Boundary element-associated factor of 32kD: Biological Overview | References
Gene name - Boundary element-associated factor of 32kD
Cytological map position - 51C2-51C2
Function - chromatin factor
Keywords - boundary elements, chromatin, position-effect variegation
Symbol - BEAF-32
FlyBase ID: FBgn0015602
Genetic map position - 2R:10,657,975..10,660,135 [+]
Classification - BESS motif, BED zinc finger
Cellular location - nuclear
|Recent literature||Mourad, R. and Cuvier, O. (2016). Computational identification of genomic features that influence 3D chromatin domain formation. PLoS Comput Biol 12: e1004908. PubMed ID: 27203237
Recent advances in long-range Hi-C contact mapping have revealed the importance of the 3D structure of chromosomes in gene expression. A current challenge is to identify the key molecular drivers of this 3D structure. Several genomic features, such as architectural proteins and functional elements, were shown to be enriched at topological domain borders using classical enrichment tests. This study proposes multiple logistic regression to identify those genomic features that positively or negatively influence domain border establishment or maintenance. The model is flexible, and can account for statistical interactions among multiple genomic features. Using both simulated and real data, the model was shown to outperform enrichment test and non-parametric models, such as random forests, for the identification of genomic features that influence domain borders. Using Drosophila Hi-C data at a very high resolution of 1 kb, the model suggests that, among architectural proteins, BEAF-32 and CP190 are the main positive drivers of 3D domain borders. In humans, this model identifies well-known architectural proteins CTCF and cohesin, as well as ZNF143 and Polycomb group proteins as positive drivers of domain borders. The model also reveals the existence of several negative drivers that counteract the presence of domain borders including P300, RXRA, BCL11A and ELK1 (Mourad, 2016).
|Jukam, D., Viets, K., Anderson, C., Zhou, C.,
DeFord, P., Yan, J., Cao, J. and Johnston, R.J. (2016). The
BEAF-32 insulator protein is required for Hippo pathway activity in the
terminal differentiation of neuronal subtypes. Development [Epub
ahead of print]. PubMed ID: 27226322
The Hippo pathway is critical for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. This study identified the BEAF-32 insulator protein as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin6 (Rh6) and represses Rhodopsin5 (Rh5) whereas in the alternate subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double negative feedback loop. It was shown that the BEAF-32 insulator is required for expression of warts and repression of melted. Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. In summary, the study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein.
|Avva, S. V. and Hart, C. M. (2016). Characterization of the Drosophila BEAF-32A and BEAF-32B insulator proteins. PLoS One 11: e0162906. PubMed ID: 27622635
Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. This study reports a further characterization of BEAF. The BESS domain alone is shown to be sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, the middle region and the BESS domain was shown to be essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, the dynamics are documented of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function.
|Afik, S., Bartok, O., Artyomov, M. N., Shishkin, A. A., Kadri, S., Hanan, M., Zhu, X., Garber, M. and Kadener, S. (2017). Defining the 5' and 3' landscape of the Drosophila transcriptome with Exo-seq and RNaseH-seq. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 28335028
Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. This study presents a simple and straightforward approach for genome-wide annotation of 5'- and 3'-RNA ends. The approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. This methodology was appled to study the impact of temperature on the Drosophila melanogaster head transcriptome. Hundreds of previously unidentified TSS and PAS were found, revealing two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result that was validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation.
|De, D., Kallappagoudar, S., Lim, J. M., Pathak, R. U. and Mishra, R. K. (2017). O-GlcNAcylation of Boundary Element Associated Factor (BEAF 32) in Drosophila melanogaster correlates with active histone marks at the promoters of its target genes. Nucleus [Epub ahead of print]. PubMed ID: 28910574
Boundary Element-Associated Factor 32 (BEAF 32) is a sequence specific DNA binding protein involved in functioning of chromatin domain boundaries in Drosophila. Several studies also show it to be involved in transcriptional regulation of a large number of genes, many of which are annotated to have cell cycle, development and differentiation related function. Since post-translational modifications (PTMs) of proteins add to their functional capacity, this study investigated the PTMs on BEAF 32. The protein is known to be phosphorylated and O-GlcNAcylated. An O-GlcNAc site was mapped at T91 of BEAF 32, and it was shown to be linked to the deposition of active histone (H3K4me3) marks at transcription start site (TSS) of associated genes. Its role as a boundary associated factor, however, does not depend on this modification. This study shows that by virtue of O-GlcNAcylation, BEAF 32 is linked to epigenetic mechanisms that activate a subset of associated genes.
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 BEAFAB-KO allele, and the BEAFA-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 wm4h 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 (BEAFAB-KO). In the process, an allele was isolated that eliminates the ability to produce the 32A protein (BEAFA-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 BEAFA-KO allele are healthy and viable, indicating that the 32B protein is sufficient for normal development. In contrast, flies homozygous for the BEAFAB-KO allele cannot be maintained as a stable line. Maternal BEAF is sufficient to obtain fertile adults, although the resulting BEAFAB-KO flies eclose 1-2 days later than their BEAFAB-KO/CyO siblings and are sickly. Also, although equal numbers of males and females are obtained, the fertility of the BEAFAB-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 BEAFAB-KO flies, although ovaries from a given BEAFAB-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 BEAFAB-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, BEAFA-KO animals are healthier than BEAFAB-KO animals, and a weaker effect on the male X chromosome was observed in BEAFA-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 BEAFAB-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 BEAFAB-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 BEAFAB-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).
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 wm4h 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 wm4h 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
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 wm4h and KV20 y alleles. These results provide strong support for a role of chromatin structure or dynamics in BEAF-dependent insulator function (Roy, 2007a).
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).
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 BEAFAB-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 BEAFAB-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 BEAFAB-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 BEAFAB-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 Toll10b 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 Toll10b 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).
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).
Understanding the relationship between genome organization and expression is central to understanding genome function. Closely apposed genes in a head-to-head orientation share the same upstream region and are likely to be co-regulated. This study identified the Drosophila BEAF-32 insulator as a cis regulatory element separating close head-to-head genes with different transcription regulation modes. The binding landscapes of the BEAF-32 insulator protein was then compared in four different Drosophila genomes, and the evolutionarily conserved presence of this protein between close adjacent genes was highlighted. Changes in binding of BEAF-32 to sites in the genome of different Drosophila species was found to correlate with alterations in genome organization caused by DNA re-arrangements or genome size expansion. The cross talk between BEAF-32 genomic distribution and genome organization contributes to new gene expression profiles, which in turn translate into specific and distinct phenotypes. The results suggest a mechanism for the establishment of differences in transcription patterns during evolution (Yang, 2012).
This study shows that the presence of BEAF-32 between close adjacent genes arranged in a head-to-head orientation correlates with different transcription regulatory patterns in the two genes of the pair in Drosophila. Close head-to-head gene pairs exist in almost all eukaryotes but it is not known whether other species also use this strategy in order to maintain independent regulation of adjacent genes. In humans, genes present in head-to-head gene pairs also show a bimodal distribution in the correlation of expression. In addition to the peak indicative of high correlation, there is also a peak of enrichment of gene pairs whose expression is not correlated. For these pairs, it is reasonable to predict the existence of regulatory mechanisms that functionally separate the two genes in order to attain the observed differential transcription (Yang, 2012).
BEAF-32 is restricted to Drosophila species and mammalian cells may use other insulator proteins to accomplish this goal. In Drosophila there are several types of insulator elements that show different genomic distributions with respect to gene. The distribution of the dCTCF insulator partially overlaps that of BEAF-32. Since CTCF is conserved between Drosophila and humans, it is possible that this protein functionally replaces BEAF-32 in maintaining differential transcription programs in genes located in close head-to-head gene pairs. When the human genome was specifically examined for the organization of close head-to-head gene pairs, those containing CTCF showed lower correlation of expression, suggesting that this mechanism may be also conserved in humans (Yang, 2012).
