InteractiveFly: GeneBrief

Boundary element-associated factor of 32kD: Biological Overview | References

The Drosophila BEAF-32A and BEAF-32B proteins bind to the heat shock factor scs' insulator and to hundreds of other sites on Drosophila chromosomes. These two proteins are encoded by the same gene. Ends-in homologous recombination was used to generate the null BEAF^AB-KO allele, and the BEAF^A-KO allele that eliminates production of only the BEAF-32A protein was also isolated. The BEAF proteins together were found to be essential, but BEAF-32B alone is sufficient to obtain viable flies. The results show that BEAF is important for both oogenesis and development. maternal or zygotic BEAF is sufficient to obtain adults, although having only maternal BEAF impairs female fertility. In the absence of all BEAF, a few fertile but sickly males are obtained. Using both a chromosomal position-effect assay and an enhancer-blocking assay, it was found that BEAF is necessary for scs' insulator function. Lack of BEAF causes a disruption of male X polytene chromosome morphology. However, no evidence was found that dosage compensation was affected. Position-effect variegation of the w^m4h allele and different variegating y transgenes was enhanced by the knockout mutation. Combined with the effects on male X polytene chromosomes, it is concluded that BEAF function affects chromatin structure or dynamics (Roy, 2007b).

Enhancers can act over large distances and are capable of activating transcription from diverse promoters. Chromatin domain insulators are thought to help prevent promiscuous interactions between enhancers and promoters by dividing chromosomes into domains such that interactions can occur within domains but cannot occur between elements located in different domains. Perhaps the best-known example that illustrates the importance of insulators is the imprinted mammalian insulator downstream of the Igf2 gene. This insulator is not methylated on the maternal chromosome, allowing binding of the CTCF protein (see Drosophila CTCF), which blocks activation of Igf2 by a downstream enhancer. The insulator is methylated on the paternal chromosome, thus preventing binding by CTCF and allowing activation of Igf2 by the downstream enhancer. Inactivation of the insulator on both chromosomes can lead to Beckwith-Wiedemann fetal overgrowth syndrome and the development of Wilms' tumor. In Drosophila, deletion of the Fab-7 insulator in the bithorax complex leads to homeotic transformation of adult abdominal segment 6 (AS6) into another copy of the more posterior AS7 (Roy, 2007b and references therein).

There are differences between insulators in certain assays, indicating that different molecular mechanisms can result in insulator activity. In addition, some insulators are composite elements with separate components responsible for blocking enhancer-promoter communication and for acting as a barrier against chromosomal position effects. It is not clear how any insulator functions at the molecular level. The various models that have been proposed include acting as promoter decoys, influencing chromatin structure or dynamics, and nuclear organization. These models are not mutually exclusive. To understand how insulators function, it is necessary to study the proteins involved in insulator activity (Roy, 2007b).

The two 32-kDa Drosophila boundary element-associated factors, BEAF-32A and BEAF-32B. These proteins are referred to together as 'BEAF' and individually as '32A' or '32B.' BEAF binds to the scs' insulator as well as to hundreds of other sites on chromosomes (Zhao, 1995; Hart, 1997). A few other genomic BEAF-binding sites have been identified, and they function as insulators in transgenic fly assays (Cuvier, 1998). This suggests that BEAF-dependent insulators are a common class of insulator in Drosophila. 32A and 32B are derived from the same gene. They have unique amino-terminal DNA-binding domains of ~80 amino acids, but the remaining 200 amino acids are encoded by a shared exon. BEAF forms complexes with itself, and this is mediated by a region near the carboxy-terminus (Hart, 1997). Because there were no mutations available in the BEAF gene, a transgene under GAL4 UAS control was designed that encodes a dominant-negative BEAF protein (Gilbert, 2006). The current study expands on that work by generating and characterizing mutations in the BEAF gene (Roy, 2007b).

Ends-in homologous recombination was used to generate a knockout mutation in the BEAF gene (BEAF^AB-KO). In the process, an allele was isolated that eliminates the ability to produce the 32A protein (BEAF^A-KO). It was found that the 32B protein is sufficient to obtain healthy, viable flies. In contrast, eliminating both BEAF proteins reveals that BEAF is essential. Both oogenesis and development are affected by a lack of BEAF. BEAF was shown to be required for the insulator activity of scs', but not for the scs insulator (which binds the Zw5 protein) or the gypsy insulator [which binds the su(Hw) protein. Evidence is provided that BEAF function affects chromatin. This confirms and extends results obtained with the dominant-negative BEAF protein and supports the hypothesis that BEAF functions by affecting chromatin structure or dynamics (Roy, 2007b).

Flies homozygous for the BEAF^A-KO allele are healthy and viable, indicating that the 32B protein is sufficient for normal development. In contrast, flies homozygous for the BEAF^AB-KO allele cannot be maintained as a stable line. Maternal BEAF is sufficient to obtain fertile adults, although the resulting BEAF^AB-KO flies eclose 1-2 days later than their BEAF^AB-KO/CyO siblings and are sickly. Also, although equal numbers of males and females are obtained, the fertility of the BEAF^AB-KO females is compromised. Crosses with these females demonstrated that zygotic BEAF is also sufficient to obtain equal numbers of fertile males and females, and fertile males can be obtained even in the absence of BEAF. However, in the absence of maternal BEAF, less than half of the embryos hatch and there is a drastic reduction in the number of adults obtained. The absence of all BEAF results in female lethality by the pharate adult stage or shortly after eclosing. In addition, driving expression of a transgene encoding a dominant-negative form of BEAF by daughterless-GAL4 leads to embryonic lethality (Gilbert, 2006). Thus BEAF plays an important role during development, particularly in females, although sickly adults can be obtained that lack BEAF (Roy, 2007b).

A number of different phenotypes were observed in ovaries from BEAF^AB-KO flies, although ovaries from a given BEAF^AB-KO female normally exhibited only one phenotype. It is concluded that BEAF plays an important role during oogenesis as well as during development. While the defects in oogenesis could be due to deregulation of genes in the absence of BEAF, it could also be at least partly related to the genetic interaction that found between BEAF and spindle-E (spn-E) (Roy, 2007a). The protein encoded by spn-E is a helicase subunit of an RNA interference complex that plays a role in oogenesis. It is of interest to note that a genetic interaction between the RNAi machinery and gypsy insulator function has been reported and that the su(Hw) insulator protein also plays a role in oogenesis. In addition, the JIL-1 histone H3 kinase plays a role in modulating chromatin structure and is essential at all stages of development as well as for oogenesis (Roy, 2007b).

The scs' insulator was originally identified because it forms a special chromatin structure that appeared to localize to one end of the heat-shock puff at 87A of polytene chromosomes. It was subsequently shown to function as an insulator in the first transgenic enhancer-blocking and position-independent expression assays to be done. This led to the identification of the BEAF proteins as scs'-binding proteins (Zhao, 1995; Hart, 1997). The importance of the BEAF-binding sites in scs' for insulator activity has been shown using both cultured cells (Zhao, 1995) and transgenic flies (Cuvier, 1998), and additional genomic BEAF-binding sites were shown to have insulator activity (Cuvier, 1998). However, it is possible that some other protein binds to these sites in vivo to confer insulator activity. It was also shown that a dominant-negative form of BEAF interferes with scs' insulator activity (Gilbert, 2006), although this protein might affect proteins in addition to BEAF. This study shows that BEAF is required for the insulator activity of scs'. Using both a position-independent expression assay and an enhancer-blocking assay, it was found that scs' loses insulator activity in the absence of BEAF protein. In the enhancer-blocking assay, the scs and gypsy insulators were tested; these lack BEAF-binding sites, and it was found that these insulators work in the absence of BEAF (Roy, 2007b).

The altered appearance of the X polytene chromosome in BEAF^AB-KO male mutant larvae provides dramatic evidence for a role for BEAF in chromatin organization. This is further supported by the PEV assays, which indicate that BEAF helps to limit heterochromatin spreading. Mutations in genes encoding other chromatin proteins have a similar effect on the male X chromosome. This includes ISWI, the catalytic subunit of multiple chromatin-remodeling complexes, including the nucleosome remodeling factor (NURF); the NURF301 subunit of NURF and the heterochromatin proteins Su(var)3-7 and HP1. This supports models in which insulators function by affecting chromatin structure or dynamics (Roy, 2007b).

