InteractiveFly: GeneBrief

Chromator: Biological Overview | References

BIOLOGICAL OVERVIEW

The chromodomain protein, Chromator, can be divided into two main domains, a NH(2)-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal. During interphase Chromator is localized to chromosomes; however, during cell division Chromator redistributes to form a macromolecular spindle matrix complex together with other nuclear proteins that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis. It has previously been demonstrated that the CTD is sufficient for targeting Chromator to the spindle matrix. This study shows that the NTD domain of Chromator is required for proper localization to chromatin during interphase and that chromosome morphology defects observed in Chromator hypomorphic mutant backgrounds can be largely rescued by expression of this domain. Furthermore, this study shows that the ChD domain can interact with histone H1 and that this interaction is necessary for correct chromatin targeting. Nonetheless, that localization to chromatin still occurs in the absence of the ChD indicates that Chromator possesses a second mechanism for chromatin association and evidence is provided that this association is mediated by other sequences residing in the NTD. Taken together these findings suggest that Chromator's chromatin functions are largely governed by the NH(2)-terminal domain whereas functions related to mitosis are mediated mainly by COOH-terminal sequences (Yao, 2012).

The chromodomain protein, Chromator, has multiple functions depending on the developmental context (Rath, 2006; Mendjan, 2006; Wasser, 2007; Ding, 2009). During interphase Chromator is localized to interband regions of Drosophila polytene chromosomes (Rath, 2004; Gortchakov, 2005) and has been demonstrated to interact with other chromosomal proteins such as the zinc-finger protein Z4 (Eggert, 2004; Gan, 2011) and the histone H3S10 kinase JIL-1 (Rath, 2006) and to contribute to the maintenance of polytene chromosome morphology (Rath, 2006). However, during cell division Chromator redistributes to form a macro molecular spindle matrix complex together with at least three other nuclear derived proteins Skeletor, Megator, and EAST (Walker, 2000; Rath, 2004; Qi, 2004; Qi, 2005). It has recently been proposed that this structure may take the form of a hydrogel-like matrix with viscoelastic properties that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis (Johansen, 2011). Evidence that Chromator may participate in spindle matrix function has been provided by mutational analysis with two loss-of-function alleles, Chro⁷¹ and Chro⁶¹² (Ding, 2009). The analysis showed that neuroblasts from Chro⁷¹/Chro⁶¹² brain squash preparations have severe microtubule spindle and chromosome segregation defects that were associated with a developmental small brain phenotype. Furthermore, time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the chromosome segregation defects were the results of incomplete alignment of chromosomes at the metaphase plate (Ding, 2009), possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase (Yao, 2012).

Chromator can be divided into two main domains, an NH₂-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal (Rath, 2004). The studies of Ding (2009) showed that the CTD of Chromator was sufficient for localization to spindles and that expression of this domain alone could partially rescue mutant spindle defects. However, the function of the NTD and whether it plays a role in targeting Chromator to chromatin was not determined. This study provides evidence that the NTD of Chromator is responsible for correct targeting to chromatin, that it interacts with histone H1, and that the chromodomain is required for these interactions (Yao, 2012).