The organization of the genome that provides the highest fitness should be selected during evolution. If co-expression of close head-to-head gene pairs provides lower fitness, selection should favor re-arrangements that result in physical or functional separation of the two genes. A comparative analysis of head-to-head gene pairs in different species revealed that these pairs are more conserved in vertebrate lineage than in Drosophila species. Drosophila has more close head-to-head gene pairs than mammals but the conservation of these pairs is 3-fold lower. This suggests that some of the head-to-head gene pairs in Drosophila arise from genome compaction rather than selection for this specific organization. For these gene pairs, maximum fitness will select for separation of the genes in order to attain differential expression of the two genes in the pair. One strategy to accomplish this is functional separation by recruiting insulator proteins. Alternatively, chromosomal re-arrangements may physically separate the two genes. However, in an already compact genome like that of Drosophila, it may be difficult to organize all non-co-expressed genes apart from each other. Thus, a strategy relying on functionally separating the members of head-to-head gene pairs may be more effective. This analysis has concentrated on close adjacent genes that are divergently transcribed because this arrangement facilitates analysis of the correlation between the location of BEAF-32 and transcription patterns of the two genes. Nevertheless, 38% of BEAF-32 binding sites associate with non head-to-head gene pairs. It is possible that BEAF-32 plays a similar role in this situation in order to control interactions between regulatory sequences located in the 3' region or introns of genes and adjacent promoters from other genes. Although information on the location of regulatory sequences in the Drosophila genome is becoming available, it is not yet known which sequences regulate which genes. In the absence of this information, it is not possible at this time to evaluate the possible role of BEAF-32 in maintaining independent regulation of genes that are far apart and not in a heat-to head orientation (Yang, 2012).
The organization of head-to-head gene pairs in both humans and Drosophila is conserved during evolution, but the two members of each pair are not precisely co-regulated. The distribution of expression correlation suggests that most gene pairs do not show either high correlation or no correlation, but rather a relative level of correlation, suggesting that they may be co-regulated in certain developmental stages or specific tissues. Coexpression is still important for the genes, but they are not co-regulated all the time. Thus, the head-to-head orientation needs to be maintained for co-expression, but it is also necessary to separate genes when they are not co-regulated. The profiles of genome distribution of different insulator proteins in different cell types suggest a certain degree of cell type specificity in both humans and Drosophila. These observations point to a role for insulators in coordinating genome organization and function during evolution (Yang, 2012).
Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. This study tested this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. Distinct combinatorial binding was found of insulator proteins to different classes of sites, and a novel type of insulator element was uncovered that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. Insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes (Schwartz, 2012).
The binding sites of insulator proteins are often taken to represent elements that partition the genome into independent regulatory domains and demarcate chromosomes into regions of 'active' and 'repressed' chromatin. The results presented in this study give little support to this view as a general principle of genome organization although it may be true in certain regions. Instead it is argued that: 1) insulator proteins bind to genomic sites in specific combinatorial patterns, 2) the properties of sites bound by key insulator proteins SU(HW) and CTCF are markedly different depending on whether the two co-bind with CP190, 3) many of the known insulator proteins sites do not function as robust enhancer blockers, and 4) at least in cultured cells the depletion of insulator proteins has a limited impact on genome-wide gene expression (Schwartz, 2012).
Classifications of combinatorial binding of insulator proteins have been described previously. These classifications relied on the overlapping of bound regions defined according to arbitrary statistical thresholds and the position of these regions relative to TSSs. Because they did not take into account the relative strengths of binding, such classifications grouped together binding sites with very different biochemical and functional properties (Schwartz, 2012).
In contrast, this study defines the persistent co-binding patterns based on the strength of binding of the associated proteins, treating regions strongly bound by a combination of proteins differently from regions at which the same proteins are detected according to a statistical threshold but where the extent of their binding is disproportional. It is argued that this approach retains the information on biochemical interrelations between the co-bound proteins and separates the sites with different functional properties. The strongest support for this argument comes from RNAi knock-down experiments which demonstrate that the effect of the loss of one insulator protein on the binding of another insulator protein is constrained to a specific class of co-bound regions. For example, the knock-down of SU(HW) results in the loss of CP190 from class 3 (gypsy-like) sites but not from class 9 (CTCF+CP190) or class 5 (BEAF-32+CP190) sites (Schwartz, 2012).
The approach to select the sites representative of each co-binding class is conservative and inevitably excluded a fraction of binding sites from downstream analyses. For example, strong SU(HW) binding sites assigned to class 14 by initial overlap comparison were not analyzed further due the uncertainty of their co-binding by CP190. It is therefore cautioned that selection of representative binding sites is not a complete genomic catalogue and readers are advised to use the ChIP-chip binding profiles, deposited to GEO and modMINE, to gauge whether their locus of interest has a strong insulator protein binding site (Schwartz, 2012).
The prevailing model in the field suggests that CP190 is recruited to different insulator elements by DNA binding proteins where it serves as a universal adapter that mediates interactions between different insulator elements. The current results present a more complex picture. First, RNAi knock-down experiments demonstrate that the binding of SU(HW) protein to class 3 (gypsy-like) sites is dependent on CP190, indicating that CP190 is not passively tethered to common sites by SU(HW) and instead plays an active role in recruitment and/or stabilization of the bound complex. Second, the sequence analysis of class 9 (CTCF+CP190) sites suggests that the binding of both proteins to these sites is likely due to the coincidence of cognate recognition sequences. Third, RNAi knock-down experiments indicate that BEAF-32 is dispensable for CP190 binding at shared sites. Clearly CP190 plays an active role in the selection of sites shared with SU(HW), CTCF or BEAF-32. It is still possible that once it co-binds, or binds sufficiently close to another insulator protein, it may mediate the trans-interactions of the bound sites. However, such interactions would have to be rather transient, at least in cultured cells, as they are not easily detected in the ChIP-chip data (Schwartz, 2012).
In Drosophila melanogaster, the dosage-compensation system that equalizes X-linked gene expression between males and females, thereby assuring that an appropriate balance is maintained between the expression of genes on the X chromosome(s) and the autosomes, is at least partially mediated by the Male-Specific Lethal (MSL) complex. This complex binds to genes with a preference for exons on the male X chromosome with a 3' bias, and it targets most expressed genes on the X chromosome. However, a number of genes are expressed but not targeted by the complex. High affinity sites seem to be responsible for initial recruitment of the complex to the X chromosome, but the targeting to and within individual genes is poorly understood. This study has extensively examined X chromosome sequence variation within five types of gene features (promoters, 5' UTRs, coding sequences, introns, 3' UTRs) and intergenic sequences, and assessed its potential involvement in dosage compensation. Presented results show that: (1) the X chromosome has a distinct sequence composition within its gene features, (2) some of the detected variation correlates with genes targeted by the MSL-complex, (3) the insulator protein BEAF-32 preferentially binds upstream of MSL-bound genes, (4) BEAF-32 and MOF co-localize in promoters, and (5) that bound genes have a distinct sequence composition that shows a 3' bias within coding sequence. Although, many strongly bound genes are close to a high affinity site neither the promoter motif nor the coding sequence signatures show any correlation to high affinity binding sites (HAS). Based on the results presented in this study, it is believed that there are sequences in the promoters and coding sequences of targeted genes that have the potential to direct the secondary spreading of the MSL-complex to nearby genes (Philip, 2012).