It is curious that only the male X chromosome is affected, whereas global structural alterations are observed in all chromosomes of males and females when a dominant-negative form of BEAF is produced in larval salivary glands (Gilbert, 2006). It is likely that the chromatin organization of the male X chromosome is especially susceptible to disruption due to some feature associated with dosage compensation. A candidate for such a feature is the hyperacetylation of lysine 16 of histone H4, which interferes with formation of 30-nm chromatin fibers (Shogren-Knaak, 2006). Evidence that the male X chromosome is more sensitive to disruption is derived from mutations in the histone H3 kinase, JIL-1. When polytene chromosomes were observed using an allelic series of JIL-1 mutations, weak mutations were found to affect mainly the male X chromosome and stronger mutations to affect all chromosomes of both males and females. Also, BEAF^A-KO animals are healthier than BEAF^AB-KO animals, and a weaker effect on the male X chromosome was observed in BEAF^A-KO animals. This suggests that the dominant negative has a stronger effect than the lack of BEAF. This is consistent with the lethal effect of producing the dominant-negative protein in embryos, whereas homozygous BEAF^AB-KO adults are obtained. It is assumed that the dominant negative has a stronger effect because it actively interferes with BEAF activity, while the gradual disappearance of maternal BEAF mitigates the effect of the knockout. Perhaps the dominant-negative protein also interferes with the function of proteins in addition to BEAF. If so, it is likely that these proteins normally interact with BEAF since the phenotypes caused by the dominant negative and by BEAF^AB-KO are similar and can be rescued by BEAF transgenes. The future identification of any such proteins should provide insight into how BEAF functions (Roy, 2007b).

This study has shown that the BEAF proteins have insulator activity. BEAF binds to hundreds of sites on polytene chromosomes (Zhao, 1995), and other genomic binding sites have insulator activity (Cuvier, 1998; Cuvier, 2002). Yet 32A is not essential, adults can be obtained with only maternal BEAF, some embryos hatch with only zygotic BEAF, and a small number of fertile males are obtained in the absence of all BEAF. This is somewhat reminiscent of mutations in the su(Hw) insulator protein, which lead to female sterility but otherwise are not lethal. BEAF is normally present at all life stages. Using several BEAF-EGFP fusion gene (GFBF) transgenic fly lines in the BEAF^AB-KO background, in which the transgene is insulated and driven by 900 bp of BEAF promoter sequences, green fluorescent BEAF was observed in all nuclei of all tissues at all life stages that were examined. If BEAF is normally ubiquitous and contributes to gene regulation by forming boundaries between hundreds of domains, why are the effects of a lack of BEAF so limited? The answer is not known at present. One possibility is that the misregulation of genes caused by malfunctioning insulators is minor enough that fitness is reduced without being immediately lethal. Another possibility that is particularly intriguing is that there could be some type of epigenetic memory mechanism, similar to what has been proposed for Polycomb group proteins. This epigenetic memory has been shown to be meiotically inheritable. Loss of this 'epigenetic memory' could be stochastic, resulting in deregulation of different genes in different individuals or clonal populations of cells. This could result in the variable timing of death in the absence of BEAF and in the single phenotype observed per ovary but different phenotypes in different ovaries. The knockout mutations described in this study will be useful tools in future studies aimed at discovering proteins that interact with BEAF and for investigating the role of BEAF in gene regulation and chromatin organization. This will ultimately lead to an understanding of the molecular mechanisms used in insulator function (Roy, 2007b).

The Drosophila Boundary Element-Associated Factors BEAF-32A and BEAF-32B affect chromatin structure

Binding sites for the Drosophila BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, a transgene was designed encoding a dominant-negative form of BEAF under GAL4 UAS control. This BID protein encompasses the BEAF self-interaction domain. Evidence is provided that BID interacts with BEAF and interferes with scs' insulator activity and that BEAF is the major target of BID in vivo. BID expression during embryogenesis is lethal, implying that BEAF is required during early development. Expression of BID in eye imaginal discs leads to a rough-eye phenotype, and this phenotype is rescued by a third copy of the BEAF gene. Expression of BID in salivary glands leads to a global disruption of polytene chromatin structure, and this disruption is largely rescued by an extra copy of BEAF. BID expression also enhances position-effect variegation (PEV) of the w^m4h allele and a yellow transgene inserted into the pericentric heterochromatin of chromosome 2R, while a third copy of the BEAF gene suppresses PEV of both genes. These results support the hypothesis that BEAF-dependent insulators function by affecting chromatin structure or dynamics (Roy, 2007a).

BEAF-32A and -32B are 32-kDa proteins derived from the same gene (Hart, 1997). They differ at their amino termini, which have different BED finger DNA-binding domains (Aravind, 2000). The carboxy-terminal two-thirds of these proteins is identical. A BESS domain is found near the carboxy termini (Bhaskar, 2002; Delattre, 2002) and is preceded by a potential leucine zipper domain. BEAF monomers interact with each other, presumably via interactions between BESS domains or leucine zippers or both. Evidence suggests that BEAF binds DNA as trimers, although larger complexes could also be involved (Hart, 1997). No other proteins copurify with BEAF, indicating that BEAF forms only stable complexes with itself (Roy, 2007a).

To gain insight into the role of the BEAF proteins in Drosophila, a transgene was constructed encoding the BEAF self-interaction domain (BID) but lacking a DNA-binding domain. This design is based on the Drosophila Emc and vertebrate Id proteins. These proteins lack DNA-binding domains and so inhibit DNA binding by their partner transcription factors by forming dimers that lack one DNA-binding domain. The BID protein should similarly inhibit DNA binding by BEAF. The BID transgene is under GAL4 UAS control, allowing expression to be driven in different patterns by different GAL4 driver fly lines. This study demonstrates that the BID protein inhibits BEAF activity and provide evidence that BEAF function influences chromatin structure or dynamics (Roy, 2007a).

Co-immunoprecipitation experiments show that the BID protein physically interacts with BEAF in vivo, and immunostaining shows that it removes BEAF from polytene chromosomes. Adding a third copy of the BEAF gene rescues the BID-associated rough-eye phenotype and disruption of polytene chromosome structure. Furthermore, BID interferes with scs' insulator function in both a position-independent expression and an enhancer-blocking assay. It is concluded that BID interferes with BEAF function by reducing the level of chromatin-associated BEAF (Roy, 2007a).

Could interactions between BID and proteins other than BEAF account for the effects of BID? No proteins copurify with BEAF, indicating that BEAF does not form stable complexes with other proteins. However, interactions between BEAF and other proteins have been reported. D1 is an abundant chromosomal protein that resembles mammalian HMGA (formerly HMG-I) proteins, except it is larger. Whereas mammalian HMGA proteins have 3 AT-hook domains, D1 has 10 (at least 6 of which should be functional). Although D1 predominantly binds to AT-rich satellite DNA sequences, it can cooperatively bind to certain DNA sequences with BEAF (Cuvier, 2002). The potential role of this in the effect of BEAF on PEV of the w^m4h and KV20 y alleles is discussed below. Another protein that interacts with BEAF is Zw5 (Blanton, 2003), a protein that binds to the scs insulator (Gaszner, 1999). This interaction could account for the apparent weak effect of BID on scs insulator activity in the enhancer-blocking assay. A protein interaction map derived from a high-throughput yeast two-hybrid screen identified five proteins that can interact with BEAF. Four of these proteins are encoded by conceptual genes, and no functional information is available. The fifth protein is katanin-60, the catalytic component of a microtubule-severing complex. The two-hybrid screen did not identify D1 or Zw5, and it is unknown if BEAF interacts with any of these five proteins in vivo. The possibility that interactions with these or other proteins contribute to the effects of the BID protein cannot be formally ruled out. But the effect of BID on the activity of the scs' insulator, the lack of effect on the gypsy insulator, the minimal effect on the scs insulator, and the rescue of the rough-eye and polytene chromosome phenotypes by a third copy of the BEAF gene suggest that BEAF is the major target of BID (Roy, 2007a).

Ubiquitous expression of BID during embryogenesis is lethal, indicating that the BEAF proteins are essential during development. It was previously shown that expression of a BEAF-32A transgene in eye imaginal discs led to a rough-eye phenotype associated with increased apoptosis (Yamaguchi, 2001). Overproduction of 32A should affect the function of insulators that require 32B DNA-binding activity, but not those that require only 32A. The BID protein should affect all BEAF-dependent insulators. On the basis of the proposed role of BEAF in insulator function, it is hypothesized that many genes are misregulated when BEAF insulator function is perturbed. This misregulation could be due in part to the transcription factor DREF. Originally proposed to regulate DNA-replication-related genes, it has more recently been proposed that DREF functions as part of a core promoter selectivity factor for TRF2-utilizing promoters. There is evidence that BEAF and DREF compete for binding to certain DNA sequences (Hart, 1999); removing BEAF would facilitate binding by DREF to these sites. It is proposed that a breakdown in gene regulation disrupts the developmental program in the developing eye, resulting in a rough-eye phenotype. In the developing embryo, this breakdown is lethal (Roy, 2007a).

