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).

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).

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).

Search PubMed for articles about Drosophila CTCF

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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

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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

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

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

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

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

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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

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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

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

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

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

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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

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

Yusufzai, T. M., Tagami, H., Nakatani, Y. and Felsenfeld, G. (2004). CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. Mol Cell 13: 291-298. Medline abstract: 14759373

Zhang, R., Burke, L. J., Rasko, J. E., Lobanenkov, V. and Renkawitz, R. (2004). Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody. Exp. Cell Res. 294: 86-93. Medline abstract: 14980504

Zhao, H. and Dean, A. (2004). An insulator blocks spreading of histone acetylation and interferes with RNA polymerase II transfer between an enhancer and gene. Nucleic Acids Res 32: 4903-4919. Medline abstract: 15371553

date revised: 25 August 2009

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