This study has thoroughly investigated X chromosome sequence variation in D. melanogaster and related this variation to the targeting of the dosage compensation complex, using frequencies of two to six base pair sequence 'words' and multivariate statistical analyses. The advantage of this approach is that it is unbiased and focused on finding sequences with predictive value, rather than merely over-represented sequences. First, the genome sequence was divided into intergenic, promoter, 5' UTR, coding, intron and 3' UTR sequences. Interestingly, there is more divergence among these six sequence types or gene features than within the sequence types on different chromosomes. The findings also show that sequences are present in promoters and coding sequence that could be involved in the spreading of the MSL-complex from the high affinity sites on the X chromosome. The coding sequences that were identified share a similar 3' bias with the MSL-complex. Further, the highest scoring promoter sequences form the target motif of the insulator protein BEAF-32, and BEAF-32 mapping data indicate that this protein binds preferentially upstream of genes strongly bound by MSL (Philip, 2012).
Different gene features are known to vary in sequence composition, but their variation is not normally taken into account in attempts to discover new sequence motifs. This study shows the extent of this sequence variation, and that coding sequences have the most distinct sequence composition followed by 5' UTRs, 3' UTRs and promoters. This has important implications for studies of sequence variation and motif discovery; when groups of sequences are compared it is important to take gene features into account (e.g. when using the MEME option of discriminative motif discovery), otherwise the results may reflect differences in gene feature composition rather than biologically relevant sequence variation (Philip, 2012).
The separate analyses of the six gene features clearly show that the sequence composition of those in the X chromosome differs from the composition of corresponding features in all other chromosomes. This distinction of the X chromosome is mainly due to differences in frequencies of various di-nucleotides, many of which have been previously found to be enriched on X . These sequences could, in principle, be involved in recruiting X chromosome-specific factors, such as the MSL-complex. Apart from being dosage-compensated in males, the X-chromosome might also be under selective forces that do not act on the autosomes. Some of the sequence variation of the X-chromosome is likely a result of its evolution as a sex chromosome. The MSL-complex is the only known protein complex involved in dosage compensation in Drosophila with an X chromosome-specific distribution. This study has focused on the sequence variation that could be related to the targeting of this complex. It has been shown that the MSL-complex is initially targeted to X by binding to so-called high affinity sites (HAS) that contain the GA-rich MSL recognition element (MRE)]. The MSL-complex can be recruited to autosomes by inserting MRE-containing high affinity sites, but the mechanism involved in the spreading of MSL to X-chromosomal genes is under debate. This study has investigated whether sequence patterns may be involved in this spreading of the MSL-complex, as discussed below (Philip, 2012).
The genome distribution of the MSL-complex has been mapped in several studies. This study used the data from (Kind, 2009) to select genes that are expressed and strongly MSL-bound, expressed and weakly MSL-bound as well as unexpressed genes. This is the only currently available dataset where mapping of several MSL-complex components and transcription in mutants/knock-downs of MSL-components was done in parallel and in the same cell-type. When merging all strongly MSL-bound expressed genes into one observation and all weakly MSL-bound expressed genes into another, it was found that all six sequence types have sequences that differ between strongly bound and weakly bound genes. It was observed that sequence variation between expressed genes strongly bound and weakly bound by MSL complex is much higher than that between expressed and unexpressed genes on chromosome X. Further, expressed genes that are weakly bound by the MSL complex group more closely to unexpressed genes than to expressed MSL-bound genes in Principal Component Analysis (PCA) score plots. Therefore, the small but significant expression difference detected between the expressed genes that are strongly bound and weakly bound by the MSL complex did not have any major correlation on the sequence variation observed between the two groups. Sequence words extracted from PCA models of intron, 3' UTR and 5' UTR sequences were more GA, CA or adenine rich, in agreement with the previous identification of CA dinucleotide repeats, runs of adenines and GA-rich MRE motif from High Affinity Sites (HAS). It is conclude that there are differences in sequences of all six features between expressed genes that are strongly bound and weakly bound by the MSL-complex. However, these results merely identify sequence words that are overrepresented in groups of genes strongly or weakly bound by MSL. In order to search for predictive sequence patterns for MSL-binding to individual genes, Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA, was applied (Philip, 2012).
Using OPLS-DA, differences were examined between features of individual genes that are strongly MSL-bound and expressed versus weakly MSL-bound and expressed, sequence words with the highest predictive power were extracted, and attempts were made to combine them into more complex motifs using the algorithm described in this study. Interestingly, both coding sequence and promoter models yielded sequence words that could be used to predict the MSL-binding status of genes excluded from the modeling. Neither nucleotide content nor expression level significantly influence these promoter and coding sequence models and the top sequence words that were identified are only weakly overrepresented on the X-chromosome. It is concluded that promoters and coding sequences contain sequence signatures that are potentially involved in the spreading of the MSL-complex from high affinity sites. In principle, there may be motifs in unbound, expressed genes that block the binding of the MSL-complex, but no evidence was obtained for such motifs (Philip, 2012).
From the promoter model a motif was extracted which could be used to predict promoters of genes strongly bound by MSL. This motif proved to correspond to the targeting motif for the insulator protein BEAF-32, which binds to hundreds of sites across the genome, generally located upstream of active genes. Although the molecular mechanisms of BEAF-32 activity are unknown, it seems to be linked with active transcription (Jiang, 2009). In order to test whether the BEAF-32 protein itself is enriched at strongly MSL-bound genes BEAF-32 ChIP-chip mapping data obtained from modENCODE was used, and it was found that BEAF-32 preferentially binds proximal to transcription start sites of genes strongly bound by MSL. This exciting link between BEAF-32 and dosage compensation is supported by the observation that beaf-32 mutants have a male-specific defect in X-chromosome morphology. Further, Laverty (2011) found that reporters inserted on the X chromosome are better able to recruit the MSL-complex if they have binding sites for GAGA and DREF factors. The DREF binding site is very similar to the BEAF-32 binding site and although DREF might be involved in dosage compensation it is possible that increased BEAF-32 recruitment is the true cause of the effects observed by Laverty. However, since DREF has not been mapped genome wide the possibility cannot be excluded that the promoter motif correlate better with DREF. BEAF-32 is associated with active transcription and might facilitate the MSL-complex targeting of active genes. Since MSL-complex bound genes show MOF binding in the promoter and MOF clearly co-localizes with BEAF-32, it is hypothesized that BEAF-32 and MOF interact in promoters of MSL-complex bound genes. BEAF-32 is a DNA-binding protein and might recruit MOF to active genes on the X-chromosome, genes that are then targeted by the MSL-complex. However, further experimental efforts are needed to understand the link uncovered in this study between BEAF-32 and the MSL-complex (Philip, 2012).
The finding of sequence patterns that are predictive of MSL-binding genes within coding sequences is intriguing. Scoring the sequence words only in the transcribed strand or the correct frame did not improve the coding sequence model, suggesting that the relationships are not attributable to (for instance) specific variations in amino acid composition. Neither was any codon usage bias between strongly bound and weakly bound expressed genes found, nor any model correlation with expression and AT-content. However, Orthogonal Partial Least Squares Discriminant Analysis found that bound coding sequences are rich in AG di-nucleotides, which have been previously reported to be abundant in dosage-compensated chromosomes (Philip, 2012).
The MSL-complex binds to genes with a preference for exons. The relatively low binding to introns might suggest that the complex targets spliced RNA transcripts. However, it was recently found that the complex targets chromatin rather than transcribed RNA. The exon specificity could be explained by various chromatin factors, nucleosome density and/or sequence specificity. Variations in nucleosome density may partially explain the exon bias, as it is higher in exons and thus may provide more targets for H4K16 acetylation, a modification that is strongly linked to the MSL-complex. In addition, the MSL-complex binding profile clearly shows that it binds most strongly towards the 3' end of genes. Accordingly, the models predicted the MSL-binding status of genes better from the 3' thirds than from the 5' thirds of the coding sequences. This is in contrast to the lack of 3' bias of the [G(GC)N]4 motif reported in another study. Taken together, these results strongly indicate that the MSL-complex distribution within genes on the X-chromosome is influenced by the primary DNA sequence (Philip, 2012).