BEAF and the D1 protein can cooperatively bind to DNA (Cuvier, 2002). However, their patterns of immunolocalization on polytene chromosomes are largely distinct. D1 binds an AT-rich sequence and largely immunolocalizes to heterochromatin, especially the AT-rich 1.672 and 1.688 g/cm³ satellites. These satellites are found in the pericentromeric heterochromatin of the X and Y chromosomes and of chromosome 4. BEAF binds to several hundred sites in euchromatin (Zhao, 1995). Despite their largely distinct chromosomal distributions, BEAF and D1 likely interact at the bases of the X, 2L, and 2R chromosome arms, where several hundred dispersed copies of a sequence (BE28) that has both BEAF- and D1-binding sites are found (Cuvier, 1998; Cuvier, 2002). The w^m4h gene is located near the base of the X chromosome, and the 1.688 g/cm³ satellite is a component of the pericentromeric heterochromatin in this region. Interfering with D1 function suppresses w^m4h variegation. The BE28 repeats could be locations where BEAF and D1 normally interact to create a transition zone that is checkered with heterochromatin and euchromatin islands. Perhaps BID enhances the PEV of w^m4h by allowing D1-associated heterochromatin to spread farther, while extra BEAF blocks the spread. This could also occur for the KV20 y transgene, although 2R does not have high concentrations of the 1.672 or 1.688 g/cm³ satellites. Alternative possibilities include direct suppression of variegation by BEAF by some other currently unknown mechanism, or indirect suppression by affecting the activity or gene expression of other chromatin proteins that directly affect variegation (Roy, 2007a).

The mechanism leading to disruption of polytene chromosome structure by BID is not known. It is possible that the D1 protein is involved, although as pointed out above, D1 is mainly associated with satellite heterochromatin and BEAF is mainly found on euchromatin. Furthermore, the chromosomes look puffy, not condensed like heterochromatin. It is possible that underreplication of the chromosomes could be involved, but that cannot account for the loss of banding patterns. Also, no effect on replication was apparent in examination of mitotic figures in larval brain squashes. It has been shown in vertebrates and yeast that covalent histone modifications can differ on either side of insulators or barrier elements. Perhaps impairing BEAF function allows these modifications to spread farther in a stochastic manner. Then individual chromosomes in the polytene bundle could have different patterns of histone modifications over the same sequences, causing a loss of banding and coherence between chromosomes. Similar phenotypes are observed in the presence of mutations known to affect proteins that act on chromatin. Examples include the JIL-1 histone H3 Ser10 kinase, the chromatin-remodeling factor ISWI, SU(VAR)2-10, and the Z4 interband-specific protein. In all cases, the cause of the loss of polytene chromosome morphology remains unknown (Roy, 2007a and references therein).

Some models propose that insulators limit communication between regulatory elements and promoters located in different domains by affecting chromatin structure or dynamics. Inhibiting the ability of BEAF to associate with chromatin leads to a global disruption of polytene chromosome structure and enhances PEV of the w^m4h and KV20 y alleles. These results provide strong support for a role of chromatin structure or dynamics in BEAF-dependent insulator function (Roy, 2007a).

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 (See Drosophila SUMO) 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).

Genome-wide mapping of Boundary Element-Associated Factor (BEAF) binding sites in Drosophila melanogaster links BEAF to transcription

Insulator elements play a role in gene regulation that is potentially linked to nuclear organization. Boundary element-associated factors (BEAFs) 32A and 32B associate with hundreds of sites on Drosophila polytene chromosomes. DNA isolated by chromatin immunoprecipitation has been hybridized to genome tiling microarrays to construct a genome-wide map of BEAF binding locations. A distinct difference in the association of 32A and 32B with chromatin was noted. 1,820 BEAF peaks were identified and it was found that more than 85% were less than 300 bp from transcription start sites. Half are between head-to-head gene pairs. BEAF-associated genes are transcriptionally active as judged by the presence of RNA polymerase II, dimethylated histone H3 K4, and the alternative histone H3.3. Forty percent of these genes are also associated with the polymerase negative elongation factor NELF. Like NELF-associated genes, most BEAF-associated genes are highly expressed. Using quantitative reverse transcription-PCR, it was found that the expression levels of most BEAF-associated genes decrease in embryos and cultured cells lacking BEAF. These results provide an unexpected link between BEAF and transcription, suggesting that BEAF plays a role in maintaining most associated promoter regions in an environment that facilitates high transcription levels (Jiang, 2009).

Using three different antibodies to perform ChIP-chip, BEAF was localized to 1,820 regions in the Drosophila genome. This is in agreement with the broad distribution seen by immunostaining polytene chromosomes. There was a clear difference between the association of 32A and 32B with chromosomes. 32B gave robust peaks, while 32A gave smaller peaks. By peak selection criteria, only about 40% of the regions with 32B peaks also have 32A peaks. In contrast, more than 95% of the regions with 32A peaks also have 32B peaks. The dominant role of 32B in binding to chromosomes is consistent with results showing that flies producing only the 32B protein are viable but flies lacking both forms of BEAF are not. Also, the BEAF^AB-KO null mutation can be rescued with a 32B transgene but not with a 32A transgene, yet 32A is presumably performing an important function. Both 32A and 32B are highly conserved in all 12 sequenced Drosophila species, representing more than 40 million years of evolution (Jiang, 2009).

In addition to the peaks that were counted as genuine BEAF binding regions, there are other, lower, peaks with an false discovery rate (FDR) of less than 5% that are present in three or four of the data sets. An example of this is found at scs. BEAF has been reported to indirectly associate with scs by interactions with Zw5, which directly binds scs. An intriguing possibility for future investigation is that these peaks represent interactions between BEAF and other chromatin-associated proteins such as Zw5. Although the number of these peaks was not tabulated, there are certainly hundreds of them in the data. If they indeed represent the formation of chromatin loops by heterologous interactions, then investigating this phenomenon will provide valuable insight into nuclear organization (Jiang, 2009).

It was confirmed that BEAF binds to the identified regions by a combination of PCR and EMSA experiments. In agreement with previous footprinting, CGATA mutagenesis, and dual-core results, the results support the view that clusters of CGATA motifs (for 32B) and CGTGA motifs (for 32A) play a role in binding at some sites. However, they also indicate that this view is too simplistic. While 32B appears to have a preference for two of three CGATA motifs being arranged as + - inverted repeats, binding sites rarely look like the two in scs' and no rules relating spacing and orientations of motifs to binding affinity in single sites emerged. Only about 25% of 1,720 dual cores correspond to the BEAF peaks described in this study. The FlyEnhancer program was used with the more stringent definition for a single potential 32B binding element of three CGATA elements in a 100-bp window, with a DRE counted as a single CGATA, and more than 2,800 clusters were found in the Drosophila genome. Most of these sites are not included in the set of BEAF peaks that met the current selection criteria. The lack of strong binding of BEAF to these regions suggests that these motif clusters are not organized properly or are, for some reason, inaccessible to BEAF. In addition, examination of peak sequences indicates that these motifs are not necessary for binding by BEAF. Other, unknown, sequence features must play a role at many sites. Using the MEME programs did not help identify consensus sequences. Presumably, this is because BEAF binds to short motifs with variable spacing and orientations between motifs, rather than a long, contiguous sequence. Refining models of BEAF binding sites so that they can be identified by inspection of DNA sequences will require performing additional experiments such as footprinting assays and perhaps identification of partner proteins (Jiang, 2009).

The centers of BEAF peaks show a striking clustering near annotated TSSs. In addition, about half of the peaks are between head-to-head gene pairs so that the 1,820 peak centers are located within -500 bp to +200 bp of the TSSs of 2,305 genes. The scs' insulator is one example of a head-to-head gene pair associated with a BEAF peak. It has two BEAF binding sites, one near each TSS. One is a high-affinity binding site that gives a prominent shift, and the other is a low-affinity binding site that gives a weak shift under the EMSA conditions that were used. This could be a common theme. More than half of the 434 dual-core regions that correspond to BEAF peaks are between head-to-head gene pairs, suggesting that there are BEAF binding sites by both TSSs of these gene pairs. This possibility cannot be evaluated outside of the context of dual cores at this time because BEAF binding sites cannot be unambiguously identified based on DNA sequence (Jiang, 2009).

A comparison with published data indicates that the majority of BEAF-associated genes are transcriptionally active or poised for activation and are highly expressed. One concern is that the Pol II, histone, and gene expression data came from a variety of tissues and cultured cells. However, the results consistently indicate that BEAF-associated genes are active. In fact, 70% of the BEAF-associated genes identified are in the upper half of the genes ranked by expression levels in both brain and testis. This suggests that most BEAF-associated genes are expressed at high levels in a wide range of tissues, perhaps even ubiquitously. Based on this, BEAF is likely to constitutively bind to most sites, as has been reported for Su(Hw). Therefore, the comparisons of these data to the BEAF peak data are likely to be relevant. This linkage of BEAF, TSSs, and high expression levels was not anticipated (Jiang, 2009).

