splinter, E., et al. (2006) CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus. Genes Dev 20: 2349-2354. Medline abstract: 16951251

Szabo, P., Tang, S. H., Rentsendorj, A., Pfeifer, G. P. and Mann, J. R. (2000). Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function. Curr Biol 10: 607-610. Medline abstract: 10837224

Tanimoto, K., Sugiura, A., Omori, A., Felsenfeld, G., Engel, J. D. and Fukamizu, A. (2003). Human β-globin locus control region HS5 contains CTCF- and developmental stage-dependent enhancer-blocking activity in erythroid cells. Mol Cell Biol 23: 8946-8952. Medline abstract: 14645507

Vostrov, A. A. and Quitschke, W. W. (1997). The zinc finger protein CTCF binds to the APBß domain of the amyloid β-protein precursor promoter. Evidence for a role in transcriptional activation. J. Biol. Chem. 272: 33353-33359. Medline abstract: 9407128

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Xie. X., et al. (2007). Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites. Proc. Natl. Acad. Sci. 104: 7145-7150. Medline abstract: 17442748

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date revised: 15 December 2011

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