The MSL-complex evidently targets a limited number of High Affinity Sites along the X-chromosome. Although, many strongly bound genes are close to a HAS neither the promoter motif nor the coding sequence signatures found in this study show any correlation to HAS. Based on the results presented in this study, it is believed that there are sequences in the promoters and coding sequences of targeted genes that have the potential to direct the secondary spreading of the complex to nearby genes. However, a number of genes are dosage-compensated by MSL-independent mechanisms and expression on the X-chromosome is only reduced to ~80% of wild type levels in males when msl genes are mutated or knocked down using RNAi. Apart from the dosage compensation mediated by the MSL-complex there is evidence for a more general buffering system that targets haploid regions in the genome. So other, as yet unknown, factors are likely involved in compensating the X chromosome and these factors could potentially act on a number of levels, such as transcription regulation, mRNA export, mRNA stability and translation. The observed optimal codon usage on the X-chromosome likely represents compensation on the translational level. However, even if additional factors involved in dosage compensation remain to be discovered, this study shows that there are plenty of sequences within all types of gene features that could act as X-targeting elements (Philip, 2012).
Editors Note: The following article by Gurudatta et al (2012) has been superseded by an article buy Craig Hart 'Do the BEAF insulator proteins regulate genes involved in cell polarity and neoplastic growth?' . PubMed ID: 24211761 . A role for BEAF in cell polarity and neoplastic growth is not observed with the null BEAFAB-KO allele (which is null, despite the claim by Gurudatta et al. that it is a hypomorph). Their claims are based on analysis of a chromosome with second-site mutations in addition to the null BEAFNP6377 mutation, and these confounded the analysis.
Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. This study found that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, a genome-wide analysis of BEAF-32 localization was carried out in wing imaginal disc cells. Mutation of BEAF-32 results in misregulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Upregulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Misregulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants (Gurudatta, 2012).
The BEAF-32 insulator protein is predominantly enriched near transcription start sites and associates with highly expressed genes. The role of this protein in transcription has only been examined previously at a specific subset of genes. Knockdown of BEAF-32 in S2 cells using RNAi results in reduced transcription of five out of six genes tested, whereas loss of BEAF-32 in Drosophila embryos carrying the BEAFAB-KO allele results in reduction of transcription of 19 out of 23 genes tested. Analysis of flies carrying the BEAFAB-KO mutation suggests that BEAF-32B is required for viability of adult files. This allele is female sterile and the maternal BEAF-32 protein is sufficient to drive development to adult. Additionally, these flies show abnormal X polytene chromosome morphology but structural defects are not pronounced, suggesting BEAFAB-KO could be a hypomorphic allele. In support of this, these mutants appear to have residual BEAF-32 protein as detected by western blot analysis. This study examined the effect of BEAF-32 on the transcriptome of wing imaginal disc cells by analyzing a null allele of BEAF-32 and shows that loss of this protein has a dramatic effect on transcription in wing imaginal disc cells, resulting in up-regulation and down-regulation of a large number of genes and suggesting a general role for this protein in gene expression (Gurudatta, 2012).
The mechanisms by which BEAF-32 affects gene expression during wing development are not clear. Based on the results described in this study it appears that BEAF-32 does not act as a classical transcription factor, since lack of BEAF-32 results in both up- and down-regulation of bound genes without an obvious correlation to levels of RNAPII or silencing histone modifications. It is then likely that, in spite of its location close to TSSs, BEAF-32 plays a role more directly related to its hypothesized function as a chromatin insulator. In mammalian cells the insulator protein CTCF has been identified in complexes associated with active RNAPII and has been shown to help target enhancers to the appropriate promoter. It is possible that BEAF-32 plays a similar role in Drosophila, helping recruit genes involved in specific cellular processes to transcription factories. In the absence of BEAF-32, these genes may fail to be recruited to these factories or may be recruited to alternative subnuclear compartments by other insulator proteins, resulting in the observed down- or up-regulation of their expression (Gurudatta, 2012).
Loss of function mutations in BEAF-32 result in developmental abnormalities characterized by neoplastic growth. This phenotype is similar to that of mutations in the lethal giant larvae and scrib genes. The loss of function BEAF-32NP6377 allele shows defects in development, cell identity and patterning. The wing imaginal discs lack features such as notum or pouch cells. The discs also lack patterning features such as anterior posterior axis (A-P) or markers such as wingless, normally expressed by organizer cells on the A-P axis. This study identified genes whose expression is affected by a factor of 1.5-fold or more in BEAF-32 mutants and examined the role of the encoded proteins in the context of these phenotypes. If only those genes that are bound by BEAF-32 are considered, that are likely direct targets of regulation by this protein, the nature of the affected genes can explain the observed phenotypes. These include genes involved in cell polarity, various signaling pathways, cell survival, cell patterning, and amino acid metabolism. Mechanisms involved in the establishment of cell-polarity are responsible not only for the diversification of cell shape but also participate in the regulation of asymmetric cell divisions of stem cells that are crucial for their correct self-renewal and differentiation. Disruption of cell polarity is a hallmark of cancer, and its establishment requires localization of the Crumbs, Stardust, Par6, Bazooka and aPKCs proteins to the apical membrane to specify the apical domain. The up-regulation of Bazooka in BEAF-32 mutant imaginal tissue could compete with aPkc for binding to PAR6, resulting in loss of epithelial morphogenesis. The Drosophila cell polarity proteins Scrib, Dlg and Lethal giant larvae (Lgl) are powerful tumor suppressors and loss of these proteins cause neoplastic transformation. These results suggest that mutation of BEAF-32 could affect cell polarity, epithelial morphology and Dlg localization. Perturbation of cell morphology is also known to activate the JNK pathway. Molecular profiling of BEAF-32 mutant tissue also shows misregulation of key mitogenic pathways, including Insulin growth factor, CDK5, Integrin, FAK, ERK/MAP kinase and JNK signaling. Insulin growth factor signaling is crucial for malignant transformation. The regulation of the IGF receptor is a link between oncogene and tumor suppressor pathways that has substantial impact on metabolic and proliferative pathways. Key molecules in these pathways are Insulin receptor 1 and p70-S6 kinase. These signaling events can activate other pathways such as PI3 kinase, AKT, PTEN and MAP kinase in BEAF-32 mutant cells. Activation of the JNK pathway in mutant cells appears to promote a proliferative role as suggested by the over-expression of unpaired 3 ligand and the target gene Socs35. The activated pathways causing increased cell proliferation could increase cell survival by counteracting hypoxia or oxidative stress through the activation of antioxidant response element (ARE) dependent genes or by inducing DNA double strand break response genes (Gurudatta, 2012).
Mutations in other insulator proteins, such as dCTCF and Mod(mdg4), result in homeotic transformations. The only other insulator protein that shows a strong tumor like growth phenotype is l(3)mbt, which has recently been shown to be required for CTCF/CP190-mediated function of the Fab8 insulato. However, unlike l(3)mbt, which plays a role in hyperplastic cell growth by activating Hippo pathway target genes, BEAF-32 mutations result in neoplastic overgrowth. BEAF-32 proteins are restricted to Drosophila species and have not been identified in higher organism. Nevertheless, a possible role of insulators in the expression of tumor suppressor genes in mammalian cells should be explored further (Gurudatta, 2012).
dREAM complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts, the distribution and localization of Drosophila E2F and dREAM proteins were examined. This study reports a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains (Korenjak, 2014).