In support of the link between BEAF and transcription, RT-PCR results with the BEAF^AB-KO null mutation and siRNA in cultured cells indicate that BEAF is important for most BEAF-associated genes to maintain their expression levels. In the absence of BEAF, expression levels typically drop two- to fourfold. An exception to this appears to be activation of BEAF-associated genes regulated by DREF. However, the results with BEAF^AB-KO embryos are not as clear on this point as previous results obtained with siRNA in cultured cells and the production of a dominant-negative form of BEAF in embryos. Perhaps prolonged growth without BEAF in the BEAF^AB-KO embryos allowed repressive effects to dominate. This would be consistent with BEAF helping to keep promoter regions in an open configuration even at promoters where it competes with DREF (Jiang, 2009).

The location of BEAF near TSSs of active genes is reminiscent of results recently reported for NELF, the negative regulator of elongation by Pol II. 40% of BEAF-associated genes were also associated with NELF. Data indicate that NELF plays a role in pausing by Pol II and that this stimulates transcription levels by inhibiting promoter-proximal nucleosome assembly. Compared to the genome as a whole, NELF-associated genes are nearly threefold enriched for genes with paused Pol II as opposed to active Pol II in both Toll^10b embryos and S2 cells. In contrast, BEAF-associated genes are about 1.1-fold enriched for genes with active Pol II as opposed to paused Pol II in both Toll^10b embryos and S2 cells. This suggests that BEAF and NELF are functionally distinct. Perhaps their colocalization at a large number of genes provides complementary mechanisms of ensuring that the promoters of those genes are accessible to Pol II (Jiang, 2009).

Comparison of the data for BEAF with data for the Su(Hw) and CTCF insulator proteins indicates that BEAF does not colocalize with Su(Hw) and rarely colocalizes with CTCF. In fact, BEAF mainly localizes near TSSs while Su(Hw) and CTCF are usually found kilobases away from TSSs. In light of the possibility that the numerous minor BEAF peaks that were not included in this analysis might represent indirect interactions of BEAF with DNA via interactions with other proteins, minor peaks in the Adh region were checked to see if they colocalized with Su(Hw) (60 peaks) or CTCF (18 peaks). Of 18 minor BEAF peaks in this region, 4 were within 1 kb of CTCF peaks. This indicates that BEAF and Su(Hw) do not physically interact. The results for CTCF are ambiguous, indicating that BEAF and CTCF might interact at a minor subset of CTCF binding sites. However, it is clear that if minor BEAF peaks represent interactions with other DNA binding proteins, neither Su(Hw) nor CTCF is a major target (Jiang, 2009).

While it has been known for some time that transcripts emanate from heat shock factor boundary element scs', the relationship between this and BEAF binding is unknown. The results indicate that BEAF normally binds near TSSs, suggesting that BEAF is performing the same function at scs' as it is at the majority of its sites of association. In fact, like scs', many BEAF binding regions are between closely spaced head-to-head gene pairs. The data are not consistent with the model in which BEAF insulates these adjacent promoter regions from each other. Instead, the data suggest that BEAF helps to maintain most associated promoter regions in an environment that facilitates transcription. Insulator activity in transgene assays might be a consequence of this local open chromatin configuration. This is similar to the promoter decoy model of insulator function proposed by Geyer (1997). The accessible BEAF-associated promoter might trap upstream regulatory elements so that they do not affect downstream reporter transgenes. There are a variety of possible mechanisms that could be involved by which BEAF might affect promoter accessibility by positively or negatively influencing nucleosome modifications, structure, or positioning. Another interesting possibility is related to the report that nuclear matrix preparations retain 25% of BEAF. Both Su(Hw) and CTCF have also been reported to be retained in nuclear matrix preparations, leading to the proposal that they function in part by organizing chromatin into loop domains. It is possible that BEAF also organizes chromatin loop domains, but given its proximity to TSSs and relationship with gene expression, perhaps the nuclear matrix association is caused by BEAF-mediated targeting of promoters to transcription factories (Jiang, 2009).

If BEAF is performing the same function at scs' as at other sites, why did the expression levels of the genes in scs' remain the same in the absence of BEAF? scs' localizes to one end of an hsp70 domain, and recent results indicate that heat shock leads to a rapid, transcription-independent loss of nucleosomes over this domain that stops at scs'. Depletion of BEAF by siRNA did not allow nucleosome loss to spread further, indicating that BEAF is not directly responsible for blocking the heat shock-induced nucleosome loss. Perhaps the promoters in scs' are responsible. The ability to rapidly lose nucleosomes suggests that the chromatin of the hsp70 domain is readily accessible. According to this reasoning, BEAF might then be redundant for keeping the promoters in scs' in an open configuration. One way to test this would be to determine if transcripts initiate from scs' in a transgenic context and, if so, if these transcript levels drop in the absence of BEAF (Jiang, 2009).

The results presented in this study link BEAF to TSSs and highly expressed genes. They also provide the suggestion that interactions between BEAF and other chromatin-bound proteins could be widespread and contribute to nuclear organization. Future studies aimed at elucidating the relationships between BEAF and transcription and between BEAF and nuclear organization and the different roles of 32A and 32B will provide valuable insight into nuclear function (Jiang, 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).

The DRE motif is a key component in the expression regulation of the importin-β encoding Ketel gene in Drosophila

Importin-β, encoded by the Ketel gene in Drosophila, is a key component of nuclear protein import, the formation of the spindle microtubules and the assembly of the nuclear envelope. The Drosophila embryos rely on the maternal importin-β dowry at the beginning of their life. Expression of the zygotic Ketel gene commences during gastrulation in every cell and while the expression is maintained in the mitotically active diploid cells it ceases in the non-dividing larval cells in which nuclear protein import is assured by the long persisting importin-β molecules. How is the expression of the Ketel gene regulated? In silico analysis revealed several conserved transcription factor binding sequences in the Ketel gene promoter. Reporter genes in which different segments of the promoter ensured transient expression of the luciferase gene in S2 cells identified the sequences required for normal Ketel gene expression level. Gel retardation and band shift assays revealed that the DREF and the CFDD transcription factors play key roles in the regulation of Ketel gene expression. Transgenic LacZ reporter genes revealed the sequences that ensure tissue-specific gene expression. Apparently, the regulation of Ketel gene expression depends largely on a DRE motif and action of the DREF, CFDD, CF2-II and BEAF transcription factors (Villanyi, 2008).

The Ketel gene has been known to be expressed (1) in the egg primordia to provide importin-β for oogenesis and early embryogenesis, (2) in every cell of the gastrulating embryo and (3) in the diploid cells during larval life, but not in the polytenic larval cells. The polytenic cells need relatively few importin-β molecules to accomplish nuclear protein import, and this duty is accomplished by the unusually long-lived importin-β molecules, some of which are maternally provided others are produced during early gastrulation (Villanyi, 2008). Intensive expression of the Ketel gene in the diploid cells is comprehensible since these cells need importin-β not only for nuclear protein import but also for the formation of the spindle microtubules and the reassembly of the nuclear envelope at the end of mitosis. The aim of the present study was to understand the mechanisms that ensure the characteristic expression pattern of the Ketel gene. To achieve this goal, cis-acting control elements were examined that are engaged in (1) the proper loading of the egg cytoplasm with the Ketel gene products, (2) the regulation of the all-over type of importin-β production during gastrulation and (3) controlling tissue-specific expression of the Ketel gene during the later stages of development (Villanyi, 2008).

Computer analysis revealed several evolutionarily conserved transcription factor binding sites in the Ketel promoter of which only the CF2-II, the CFDD, the DREF and perhaps the BEAF binding sites are of relevance. The CFDD, the DREF and the BEAF transcription factors have been known to be involved in the expression regulation of a number of genes engaged in cell cycle regulation. In fact, the CFDD binding sites are commonly present in the promoters of a number of DNA replication-related genes like PCNA and DREF. Since importin-β is required for spindle formation and nuclear envelope assembly, which are essential events in cell proliferation, it may not be surprising that the expression of the Ketel gene is regulated by the same transcription factors that control the expression of several genes engaged in cell cycle regulation (Villanyi, 2008).

Transient expression of a luciferase reporter gene in S2 cells clearly showed that all the sequences which regulate Ketel gene expression reside within a 750 bp sequence towards the 5' region of the Ketel gene. The 'active' transcription factor binding sequences within the region were identified in gel-shift experiments, and the sequences that ensure tissue-specific expression of the Ketel gene were determined through the analysis of the expression patterns of LacZ reporter transgenes (Villanyi, 2008).

It appears that the presence of an approximately 140 bp long sequence around the transcription start site is sufficient for a basic expression of the Ketel gene in the gonial cells. The simultaneous presence of two sequences is required for the expression of the Ketel gene in the nurse cells and for the loading of the egg cell cytoplasm with the Ketel gene products: a CFDD binding site in the first intron (around +247) and the DRE motif around −74. (Note that the importin-β-related maternal effect depends on the expression of the Ketel gene in the germ line components of the egg primordia) Removal of either of these sequences leads to an absence of Ketel gene expression in the nurse cells. Similarly, the concurrent presence of the DRE motif at −74 and the CFDD site(s) around −250 is necessary for the expression of the Ketel gene in every cell of the gastrulating embryo. Removal of any of these sequences abolishes Ketel gene expression during early gastrulation. It appears that cooperative binding of transcription factors to the DRE motif and to either of the CFDD recognition sites establishes favourable conditions for tissue-specific expression of the Ketel gene. A CF2-II binding site around −483 is sufficient and necessary for the expression of the Ketel gene in the diploid cells of the imaginal discs, the neuroblasts and the follicle epithelium. CF2-II has been reported to be expressed in the follicle cells and seems to be the only candidate to control Ketel gene expression in the imaginal disc cells and in the neuroblasts (Villanyi, 2008).

Interestingly, none of the six different types of LacZ reporter transgenes are expressed in any polytenic larval cell types. One possible explanation could be the different modes of action of DREF in the larval and in the diploid cells: DREF does not displace BEAF from the DRE motif in the larval cells and, thus, an insulator can form which blocks transcription of the Ketel gene. Three BEAF binding sites are necessary for the formation of an insulator, and the promoter of the Ketel gene contains three BEAF binding sites, one of which is part of the DRE motif. In the diploid cells, where DREF binds to the DRE motif and competes with BEAF, the insulator cannot form and, hence, there is no block to prevent expression of the Ketel gene. However, the above model is rather unlikely since when the DRE motif, and along with it one of the BEAF binding sites, is abolished the BEAF insulator cannot form. Yet, the Ketel gene is not expressed in the larval cells. The lack of Ketel gene expression in the larval cells can also be explained by the absence of CF2-II transcription factor in that cell type. Further studies are needed to ascertain whether this assumption is correct (Villanyi, 2008).

In summary, the DRE motif is a key component in the regulation of Ketel gene expression: transcription factors that bind to the DRE motif interact with different CFDDs, which are bound to different CFDD binding sites, ensuring tissue-specific expression of the gene. The DRE motif and the CFDD sites are commonly present in the promoter of several genes engaged in DNA replication and cell cycle control, and interaction of DREF and CFDD could be a key component in the regulation of those genes as well (Villanyi, 2008).

BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved Dual-Core binding sites

Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. This study combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element-associated factor (BEAF) subfamily. A dual-core BEAF binding signature was identified at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core-associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. These results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle (Emberly, 2008).

Chromatin insulators/boundary elements (BEs) are defined as sequences able to insulate a transgene from its chromosomal context and to block promiscuous enhancer-promoter interactions or heterochromatin spreading. These elements are thought to subdivide the genome into functional chromosome domains, through their ability to cluster DNA loops and to control the deposition of histone epigenetic marks to regulate chromatin accessibility for gene expression (Emberly, 2008).

No common signature and/or mechanism of action has been identified among characterized insulators/boundary elements. Rather, several factors confer insulating activity by targeting different DNA consensus sequences in the known insulators. In Drosophila, insulating factors include dCTCF, Zw5, BEAF, and the well-characterized suppressor of Hairy-wing (Su(Hw)), which targets hundreds of distinct, largely uncharacterized genomic sites. Whether each of these factors and subfamily of insulators might possess distinct cellular functions is of particular interest (Emberly, 2008).

BEAF blocks both enhancer-promoter communication and repression by heterochromatin, as shown using reporter transgenes. This insulating activity of BEAF was also evidenced by a genetic screen in yeast, confirming that, unlike de-silencing activity, BEAF binding sites must bracket a transgene for insulation. The hundreds of BEAF binding sites have not been characterized in situ, however, and the cellular function of BEAF remains to be elucidated in vivo (Emberly, 2008).

This study has combined computational and experimental approaches to address the function of BEAF binding sites in vivo. 1,720 BEAF dual-core elements have been identified genome-wide that share an unusual organization conserved over 600 bp. The dual-core signature consists of five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. BEAF dual-cores juxtapose to hundreds of genes highly enriched in gene annotations regulating chromosome organization/segregation and the cell cycle. Accordingly, BEAF depletion leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses further show that dual-cores regulate the expression of key cell-cycle genes including cdk7 and mei-S332. These results are also reproduced in embryos expressing truncated beaf mutants, which abolish the proper targeting of BEAF to dual-cores and its insulating activity. Chromatin immunoprecipitation analyses show that BEAF acts by restricting the deposition of methylated H3K9 marks in dual-cores. The data reveal a new role for BEAF in regulating chromosome organization/segregation and the cell cycle through its binding to highly conserved chromatin dual-cores (Emberly, 2008).

The DNA-binding activity of BEAF has been well-characterized in vitro. Each subunit of the BEAF complex targets one CGATA motif. Point mutations within this consensus abolish both its binding and insulating activities. Clusters of three to four CGATA motifs can create high-affinity BEAF in vitro binding sites, which are called single elements. A computational scan of the Drosophila genome revealed thousands of single elements, yet immunostaining analysis demonstrated that they were not good predictors for BEAF binding in vivo. For example, Chromosome 4 was found to contain hundreds of single elements, yet immunodetection analysis showed only three major BEAF signals on this chromosome. Interestingly, statistical analysis showed that single elements were often organized in a pair-wise configuration. Genome-wide, 988 single elements form 494 so-called 'dual-cores,' which harbor two separate clusters of three CGATAs, a statistically significant result. Moreover, 1,226 additional 'dual-core-like' elements have a second cluster of two (instead of three) CGATAs. These elements include all characterized BEAF insulators whose activity involves a second, lower-affinity CGATA cluster (K_d ~ 400-600 pM) where BEAF binding is abolished when the first high-affinity cluster is mutated (Emberly, 2008).

Detailed analysis by alignment of all 1,720 dual-core and dual-core-like elements showed a highly organized distribution of their 12,058 CGATAs, which preferentially segregate into two clusters separated by spacers of approximately 200 bp. For scs' and other characterized BEAF insulators, these spacers were found to be relatively AT-rich. Scanning the 1,720 dual-cores for A+T content showed that they all harbor significant AT-rich sequences in their spacers. The remarkably conserved organization of dual-cores indicates that they likely correspond to a highly specific BEAF-binding signature (Emberly, 2008).

Genome-wide ChIP-on-chip analysis detects approximately 1,800 significant BEAF binding sites, suggesting that the dual-core database presented in this study encompasses most in vivo BEAF binding sites. The few (<100) additional peaks not included in the database but detected by ChIP-on-chip analysis may correspond to elements initially scored as single elements but whose organization is close to that of dual-cores. These rare exceptions are in part due to the computer stringency of the dual-core signature. For example, BEAF-1255 can be bound by BEAF in vivo, yet this element could not be scored as a dual-core because one out of five of its clustered CGATA motifs lies 3 bp outside the defined 100-bp window. Furthermore, approximately 10% of the minor BEAF sites are found in regions lacking any CGATA motifs, including the scs insulator. Since this region is not directly bound by BEAF, it is thus possible that some of the minor BEAF peaks are due to indirect interactions between BEAF and other insulator proteins, as previously suggested for the scs'-scs pair of insulators. Other protein-protein interactions that regulate BEAF binding could also involve the splicing variant of the beaf gene itself, called BEAF-32A, which does not harbor the BEAF DNA-binding domain that recognizes clustered CGATA motifs. ChIP-on-chip analysis using antibodies that also recognize this isoform showed no additional major peaks, indicating that dual-cores constitute the main binding sites for both BEAF isoforms. Finally, it is noted that the BEAF-32A isoform is unlikely to play a major role in the activities described in this study, as its binding is dispensable for the insulating function of BEAF, and its expression is not essential for the development of embryos into adult flies. Taken together, these results show that the BEAF dual-core signature is a bona fide mark that identifies a cis-regulatory element that regulates the expression of nearby genes (Emberly, 2008).