The large number of E2F proteins has precluded all attempts to generate a comprehensive set of E2F binding sites in the human genome. This study has taken advantage of the simplicity of the Drosophila E2F family to examine the genome-wide distribution of E2F proteins. An unexpected finding from this analysis is that dE2F proteins strongly co-localize with insulator-binding proteins. The extent of overlap between dE2F2/dREAM and CP190 binding sites is comparable to the co-localization previously described for dCTCF and CP190, which are both required for insulator function at common binding sites. Furthermore, the co-localization between dREAM and Beaf-32 is even greater than that observed for Beaf-32 and CP190. This striking overlap of dREAM binding sites with proteins involved in nuclear architecture has exciting new implications for the function of dREAM complexes (Korenjak, 2014).
An interesting feature of dREAM bound genes is their strong enrichment in divergently paired genes (DPGs), genes that are transcribed in opposite direction, with their TSS separated by less than 1000 bp. Moreover, the set of genes de-regulated upon loss of dE2F2/dREAM include mostly DPGs that are differentially expressed, with one gene of the pair being stably repressed whereas its partner is actively transcribed. Inactivation of dREAM complex subunits or CP190 results in the loss of transcriptional integrity at these differentially expressed DPGs (Korenjak, 2014).
Several different models could account for the observed transcriptional up-regulation of the stably repressed and down-regulation of the actively expressed gene. First, dREAM/CP190/Beaf-32 sites might act as boundary elements at DPGs, separating an active from a repressed chromatin domain. Genome-wide binding maps for insulator-binding proteins revealed enriched binding to DPGs. In addition, these studies have shown that CP190 and Beaf-32 binding are significantly enriched at differentially expressed DPGs. The exact role of insulator-binding proteins at differentially expressed DPGs is still unclear. A recent study has shown that, upon inactivation of the SOX14 transcription regulator, DPGs that lack Beaf-32 binding show a significantly higher likelihood of concerted de-regulation of the two genes within a pair (up or down) than when Beaf-32 is present at the DPG. This suggests a role for Beaf-32 in the maintenance of independent regulation of gene expression at DPGs, consistent with a function as boundary factor. Moreover, CP190 binding sites are commonly found at the borders of large H3K27me3 domains, which are a hallmark of Polycomb-mediated silencing. These studies further show that inactivation of CP190 can, at a subset of regions, result in local spreading of H3K27me3 beyond the CP190 binding site. Although the repressive mechanisms might vary at different dREAM-regulated DPGs, several of the stably repressed genes display H3K27me3 over the length of the gene body. The possibility of dREAM and CP190 being important for the physical separation of distinct chromatin domains was tested by assessing the distribution of H3K27me3 over selected gene pairs. In agreement with the observed de-repression of the inactive gene, mutant animals displayed loss of H3K27me3 in the gene body. However, spreading of the mark into the active gene was not observed, suggesting that the down-regulation is achieved by a different mechanism or the level of reduction in gene expression is below the detection limit of our H3K27me3 ChIP (Korenjak, 2014).
Second, dREAM, together with CP190 and possibly Beaf-32, may be involved in the silencing of stably repressed genes. The repressive mechanism might include specific activities provided by CP190/Beaf-32. Both CP190 and Beaf-32 have been shown to be critical for the establishment of long-range chromatin interactions through looping mechanisms, which could be utilized to physically separate a stably repressed gene from the surrounding transcriptionally active chromatin environment. It is intriguing to speculate that dREAM, either by facilitating chromatin association of CP190 or even more directly, could be involved in the formation of these chromatin loops. Interestingly, the CP190 and Beaf-32 binding sites involved in long-range chromatin interactions are also prominent dREAM binding sites. Alternatively, Beaf-32 is known to compete for DNA binding with the transcriptional activator DNA replication-related element factor (DREF) (70). It has further been shown that, upon inactivation of Beaf-32, bound genes are specifically de-repressed when they also contain a DREF consensus site. In this scenario, loss of the repressive mechanism by inactivation of dREAM or CP190/Beaf-32 might either generate a vacant binding site for a transcription activator or result in the re-distribution of the general transcription machinery or a specific transcription activator from the actively expressed to the repressed gene (Korenjak, 2014).
Third, the possibility cannot formally be ruled out that dREAM acts on both components of differentially expressed DPGs, serving as a repressor for one and as an activator for the other gene. dREAM has been implicated in transcriptional repression as well as activation. However, dREAM complexes containing dE2F2 do not appear to be involved in gene activation and, based on genome-wide binding studies for dREAM subunits, there is no evidence for the presence of two independent dREAM peaks at differentially expressed DPGs (Korenjak, 2014).
Genome-wide association studies have identified a large number of binding sites for insulator-binding proteins, but only a few studies have addressed the potential enhancer-blocking activity of these DNA fragments. In these experiments, CP190 and dCTCF co-bound regions display strong enhancer-blocking activity compared to Su(Hw) bound sites. Moreover, it has been shown that sites associated with CP190 or CP190 and Beaf-32 exhibit strong enhancer-blocking activity, whereas sites occupied by any other combination of insulator-binding proteins show only weak or no enhancer blocking. In agreement with the importance of CP190 for the definition of elements with enhancer-blocking activity, dREAM-CP190 co-bound regions display robust enhancer-blocking in a cell-based assay system. The notion that dREAM functions as an enhancer-blocker may explain why a complex that is best known as a transcriptional repressor is almost exclusively found in euchromatic regions (Korenjak, 2014).
The dREAM subunits Mip130 and Mip120 are important for the observed enhancer-blocking activity, but the underlying mechanism is unclear. Work in Drosophila suggests that the propagation of a nucleosome-free region in insulator elements is required for enhancer blocking. It is possible that dREAM is needed for the establishment of nucleosome-free regions through recruitment of chromatin modifying activities or their maintenance through binding and stabilization of these regions, which in turn might be important for the recruitment of CP190 (Korenjak, 2014).
A detailed analysis of the enhancer-blocking function of the 1A2 insulator, which harbors Su(Hw) binding sites, shows that the region adjacent to the Su(Hw) sites is important for full enhancer-blocking activity of 1A2, even though this element by itself lacks any activity. It is conceivable that dREAM binding sites fulfill a similar 'facilitator' function for CP190 and/or Beaf-32 (Korenjak, 2014).
Although cell-based assays have been effectively used to measure the enhancer-blocking activity of characterized insulator elements, transfected plasmids are only partially chromatinized. In order to address the enhancer-blocking function of dREAM/CP190-bound regions in more detail and dissect the underlying mechanism it will, therefore, be interesting to test the identified elements in an in vivo enhancer-blocking assay (Korenjak, 2014).
The underlying mechanism(s) by which dREAM cooperates with CP190 is likely to be connected with the ability of dREAM to help establish or maintain CP190 chromatin association. Interestingly, CP190, but not Beaf-32 chromatin binding was reduced in dE2f2 mutant animals. Beaf-32 can bind to DNA in a sequence-specific manner through recognition of the CGATA motif. In contrast, CP190 does not possess known DNA-binding activity and is thought to get recruited indirectly by DNA-binding factors. This view is based on physical and functional interactions with sequence-specific insulator-binding proteins as well as the high degree of co-localization of these factors in genomic binding studies. Despite the high degree of overlap in their genomic binding profiles, the significance of Beaf-32 for CP190 chromatin association is unclear. Recent studies found contrary results regarding the significance of Beaf-32 for CP190 recruitment. CP190 chromatin association is reduced, however, in ctcf mutant animals and upon dCTCF knockdown in tissue culture cells. A recent study in Drosophila cells has shown that CP190 associates with the majority of its binding sites in distinct combinatorial patterns with other insulator-binding proteins. At a subset of binding sites, however, it does not co-localize with any known DNA-binding protein, suggesting that CP190 either has intrinsic DNA-binding activity or depends on a not yet identified factor for recruitment. Given the physical interaction between dREAM complex subunits and CP190, it is speculated that dREAM complexes may be directly involved in the recruitment of CP190 to these sites (Korenjak, 2014).