Results of experiments using both BEAF depletion in tissue culture cells and BID expression in vivo provide clear evidence for specific functions of the BEAF dual-cores, reflected by a selective association with genes that control cell-cycle and/or chromosome organization/segregation. The competition between DREF and BEAF for binding to nested consensus sequences is also supported by ChIP analyses showing that DREF targets' identical sites clearly enriched nearby genes associated with the cell cycle and chromosome dynamic GOs. Thus, while DREF levels increase at the G1/S transition to activate mei-S332 and cdk7 within the appropriate window for cell-cycle progression BEAF may further facilitate this activation by restricting the deposition of H3K9me marks. Indeed, over-expressing BEAF was shown to reduce the phenotypes related to cell-cycle progression in flies that over-express DREF, supporting a role for BEAF in controlling the cell cycle. Such a model is also supported by the observation that anacardic acid treatment strongly represses these genes in BEAF-depleted cells and that mutation of the BEAF-binding site in a dual-core results in a local increase in H3K9m3 levels. In addition, computer analysis of micro-array expression data for Drosophila embryos during early development shows that the 545 genes associated with dual-cores are positively correlated with beaf expression, in contrast to genes unlinked to these elements. This strict correlation further indicates that BEAF has a global positive role on gene expression genome-wide, and similar analyses did not reveal any significant correlation change between genes whose TSS is closely juxtaposed (<100 bp) to dual-cores, including snf or cdk7, compared to genes whose TSS is more distant (500 bp). Accordingly, the cell-cycle and chromosome dynamics GOs that include cdk7 and mei-S332 are enriched for positively correlated genes. These results show that BEAF could play an important role in chromosome organization during the cell cycle through a regulated switch involving the BEAF-DREF competition: According to such a mechanism, BEAF would restrict the deposition of H3K9me3, allowing dual-core-associated genes to remain in a potentially active state, while controlling the time of activation of cell-cycle GOs by DREF. Accordingly, BEAF depletion leads to down-regulation of genes associated with a dual-core lacking a DREF element (CG10946, ras, CG1430, Janus, CG1444), but to increased expression of CG32676, mei-S332, cdk7, CG10944, and ser, which are under the control of DREF-associated dual-cores. In the latter case, the apparent contradiction between the positive (restriction of H3K9me3 deposition) and negative effects of BEAF can be reconciled by the results showing that BEAF controls the activation of these genes by DREF. BEAF depletion relieves the competition for binding by DREF, leading to the increased expression of cdk7 or mei-S3332 in spite of an increased deposition of H3K9me3 marks under these conditions. Mutating the DREF or BEAF binding sites of DREF-associated dual-cores allows for distinguishing between these different effects on the expression of linked genes (Emberly, 2008).

It is intriguing that the spacers of dual-cores are well-conserved. One possibility is that they may be preferentially bound by a nucleosome, as recently shown for CTCF insulators (Fu, 2008). Supporting this idea, the known dual core-spacers correspond to nuclease-resistant 'cores', between two nuclease-hypersensitive sites (BE76, scs'), where a nucleosome may be present. Indeed, it was found that dual core-spacers fall within predicted nucleosome-positioning sequence (NPS) databases, as indicated by NPS/dual-core sequence alignments, possibly accounting for the conserved organization of dual-cores. These results further suggest that the cooperative binding of BEAF across these AT-rich spacers may be important for BEAF function. Indeed, expression of BID, which prevents its cooperative binding across the spacers, mimics the effect of BEAF depletion on the expression of dual-core-associated genes, as also found by mutagenesis of two CGATA motifs from one dual-core cluster. However, BEAF still efficiently binds in vivo to the few dual-cores that harbor a shorter spacer (<150 bp), indicating that the conserved dual-core-spacer is dispensable for BEAF binding. Recent reports have shown that gene expression is differentially regulated through nucleosome positioning in several species. Positioned nucleosomes may restrict promoter accessibility in yeast, and pausing of RNA polymerase facing the +1 nucleosome may be regulated through nucleosome positioning in Drosophila. Similarly, dual-cores are also closely associated with TSSs, and a potential link to nucleosome positioning strengthens the view that BEAF may regulate chromatin accessibility for gene expression through a restriction of the deposition of methylated H3K9 marks into dual-cores (Emberly, 2008).

The model whereby dual-cores regulate the deposition of specific epigenetic marks is in agreement with the activity of other known insulators. Variations in H3K9me3 levels might affect the interplay between the deposition of H3K9me3 and acetylated histone H4 (H4Ac) marks. However, no variation in the deposition of H4Ac could be found in dual-cores compared to control regions after BEAF depletion. This is not surprising, as BEAF has no de-silencing activity on its own. Computer analysis failed to reveal any enrichment of dual-cores near the 3'UTR of genes, and the activity of dual-cores may thus essentially play a role in regulating chromatin accessibility near promoter regions, but not within the 3' border of genes. Furthermore, the insulating activity of BEAF was demonstrated in the context of two dual-cores bracketing a transgene, and most likely also involved higher-level chromatin organization. Although not enriched near the 3'UTR of genes, dual-cores still bracket/separate groups of genes clustered within 5-15 Kbp, a genomic context that may further require insulating activity to block promiscuous enhancer-promoter interactions and involve DNA looping between distant insulators. It has recently been shown for a Su(Hw) insulator that the regulation of gene expression may further depend on its genomic environment (Soshnev, 2008). Also, other dual-cores are often found in the vicinity of genes exposed to repression by heterochromatin, and the function of BEAF may be particularly important in this context. It is proposed that the BEAF dual-cores closely linked to a restricted array of several hundred genes define a family of insulators that provide a link between chromatin organization and the cell cycle (Emberly, 2008).

Boundary Element-Associated Factor 32B connects chromatin domains to the nuclear matrix

Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the Boundary Element-Associated Factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. This report shows that BEAF is associated with the nuclear matrix and maps the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. The DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains (Pathak, 2007).

Nuclear matrix was prepared from a Drosophila embryo. Proteins of the nuclear matrix were resolved by 2D gel electrophoresis and processed for MALD-TOF MS-MS. Several proteins were identified in this way, including many known components of the nuclear matrix such as lamin, actin, and heat shock proteins. One of the moderately abundant protein spots with a molecular mass of ~42 kDa and a pI of ~6.0 was identified as BEAF-32B. The distribution of proteins in different fractions was analyzed during matrix preparation and the presence of BEAF in the matrix fraction was directly tested using Western blotting. The results show that a significant proportion of BEAF is retained in the matrix (Pathak, 2007).

BEAF-32 exists in at least two isoforms, termed 32A and 32B, differing in the amino-terminal 80 aa. No tryptic peptides representing the amino-terminal portion of BEAF-32A were detected, suggesting that either it is absent or it is present in a form or amount that is undetectable. It is known that BEAF-32B is at least four times more abundant than BEAF-32A. The monoclonal antibody used in these experiments does not distinguish the two isoforms, and hence, it is not possible to exclude the presence of the 32A isoform in the matrix. Furthermore, the region of BEAF-32B that is necessary for matrix association is common to the two isoforms of BEAF, suggesting that BEAF-32A has the potential to be present in the nuclear matrix. Therefore, this protein is referred to as BEAF throughout this report (Pathak, 2007).

BEAF has been purified from nuclear extracts. In the current extraction procedure, it was observed that while a large fraction of BEAF was soluble in different extraction buffers, 25% of the nuclear BEAF remained matrix bound. An identical extraction procedure removes all of the nuclear UBX protein, and it is totally absent from the nuclear matrix fraction. Earlier studies have suggested that some components of the nuclear matrix are released upon RNase A digestion of the matrix. This study treated the matrix preparations with RNase A and this treatment does not release BEAF from the nuclear matrix. In this respect, BEAF behaves like CTCF, the mammalian boundary protein that is associated with the nuclear matrix but is not released upon RNase treatment (Pathak, 2007).

Many nuclear proteins, including histones, show a rich variety of posttranslational modifications that play an important role in their function. BEAF has been earlier shown to be phosphorylated as evidenced by the upper band of the doublet seen in Western blots. BEAF also contains several potential sites for other posttranscriptional modifications. In order to investigate the nature of these modifications and their possible relation to matrix association, Western blot assays were performed using narrow-pH-range 2D gels. The nuclear BEAF resolves into six spots. The three upper spots shift towards a more acidic pH. Phosphorylation, myristoylation, and methylation render proteins more acidic whereas esterification makes them more basic. Some modifications such as glycosylation and prenylation alter the molecular weight of the protein but not its p. While all forms of the BEAF protein are seen in the soluble fraction, the protein form with a higher molecular weight and a more-acidic p is enriched in the matrix fraction, suggesting extensive posttranslational modifications (Pathak, 2007).

To directly test if phosphorylation of BEAF is necessary for its association with the nuclear matrix, the nuclear matrix preparation was treated with PP1. PP1 is a Mn2+-dependent phosphatase with activity towards phosphoserine/threonine residues. Its activity is inhibited by -2, which specifically interacts with the catalytic subunit of PP1. When the preparation was analyzed on a 6 to 10% gradient gel, which resolved the BEAF doublet, it was observed that the upper band of the doublet disappeared upon phosphatase treatment. However, the intensity of the lower band increased, and it remained quantitatively associated with the matrix. This shift is not observed in the presence of PP1-specific inhibitor -2, implying that the upper band is a phosphorylated form of BEAF that is dephosphorylated by PP1. It is interesting that both the phosphorylated and dephosphorylated forms are components of the nuclear matrix. TC-PTP, a phosphotyrosine-specific protein phosphatase, was also tested to check for tyrosine-specific phosphorylation. Treatment with TC-PTP did not affect BEAF mobility or levels, suggesting that BEAF is not phosphorylated on tyrosine residues (Pathak, 2007).