Interestingly, the strong enrichment of DPGs among dREAM-bound genes is conserved in human cells, but, to date, CTCF is the only known mammalian ortholog of fly insulator-binding proteins. Based on the extensive co-localization among insulator-binding proteins in the fly genome and the presence of CP190 as a common insulator co-factor, which suggests a shared mechanism, it has been proposed that the functions of the Drosophila proteins were integrated in CTCF. CP190 is a chromatin architectural protein, and it is conceivable that another protein with similar properties has adopted its role. Interestingly, the function of mammalian CTCF, including its role in chromatin looping, has been intimately linked to the Cohesin complex. Remarkably, inactivation of pRB in mammalian cells results in reduced chromatin association of Cohesin and the functionally related Condensin II-subunit Cap-D3. Furthermore, pRB physically interacts with Condensin II-subunits in fly and human cells and RBF1 co-localizes extensively with Cap-D3 on polytene chromosomes, raising the fascinating possibility that these specialized architectural protein complexes have taken over a CP190-related role in higher organisms (Korenjak, 2014).
Over the past years, several studies have shown that a variety of different proteins co-localize with insulator-binding proteins and/or contribute to insulator function. These factors range from different combinations of insulator proteins to factors like L(3)MBT, Topoisomerase II, the ubiquitin ligase dTopors, Ago2, the Rm62 helicase and exosome components. Further studies are clearly needed to determine how dREAM function is integrated with the array of factors acting in concert with insulator-binding proteins and to delineate a potential role of dREAM complexes in the organization of chromosome architecture (Korenjak, 2014).
Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. This study investigated the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and developed a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. BEAF32 was shown to be able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. A general model is proposed for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division (Vogelmann, 2014 PubMed).
The physical organization of eukaryotic chromosomes is key for a large number of cellular processes, including DNA replication, repair and transcription. Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription by two independent modes of action: 'enhancer blocking' insulators (EB insulators) interfere with communications between regulatory elements and promoters, whereas 'barrier' insulators prevent the spread of silenced chromatin states into neighboring regions. Recently, insulator elements have been implicated in chromosome architecture and transcription regulation through their predicted binding to thousands of sites genome-wide. For instance, insulators were shown to regulate transcription of distinct gene ontologies, to separate distinct epigenetic chromatin states, and to recruit H3K27me3 domains to Polycomb bodies (Vogelmann, 2014).
In Drosophila, five insulator families have been identified, that differ by their DNA-binding protein (insulator binding protein, or IBP): Suppressor of Hairy-wing [Su(Hw)], boundary element-associated factor (BEAF32), Zeste-white 5 (Zw5), the GAGA factor (GAF), and dCTCF, a distant sequence homologue of mammalian CTCF. Two BEAF32 isoforms exist (BEAF32A and BEAF32B). This paper, considers BEAF32B (which will be referred to as BEAF32). BEAF32B represents more than 95% of the binding peaks detected by chip-seq in cell lines, BEAF32A binding does not play a role in the insulating function of BEAF , and BEAF32A expression is not essential for the development of embryos in adult flies. IBPs are often necessary but not sufficient to ensure insulation activity at a specific locus, and several insulator co-factors have been shown to be additionally required. Particularly, Centrosomal Protein 190 (CP190), a protein originally described for its ability to bind to the centrosome during mitosis, was shown to play a crucial role in the insulation function of various IBPs (Vogelmann, 2014).
Insulator proteins often associate in clusters of overlapping binding sites more often than would be expected by chance, suggesting that these factors often bind as a complex to the same genetic locus. For instance, BEAF32, dCTCF and CP190 binding sites most often cluster with at least another factor (~70, ~77 and >90%, respectively). In addition, insulators show a large compositional complexity, as demonstrated by the frequencies of binding of different combinations of insulator associated proteins: CP190 associates with its most common partner BEAF32 (~50%), but also to a lesser extent to dCTCF and Su(HW), while BEAF32, dCTCF, and CP190 cluster together in >15% of CP190 binding sites. This compositional complexity may be key to understanding the locus-specific functions of insulators (Vogelmann, 2014).
A critical feature of Drosophila and vertebrate insulators is their ability to form specific long-range DNA interactions (hereafter LRIs). Three-dimensional loops have been implicated in all levels of chromatin organization ranging from kb-size loops to larger intra-chromosomal loops hundreds of kb in size. To date, it is unclear what factors provide the physical interactions required for the formation and regulation of LRIs. In addition to binding the specific insulator sequences, IBPs have been proposed to be sufficient to bridge two distant DNA molecules. However, other factors such as CP190, Mod(mdg4), or cohesin have been implicated in the formation of LRIs (Vogelmann, 2014).
The observation that most CP190 binding sites co-localize with insulator binding proteins (>90%) prompted the hypothesis that CP190 is a common regulator of different insulator classes. CP190 is composed of a BTB (bric-a-brac, tramrack, and broad complex)/POZ (poxvirus and zinc-finger) domain, four predicted C2H2 zinc-finger motifs, and an E-rich, C-terminal region. Importantly, CP190 has been recently shown to preferentially mark chromatin domain barriers. These barriers are also heavily bound by other insulator proteins, such as BEAF32, dCTCF and to a lesser extent Su(HW), and have been shown to often form LRIs. Overall, these data suggest a role for CP190 in participating in the three dimensional folding of the genome by the formation of long-range interactions (Vogelmann, 2014).
Surprisingly, a second factor, called Chromator, was also shown to be overrepresented at physical domain barriers. During mitosis, Chromator forms a molecular spindle matrix with other nuclear-derived proteins (Skeletor and Megator). In contrast, during interphase Chromator localizes to inter-band regions of polytene chromosomes and plays a role in their structural regulation as well as in transcriptional regulation. Chromator can be divided into two main domains, a C-terminal domain containing a nuclear localization signal, and an N-terminal domain containing a chromo-domain (ChD) required for proper localization to chromatin during interphase (Vogelmann, 2014).
This study investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means. A unique assay was developed to determine what factors are necessary and essential for the formation of long-range DNA interactions, and BEAF32 was shown to be necessary but not sufficient to bridge long-range interactions. In contrast, addition of CP190 or Chromator is sufficient to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several IBPs. A general model is proposed for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). The multiplicity of interactions between insulator binding and associated proteins could thus explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators (Vogelmann, 2014).
Chromatin insulators promote higher-order nuclear organization through the establishment and maintenance of distinct transcriptional domains. Notably, this activity requires the formation of barriers between chromatin domains and the establishment of specific LRIs. In this paper, this paper has investigated the molecular mechanism by which insulator proteins bind DNA, interact with each other and form long-range contacts (Vogelmann, 2014).
Recently, genome-wide approaches have been used to investigate the roles of different insulator types in genome organization. Insulators enriched in both BEAF32 and CP190 are implicated in the segregation of differentially expressed genes and in delimiting the boundaries of silenced chromatin. Notably, BEAF32 and CP190 are often found to bind jointly to the same genetic locus (>50% of CP190 binding sites contain BEAF32). However, the molecular origin of this genome-wide co-localization was unknown as there was no direct proof of interaction between these proteins. This study has shown that BEAF32 is able to interact specifically with CP190 in vitro and in vivo. In particular, this interaction was shown to be mediated by the C-terminal domain of CP190, with no implication of the C2H2 zinc-finger or the BTB/POZ domains, consistent with previous studies showing that the N-terminus of CP190 was not essential for its association with BEAF32 in vivo. BEAF32 interacts specifically and cooperatively with DNA fragments containing CGATA motifs, consistent with previous observations. In contrast, the binding of CP190 to DNA showed lower affinity and no specificity and required its N-terminal domain (containing four C2H2 zinc-fingers). Overall, these data suggest that one pathway for CP190 recruitment to DNA genome-wide requires specific interactions of its C-terminal domain with BEAF32. Other factors, such as GAF, are likely also involved in the recruitment of CP190 to chromatin, explaining why RNAi depletion of BEAF32 does not lead to the dissociation of CP190 from an insulator binding class containing high quantities of BEAF32 and CP190. It is possible that post-translational modifications in CP190 may also allow it to bind DNA directly and specifically, providing a second pathway for locus-specific localization (Vogelmann, 2014).