BEAF contains several potential glycosylation sites, particularly clustered in the middle region of the protein. WGA binding was used to check if BEAF is glycosylated. WGA is known to specifically bind terminal N-acetylglucosamine (GlcNAc) moieties and GlcNAc oligomers and has been extensively used to isolate glycosylated proteins. Both the unphosphorylated and phosphorylated forms of BEAF from nuclear extracts are retained on the WGA column, showing that BEAF is indeed glycosylated, irrespective of its phosphorylation status. The nuclear matrix was solubilized by adding denaturant to the matrix preparation, the soluble proteins were dialyzed, binding to WGA-Sepharose beads was tested. BEAF, which does not bind to Sepharose alone, was found to bind WGA-Sepharose, indicating that both free and matrix-bound forms of BEAF are glycosylated. To further explore any link between the glycosylation and matrix association, the endogenous glycosylation of proteins was interfered with using alloxan, an inhibitor of O-GlcNAc transferase, in S2 cells. The cell lysate and nuclear matrix preparation of alloxan-treated cells were analyzed by Western blot assays with anti-BEAF antibody. With increasing concentrations of alloxan, several smaller bands of BEAF appear in the Western blot analysis. Since bacterially expressed BEAF that is likely to be unmodified shows the same mobility as does nuclear BEAF, the smaller peptides may be degradation products of BEAF. It is possible that lack of glycosylation destabilizes BEAF, leading to smaller peptides. Importantly, these peptides are absent in the nuclear matrix prepared from alloxan-treated cells, indicating that unglycosylated BEAF is unstable and does not bind to nuclear matrix. However, these results do not rule out the possibility that unglycosylated BEAF that is still bound to the matrix is more stable due to its association with matrix components (Pathak, 2007).

To further analyze the association of BEAF with the nuclear matrix and to ascertain whether a known partner of BEAF, ZW5, is also present in the matrix, a Western blot analysis of total nuclear protein and the nuclear matrix fraction was carried out. Equal amounts of protein from nuclei and nuclear matrix were loaded onto an SDS-polyacrylamide gel, blotted, and probed with monoclonal anti-BEAF and anti-ZW5 antibodies. ZW5 is the boundary-interacting protein that binds to scs, and like BEAF, ZW5 was also found in the matrix fraction, while several other nuclear proteins such as histone H3 and ABID-B were absent. Similar results were obtained when matrix was prepared from the embryos or S2 cells (Pathak, 2007).

In order to study the distribution of BEAF and ZW5 in the nucleus, S2 cells were spun onto glass slides and extracted to reveal the nuclear matrix. These in situ matrix preparations were stained with antibodies to BEAF, ZW5, lamin, and ABID-B. As expected, lamin showed a perinuclear rim staining that was retained in the salt-extracted matrix preparations, while ABID-B was lost during salt extractions. Prior to extraction, in the nucleus as such, both ABID-B and BEAF are present. Both BEAF and ZW5 were retained in the nuclear matrix. While ZW5 appears to be less abundant in the matrix, most of it colocalizes with BEAF. It is estimated that about 50% of BEAF and of ZW5 colocalize in the nucleus. However, in the nuclear matrix preparations, almost 95% of ZW5 colocalizes with BEAF, consistent with the earlier observation that these two proteins interact with one another in vivo. These data clearly show that BEAF-32 and ZW5 are associated in the context of the nuclear matrix. Immunofluorescence data also demonstrate that these proteins are unevenly distributed in the matrix and are not associated with prominent structures such as the nuclear lamina or nucleolus (Pathak, 2007).

BEAF has three distinct domains: the amino-terminal BED finger domain (amino acids 27 to 77, with DNA binding activity), the carboxy-terminal BESS domain (amino acids 237 to 276, with protein-protein interaction and trimerization activity), and the middle region coiled-coil domain (amino acids 203 to 223). To identify the region of the protein required for association with the nuclear matrix, full-length protein, the three distinct domains of BEAF-32B, and three other overlapping fragments were individually tagged with a Flag epitope and were expressed as N-terminal Flag fusions under the control of the Polycomb promoter in S2 cells. The expression of the recombinant fusion proteins was confirmed in S2 cell lysate by Western blot assays using anti-Flag antibody. All the constructs were found to be expressed at relatively equal levels except for the amino-terminal fusion, which could not be detected on Western blots, possibly due to its small size of 75 aa. However, the N-terminal fusion was visible in immunofluorescence experiments (Pathak, 2007).

Nuclear matrix was prepared from cells transfected with each of these constructs. Western blot analysis with anti-Flag antibody reveals that the full-length protein and the middle-region (aa 83 to 224) constructs are retained in the nuclear matrix. The endogenous BEAF was retained in matrix preparations from all samples. It is interesting that this middle region contains the coiled-coil domain (aa 203 to 223) and a shorter middle-region construct (middle region-short, aa 140 to 224) that also contains this domain is matrix bound. The construct with the coiled-coil domain and the C terminus (aa 190 to 282) is also matrix associated, although with much lower efficiency. Taken together, it appears that aa 140 to aa 224 are sufficient for targeting BEAF-32 to the nuclear matrix (Pathak, 2007).

The transfected S2 cells and matrix prepared from them was immunostained with anti-Flag antibody. Rull-length protein, N terminus, middle region, and middle region 2 are localized in the nucleus, whereas the C terminus, the C terminus with the coiled coil, and the middle region without the coiled coil are cytosolic. Thus, the coiled-coil region alone is not sufficient for nuclear localization but may be necessary because the middle region without the coiled coil is also cytosolic (Pathak, 2007).

Immunostaining confirms the findings of the Western blot analysis and shows that the region corresponding to aa 140 to 224 is responsible for nuclear matrix targeting. The N terminus (the DNA binding domain) is also independently capable of localizing to the nucleus but is not retained in the matrix. If the coiled-coil region is included with the C terminus, the protein is unable to localize into the nucleus efficiently but the small amount of protein that does enter the nucleus associates with nuclear matrix (Pathak, 2007).

Interestingly, the region of BEAF that appears to play a key role in its localization to the nuclear matrix contains most of the serine/threonine residues that have a high potential to become phosphorylated as well as glycosylated. When expressed separately as a Flag fusion, this middle region also shows lower mobility, indicating that it may be glycosylated. It is likely that these posttranslational modifications are important in the matrix association of BEAF. Further, this region is common to the two isoforms of BEAF, suggesting that BEAF-32A also has the potential to be a nuclear matrix component, even though this isoform was not detected in the proteomic analysis (Pathak, 2007).

These results described that BEAF is associated with the nuclear matrix. Since BEAF binds to several sequences that have boundary activity, the hypothesis was tested that these target sequences are also matrix bound. The presence of four different loci from the Drosophila genome in nuclear matrix preparations was detected by quantitative PCR and immuno-FSH. Two different sequences known to be targets of BEAF were tested. First, a 111-bp amplicon from BE28, a moderately repeated 1.2-kb DNA sequence that functions as a BEAF-dependent boundary element in transgenic flies, was chosen. This amplicon encompasses the BEAF binding sites. The second BEAF target is a 97-bp amplicon from the scs' boundary element including the BEAF binding sites. A 105-bp amplicon from an exon of the BEAF protein coding region with no predicted MAR was chosen as a negative control. Finally, a 192-bp amplicon from the well-characterized MAR from the histone gene cluster was chosen as a positive control (Pathak, 2007).

Nuclear matrix were prepared using EcoR and Hind in place of DNase for digestion prior to extraction. The DNA fragments (operationally defined MARs) obtained from the nuclear matrix preparation were used for quantitative real-time PCR. The results show that ~10% of the BEAF coding region amplicon is retained in the matrix, reflecting a background-level presence or transient matrix association due to transcriptional activity (which may involve indirect association with the matrix). In contrast, >50% of the BEAF target sequences (BE28 as well as scs') and the positive-control His-MAR are present in the matrix under these conditions (Pathak, 2007).

Retention of a high proportion of the BEAF target sequences (equivalent to a known MAR) suggests specific association with the nuclear matrix. If the BEAF target sequences are MARs and BEAF itself is a matrix component, they would be predicted to colocalize in nuclear matrix preparations. Immuno-FSH was performed on matrix preparations of S2 cells with BEAF antibody and BE28/scs' fluorescently labeled probes. Intensely staining spots of BE28 probe were seen to be clearly retained in the matrix. BEAF protein, in contrast, shows a more elaborate staining. Using optical sectioning, it was confirmed that the BE28 spots are located in the interior of the nucleus. Scs' too is detected in the matrix preparation. Matrix preparations doubly stained with BE28 and BEAF were examined by confocal microscopy for colocalization of the two signals using the cross-hair function. The weighted colocalization coefficient was calculated for selected regions where the BE28 signal was detected. It was observed that >70% of the BE28 signal and >75% of the scs' signal colocalized with the BEAF. The data suggest that all BE28 sites overlap with BEAF signals but that BEAF has several other target sites and gives a more disperse pattern (Pathak, 2007).

Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype

DNA replication-related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication- and proliferation-related genes in Drosophila. While the effects on the mutation in the DNA replication-related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild-type DREF activity in developing tissues have hitherto remained unclear. DREF was over-expressed in the wing imaginal disc using a GAL4-UAS targeted expression system in Drosophila. Over-expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF-induced notching wing phenotype. Furthermore, this was also the case with co-expression of baculovirus P35, a caspase inhibitor. In addition, over-expression of the 32 kDa boundary element-associated factor (BEAF-32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. This is the first evidence for a genetic interaction between DREF and BEAF-32. It is concluded that the DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc (Yoshida, 2001).

An antagonistic relationship between the boundary element-associated factor BEAF and DREF

Boundary elements interfere with communication between enhancers and promoters, but only when interposed. Understanding this activity will require identifying the proteins involved. The boundary element-associated factor BEAF is one protein that is implicated in boundary element function. Three genomic fragments (scs', BE76 and BE28) containing BEAF binding sites function as boundary elements in transgenic Drosophila, suggesting that this is an intrinsic property of the numerous genomic regions to which BEAF binds. To characterize additional proteins that interact with boundary elements, a protein was isolated that binds to two of these boundary elements (BE76 and BE28); and it was identified as the transcription factor DREF. Evidence is presented that BEAF and DREF compete for binding to overlapping binding sites, and that this competition occurs in vivo. DREF is believed to regulate genes whose products are involved in DNA replication and cell proliferation, suggesting that the activation of transcription predicted to result from the displacement of BEAF by DREF might be limited to certain rapidly proliferating tissues. This is the first suggestion that the activity of a subset of boundary elements might be regulated (Hart, 1999).

The phylogenetic distribution of non-CTCF insulator proteins is limited to insects and reveals that BEAF-32 is Drosophila lineage specific

Chromatin insulators are DNA sequences found in eukaryotes that may organize genomes into chromatin domains by blocking enhancer-promoter interactions and preventing heterochromatin spreading. Considering that insulators play important roles in organizing higher order chromatin structure and modulating gene expression, very little is known about their phylogenetic distribution. To date, six insulators and their associated proteins have been characterized, including Su(Hw), Zw5, CTCF, GAF, Mod(mdg4), and BEAF-32. However, all insulator proteins, with the exception of CTCF, which has also been identified in vertebrates and worms, have been exclusively described in Drosophila melanogaster. This work performed database searches utilizing each D. melanogaster insulator protein as a query to find orthologs in other organisms, revealing that except for CTCF all known insulator proteins are restricted to insects. In particular, the boundary element-associated factor of 32 kDa (BEAF-32), which binds to thousands of sites throughout the genome, was only found in the Drosophila lineage. Accordingly, a significant bias of BEAF-32 binding sites was found in relation to transcription start sites (TSSs) in D. melanogaster but not in Anopheles gambiae, Apis mellifera, or Tribolium castaneum. These data suggest that DNA binding proteins such as BEAF-32 may have a dramatic impact in the genome of single evolutionary lineages. A more thorough evaluation of the phylogenetic distribution of insulator proteins will allow for a better understanding of whether the mechanism by which these proteins exert their function is conserved across phyla and their impact in genome evolution (Schoborg, 2010).

Search PubMed for articles about Drosophila BEAF

Aravind, L. (2000). The BED finger, a novel DNA-binding domain in chromatin-boundary-element-binding proteins and transposases. Trends Biochem. Sci. 25: 421-423. PubMed Citation: 10973053

Bhaskar, V. and Courey, A. J. (2002). The MADF-BESS domain factor Dip3 potentiates synergistic activation by Dorsal and Twist. Gene 299: 173-184. PubMed Citation: 12459265

Blanton, J., Gaszner, M. and Schedl, P. (2003). Protein:protein interactions and the pairing of boundary elements in vivo. Genes Dev. 17(5): 664-75. PubMed Citation: 12629048

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

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

Cuvier, O., Hart, C. M. and Laemmli, U. K. (1998). dentification of a class of chromatin boundary elements. Mol. Cell. Biol. 18: 7478-7486. PubMed Citation: 9819433

Cuvier, O., Hart, C. M., Kas, E. and Laemmli, U. K. (2002). Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains. Chromosoma 110: 519-531. PubMed Citation: 12068969

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

Delattre, M., Spierer, A., Hulo, N. and Spierer, P. (2002). A new gene in Drosophila melanogaster, Ravus, the phantom of the modifier of position-effect variegation Su(var)3-7. Int. J. Dev. Biol. 46: 167-171. PubMed Citation: 11902679

Emberly, E., et al. (2008), BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dual-core binding sites. PLoS Biol. 6(12): 2896-910. PubMed Citation: 19108610

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. 2008. doi/ 10.1371/journal.pgen.1000138. PubMed Citation: 18654629

Gaszner, M., Vazquez, J. and Schedl, P. (1999). The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer-promoter interaction. Genes Dev. 13: 2098-2107. PubMed Citation: 10465787

Geyer, P. K. (1997). The role of insulator elements in defining domains of gene expression. Curr Opin Genet Dev. 7(2): 242-8. PubMed Citation: 9115431

Gilbert, M. K., Tan, Y. Y. and Hart, C. M. (2006). The Drosophila boundary element-associated factors BEAF-32A and BEAF-32B affect chromatin structure. Genetics 173(3): 1365-75. PubMed Citation: 16648647

Hart, C. M., Zhao, K. and Laemmli, U. K. (1997). The scs' boundary element: characterization of boundary element-associated factors. Mol. Cell. Biol. 17(2): 999-1009. PubMed Citation: 9001253

Hart, C. M., Cuvier, O. and Laemmli, U. K. (1999). Evidence for an antagonistic relationship between the boundary element-associated factor BEAF and the transcription factor DREF. Chromosoma 108(6): 375-83. PubMed Citation: 10591997

Jiang, N., Emberly, E., Cuvier, O. and Hart, C. M. (2009). Genome-wide mapping of boundary element-associated factor (BEAF) binding sites in Drosophila melanogaster links BEAF to transcription. Mol. Cell. Biol. 29(13): 3556-68. PubMed Citation: 19380483

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

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

Négre, N., et al. (2010). A comprehensive map of insulator elements for the Drosophila genome. PLoS Genet. 6(1): e1000814. PubMed Citation: 20084099

Pathak, R. U., Rangaraj, N., Kallappagoudar, S., Mishra, K. and Mishra, R. K. (2007). Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix. Mol. Cell. Biol. 27(13): 4796-806. PubMed Citation: 17485444

Roy, S., Tan, Y. Y. and Hart, C. M. (2007a). A genetic screen supports a broad role for the Drosophila insulator proteins BEAF-32A and BEAF-32B in maintaining patterns of gene expression. Mol. Genet. Genomics 277(3): 273-86. PubMed Citation: 17143631

Roy, S., Gilbert, M. K. and Hart, C. M. (2007b). Characterization of BEAF mutations isolated by homologous recombination in Drosophila. Genetics 176(2): 801-13. PubMed Citation: 17435231

Schoborg, T. A. and Labrador, M. (2010). The phylogenetic distribution of non-CTCF insulator proteins is limited to insects and reveals that BEAF-32 is Drosophila lineage specific. J. Mol. Evol. 70(1): 74-84. PubMed Citation: 20024537

Shogren-Knaak, M., et al. (2006). Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311: 844-847. PubMed Citation: 16469925

Soshnev, A. A., Li, X., Wehling, M. D. and Geyer, P. K. (2008). Context differences reveal insulator and activator functions of a Su(Hw) binding region. PLoS Genet. 2008. doi/ 10.1371/journal.pgen.1000159. PubMed Citation: 18704163

Villanyi, Z., Papp, B., Szikora, S., Boros, I. and Szabad, J. (2008). The DRE motif is a key component in the expression regulation of the importin-beta encoding Ketel gene in Drosophila. Mech. Dev. 125(9-10): 822-31. PubMed Citation: 18656533

Yamaguchi, M., et al. (2001). Ectopic expression of BEAF32A in the Drosophila eye imaginal disc inhibits differentiation of photoreceptor cells and induces apoptosis. Chromosoma 110: 313-321. PubMed Citation: 11685531

Yoshida, H., et at. (2001). Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype. Genes Cells 6(10): 877-86. PubMed Citation: 11683916

Zhao, K., Hart, C. M. and Laemmli, U. K. (1995). Visualization of chromosomal domains with boundary element-associated factor BEAF-32. Cell 81(6): 879-89. PubMed Citation: 7781065

date revised: 20 February 2010

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