In addition to acting as chromatin barriers, insulators have been typically characterized for their ability to block interactions between enhancers and promoters through the formation of long-range contacts. Here, this study has developed a fluorescence cross correlation-based assay that allowed investigation of the ability of BEAF32, CP190 and their complex to bridge specific DNA fragments, mimicking LRIs. Specific LRI can be stably formed between two DNA fragments containing BEAF32 binding sites, solely in the presence of both BEAF32 and CP190. Interestingly, LRI are displaced by competition in trans with the BTB/POZ domain of CP190, and LRIs are not observed in the presence of BEAF32 and CP190-C. Thus, both protein domains are required for the bridging activity of CP190. These data strongly suggest that the C-terminal domain is responsible for BEAF32-specific contacts whereas the N-terminal domain of CP190 is involved in the formation of LRI through CP190/CP190 contacts. The role of the N-terminal domain of CP190 in protein-protein interactions is consistent with previous studies showing that N-terminal fragments of CP190 containing the BTB/POZ domains co-localize with full-length CP190 in polytene chromosomes (Vogelmann, 2014).
BTB/POZ are a family of protein-protein interaction motifs conserved from Drosophila to mammals, and present in a variety of transcriptional regulators. BTB/POZ are found primarily at the N-terminus of proteins containing C2H2 zinc-finger motifs, and can be monomeric, dimeric, or multimeric. In fact, a recent study proposed that isolated CP190-BTB/POZ domains can exist as dimers or tetramers in solution. The oligomerization behavior of CP190-BTB/POZ could have important implications for the role and mechanism by which CP190 bridges LRIs. This study showed that the BTB/POZ domains of CP190 forms homo-dimers with a large, conserved interaction surface, consistent with these domains being responsible for the formation of the direct protein-protein interactions required for the establishment of long-range contacts. Interestingly, the oligomerization of CP190-BTB/POZ into homo-dimers implies a binary interaction between two distant DNA sequences, imposing important constraints for the mechanisms of DNA bridging by CP190 (Vogelmann, 2014).
In addition to interacting with BEAF32, CP190 is able to directly interact with other insulator binding proteins, such as dCTCF, Su(HW), and Mod(Mdg4), or with the RNA interference machinery. These interactions are usually mediated by the C-terminal domain of CP190, but a role for the C2H2 zinc-finger or the BTB/POZ domains in providing specific protein-protein contacts cannot be discarded. In fact, an interesting feature of several homo-dimeric BTB/POZ domains is their ability to recruit a multitude of protein partners using a single protein-protein binding interface. For instance, several transcriptional co-repressors (BCOR, SMRT and NCor) are able to bind with micromolar affinity (2:2 stoichiometry) to the BTB/POZ domain of BCL6, despite their low sequence homology. In this case, the mechanism of binding involves the formation of a third strand by the N-terminus of co-repressors folding onto the two strands exchanged by the BCL6-BTB/POZ monomers on their interface, with the rest of the minimal domain of interaction (10 residues) winding up along the lateral groove of the BCL6-BTB/POZ dimer. In the case of CP190, the sequence and structural features of the conserved peptide binding groove within insect CP190-BTB/POZ domains suggest that the dimer interface of CP190 may act as a protein-protein interaction platform. Thus, the ability of BTB/POZ domains to form dimers and the promiscuous binding of CP190 to different insulator binding proteins (Su(HW), dCTCF, and BEAF32) suggest not only that insulators share protein components, but also that CP190 may bridge long-range contacts involving distinct factors at each end of the DNA loop. This model is consistent with previous proposals, and with the requirement of both C- and N-terminal domains of CP190 for fly viability. Importantly, it provides a rationale for CP190 being a common factor between insulator binding proteins (Vogelmann, 2014).
CP190 frequently binds with additional insulator binding proteins (~85%), with BEAF32 and dCTCF being the most common partners (~50% and ~25%, respectively), and Su(Hw) amongst the least frequent partner (~20%). Importantly, BEAF32 does not show clustering with either dCTCF or Su(HW) in the absence of CP190 (<0.5% or ~0.1%, respectively), suggesting that the clustering of two insulator binding proteins requires CP190. The ability of CP190 to mediate LRIs between sites harboring different insulator binding proteins raises important questions: Are these LRIs specific? How is this specificity regulated? Are other factors or post-translational modifications involved in this selectivity? Future research will be needed to address these important questions (Vogelmann, 2014).
Chromator localizes to inter-band regions of polytene chromosomes and binds to the barriers of physical domains genome-wide, however the mechanism leading to these localization patterns has been lacking. Previous studies showed that BEAF32 and Chromator co-localize at some genomic sites, and suggested that these proteins may participate in the formation of a single comple. This study showed that BEAF32 directly and specifically interacts with Chromator in vivo and in vitro. This interaction is mediated by the C-terminal domain of Chromator, thus the ChD domain does not seem to be directly involved in interactions with BEAF32. The results show that Chromator possesses a reduced affinity for DNA and binds with no sequence specificity to loci displaying strong Chromator binding peaks at the site tested (Tudor-SN locus). Thus, it is suggested that specific interactions between BEAF32 and Chromator may be responsible for its recruitment to polytene inter-band regions and domain barriers. Significantly, most BEAF32 binding sites genome-wide (>90%) contain Chromator, suggesting an almost ubiquitous interaction between the two factors (Vogelmann, 2014).
Interestingly, Chromator also co-localizes with the JIL-1 kinase at polytene inter-band regions and the two proteins directly interact by their C-terminal domains. JIL-1 is an ubiquitous tandem kinase essential for Drosophila development and key in defining de-condensed domains of larval polytene chromosomes. Importantly, JIL-1 participates in a complex histone modification network that characterizes active, de-condensed chromatin, and is thought to reinforce the status of active chromatin through the phosphorylation of histone H3 at serine 10 (H3S10). Thus, BEAF32 could be responsible for the recruitment of the Chromator/JIL-1 complex to active chromatin domains to prevent heterochromatin spreading. This mechanism would be consistent with the observation that BEAF32 localizes primarily to de-condensed chromatin regions in polytene chromosomes, is implicated in the regulation of active genes and delimits the boundaries of chromatin silencing (Vogelmann, 2014).
CP190 is a common partner of BEAF32, dCTCF, and Su(HW), and has been thus proposed to play a role in the formation of long-range interactions at these insulators. On the other hand, both CP190 and Chromator have been recently shown to be massively overrepresented at barriers between transcriptional domains. This pape shows that only when CP190 or Chromator are present can long-range interactions between BEAF32-bound DNA molecules be generated. Strong evidence is provided that the formation of in vitro LRI requires three ingredients: (1) binding of BEAF32 to its specific DNA binding sites; (2) specific interactions between the C-terminal domains of CP190/Chromator and BEAF32; and (3) homo- interactions between CP190/Chromator molecules mediated by their N-terminal ends (Vogelmann, 2014).
To further investigate the roles of CP190 and Chromator in the formation of LRIs, statistically relevant contacts containing specific combinations of insulator factors were aggregated together from Hi-C data from embryos. This analysis shows a relatively high correlation between the presence of BEAF32 and both CP190 and Chromator in sites displaying a high proportion of interacting bins between distant BEAF32 sites, as compared with neighboring sites (16.9% of interacting bins for Chromator and CP190 sites). Thus, CP190 and Chromator may play a role at a subset of genetic loci by mediating and/or stabilizing interactions between BEAF32 and a distant locus bound by BEAF32 or a different insulator binding protein. Interestingly, the binding of BEAF32 to CGATA sites as multimers, and the existence of CP190-Chromator interactions suggest that long-range interactions at a single locus could involve hybrid/mixed complexes comprising at least these three factors (Vogelmann, 2014).
These observations suggest a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). Direct or indirect interactions of first layer insulator proteins with additional factors (e.g. JIL-1, NELF, mediator) are very likely involved in directing alternative activities (e.g. histone modifications, regulation of RNAPII pausing) to specific chromatin loci. This model provides a rationale for the compositional complexity of insulator sequences and for the multiplicity of functions often attributed to insulators (e.g. enhancer blocker, chromatin barrier, transcriptional regulator). Ultimately, a characterization of the locus-specific composition of insulator complexes and their locus-specific function may be required to obtain a general picture of insulator function. In mammals, CTCF is the only insulator protein identified so far, but other factors, such as cohesin have been identified as necessary and essential for the formation of CTCF-mediated long-range interactions. Mammalian CTCF contains eleven zinc-fingers, and it has been shown that different combinations of zinc-fingers could be used to bind different DNA sequences. Thus, in mammals CTCF may play the role of first layer insulator protein, whereas other factors such as cohesin or mediator may play the role of second layer insulator proteins (Vogelmann, 2014).
This model proposing different functional roles for insulator factors could also explain the mechanism by which insulators are able to help establish and reinforce the transcriptional state of chromatin domains throughout cell division. First layer proteins remain bound to chromatin at all stages of the cell cycle. In contrast, both CP190 and Chromator are chromatin-bound during interphase but display a drastic redistribution during mitosis: CP190 strongly binds to centrosomes while Chromator co-localizes to the spindle matrix. Thus, the dissociation and cellular redistribution of second layer insulator proteins during cell division would be responsible for the massive remodeling of chromosome architecture occurring during mitosis, and for the re-establishment of higher-order contacts at the onset of interphase. In contrast, first layer insulator proteins would act as anchor points for the re-establishment of higher-order interactions after mitosis, and for the maintenance of the transcriptional identity of physical domains. Thus, this model suggests distinct roles for insulator binding proteins and co-factors in actively shaping the organization of chromatin into physical domains during the cell cycle. This model is consistent with recent genome-wide data suggesting that, overall, first layer insulator proteins remain bound to their binding sites during mitosis, whereas second layer insulator proteins tend to show a large change in binding patterns. Further genome-wide and microscopy experiments will be needed to quantitatively test this model (Vogelmann, 2014).
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).
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 (Kd ~ 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).
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).
Precise transcriptional control is dependent on specific interactions of a number of regulatory elements such as promoters, enhancers and silencers. Several studies indicate that the genome in higher eukaryotes is divided into chromatin domains with functional autonomy. Chromatin domain boundaries are a class of regulatory elements that restrict enhancers to interact with appropriate promoters and prevent misregulation of genes. While several boundary elements have been identified, a rational approach to search for such elements is lacking. With a view to identifying new chromatin domain boundary elements genomic regions were examined between closely spaced but differentially expressed genes of Drosophila melanogaster. A new boundary element between myoglianin and eyeless, ME boundary, was identified that separates these two differentially expressed genes. ME boundary maps to a DNaseI hypersensitive site and acts as an enhancer blocker both in embryonic and adult stages in transgenic context. It is also reported that BEAF and GAF are the two major proteins responsible for the ME boundary function. These studies demonstrate a rational approach to search for potential boundaries in genomic regions that are well annotated (Sultana, 2011).
BEAF is the major player in the boundary function of ME boundary as evident from genetic data and the effect that BEAF has on the ME boundary is by direct binding to the ME region as is evident from the ImmunoFISH and ChIP data. It is already known that BEAF binds to the scs boundary as a heterotrimer at the CGATA sites and ME boundary has similar arrangement of the CGATA sites. Some scattered CGATA motifs are also present in the ME boundary. ChIP data shows that BEAF binds to the core region of ME where two palindromic CGATA sites and one additional CGATA site are present. The importance of these BEAF binding sites is also evident from the fact that when these sites were mutated, boundary activity of ME is lost (Sultana, 2011).
The ME boundary also contains binding sites for GAF. The pattern of GAF binding sites in ME boundary is similar to that seen in the case of Fab-7 boundary present in the bithorax complex of D. melanogaster. This prompted an examination of whether GAF has any effect on the boundary activity of ME. The results show that GAF is also a positive regulator of the ME boundary function as loss of single copy of GAF results in partial loss of the boundary function of ME. This effect is by direct binding of GAF to the ME sequence as seen in the ImmunoFISH and ChIP experiments. In case of GAF, it was observed that the effect of loss of GAF was more dramatic in female flies, which was opposite to what was see in the case of BEAF. Since both these proteins, specially GAF, regulate a large number of loci and GAF has also been implicated in dosage compensation, it is likely that the sex specific effect seen here in the case of ME boundary may be a result of complex and indirect interaction of multiple factors (Sultana, 2011).
It is shown that both BEAF and GAF are needed for ME boundary activity. However, either BEAF or GAF (Trl) mutations alone were not sufficient for the complete loss of the boundary function. Since flies with BEAFAB-KO/BEAFAB-KO;P/TrlR85 genotype were lethal, it remains an open question whether BEAF and GAF can account for the complete boundary function of ME. Synthetic lethality in the double mutant BEAFAB-KO/BEAFAB-KO;P/TrlR85 does, however, suggest that these two proteins act in combination at the key loci and that this combination is essential for viability. There might be several such loci working as boundary elements and the double mutant combination, by abolishing or weakening a number of such boundaries, would cause misregulation of associated genes and lead to lethality (Sultana, 2011).
ME boundary function is by recruitment of BEAF and GAF along with, perhaps, several other proteins although BEAF appears to be the major player as mutation in BEAF binding sites abolishes boundary function. Relatively lower level of GAF enrichment at ME, as seen in ChIP experiments, may also indicate an indirect role of this protein at this locus. Minor but distinct effect of Polycomb and trithorax group mutations on ME boundary function was observed. The data, although suggestive and preliminary, indicate that ME boundary functions by recruiting multiple proteins, mutants of which lead to a partial loss of the boundary function. This mode of boundary function is similar to the other well studied gypsy boundary which depends on large number of factors including Su(Hw), Mod(mdg4, CP190 and dTopors that associate with lamina. Boundary function of gypsy was also shown to depend on Polycomb and trithorax group of proteins. While no prominent effect was seen of CTCF or CP190 on ME boundary activity, which is expected as ME region does not contain binding sites for these proteins, genome wide ChIP studies do detect association of these factors with ME. It is possible that ME may be part of nuclear structures where multiple boundaries cluster and number of factor participate even if not by direct binding to each boundary (Sultana, 2011).
In conclusion, a rationale to look for boundary elements in short intergenic regions that separate differentially expressed genes can be applied successfully. Although expression pattern of a number of genes has not been analyzed in many organisms, analysis in other model organisms and human can be used and by homology criteria, large part of a genome can be mapped for potential boundary elements. Once a boundary region has been identified, the precise mapping of the functional boundary element can be accomplished by DNaseI hypersensitivity and transgene based assays available in model systems. Such studies will help in understanding the genomic organization and regulatory environment of genes (Sultana, 2011).
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).
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).
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
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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 ID: 12459265
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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 ID: 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 ID: 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 ID: 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 ID: 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 ID: 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 ID: 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 ID: 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 ID: 18654629
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date revised: 23 October 2014
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