This study shows that the NTD domain of Chromator is required for proper localization to chromatin and that chromosome morphology defects observed in Chromator mutant backgrounds can be largely rescued by expression of this domain. Evidence is provided that the ChD domain can interact with histone H1 suggesting that this interaction is necessary for the correct chromatin targeting. Nonetheless, that localization to chromatin still occurs in the absence of the ChD indicates that Chromator possesses a second mechanism for chromatin association, and evidence is provided that this association is mediated by other sequences residing in the NTD. Such an association could in principle be mediated by other molecular interaction partners of Chromator that also localize to chromatin such as JIL-1 or Z4. However, studies in S2 cells with RNAi mediated Chromator depletion and in JIL-1^z2 homozygous null mutant backgrounds demonstrated that neither protein was dependent on the other for its chromatin localization (Rath, 2006). The interaction of Chromator with Z4 was identified in co-immunoprecipitation experiments and the two proteins colocalize extensively at interband polytene regions (Eggert, 2004). Recently, Gan (2011) provided evidence that Chromator and Z4 may directly interact and that localization of Z4 to chromatin depends on Chromator, but not vice versa. Another candidate for mediating chromatin localization is Skeletor (Walker, 2000). The interaction between Chromator and Skeletor was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays (Rath, 2004). Immunocytochemical labeling of Drosophila embryos, S2 cells, and polytene chromosomes demonstrated that the two proteins show extensive co-localization during the cell cycle although their distributions are not identical (Rath, 2004). During interphase Chromator is localized on polytene chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. During mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure with Chromator additionally localizing to centrosomes that are devoid of Skeletor-antibody labeling. Thus, the extensive co-localization of the two proteins is compatible with a direct physical interaction; however, at present it is not known whether such an interaction occurs throughout the cell cycle or is present only at certain stages, with additional proteins mediating complex assembly at other stages (Rath, 2006). Regardless, it is likely that Chromator together with Skeletor functions in at least two different molecular complexes, one associated with the spindle matrix during mitosis and one associated with nuclear and chromatin structure during interphase (Rath, 2004). Furthermore, taken together the findings of the present study and those of Ding (2009) suggest that Chromator's chromatin functions are largely governed by the NH₂-terminal domain whereas functions related to mitosis are mediated by COOH-terminal sequences. The molecular mechanisms of how the two distinct chromatin binding affinities residing within the NH₂-terminal domain of Chromator interact to confer proper localization to interbands remains to be elucidated (Yao, 2012).

An important feature of the Chromator protein is the presence of a chromodomain, the only conserved motif found in database searches (Rath, 2004; Gortchakov, 2005). Structure determination of the prototype chromodomain has revealed a small, three-stranded antiparallel β-sheet supported by an α-helix that runs across the sheet. Classic chromodomains contain three conserved aromatic amino acids that confer binding affinity for methylated histone H3. However, several chromodomains have been identified that vary at some of these structurally important positions but that still conform well to the overall folding of the prototype chromodomain. One example of this is the chromo-shadow domain also found in HP1a that is a protein-protein interaction domain that allows HP1a to homodimerize via its α-helices. In addition, various chromodomains have been demonstrated to bind to a wide variety of proteins including transcription corepressors, remodeling ATPases, lamin B receptor, and chromatin assembly factors. Thus, relatively small sequence variations in the otherwise conserved structural scaffold of chromodomains can confer considerable variation in molecular interactions. Evidence is provided by modeling that the chromodomain of Chromator is likely to adopt the canonical chromodomain tertiary configuration very similar to the chromodomain of HP1a. However, due to amino acid substitutions at two of the three conserved aromatic amino acid positions it is not likely to bind to methylated histone H3. Rather this study provides evidence by overlay and pull down assays that it binds to the linker histone H1. A candidate region for providing such a binding fold or surface is the additional α-helical stretch found in the chromodomain of Chromator just prior to the main α-helix of the chromodomain structure. In future experiments it will be of interest to further determine the structural basis for the interaction of Chromator with histone H1 and specifically how the chromodomain contributes to Chromator's role in nucleosome and chromatin organization (Yao, 2012).

Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture

Genome-wide studies has identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). It is hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. It was determined that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, the interactions of each enhancer class were mapped utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity (Cubenas-Potts, 2017).

This study characterize the protein occupancy, chromatin interactions and architecture profiles for the two enhancer classes found in Drosophila. Each enhancer class has distinct H3K4 methylation states, is bound by both common and distinct architectural proteins, and is involved in distinct types of chromatin interactions. First, it was established that hkCP enhancers exclusively bind CAP-H2, Chromator, DREF and Z4, while dCP enhancers do not and are preferentially enriched for but not exclusively bound by Fs(1)h-L and Rad21. In addition, hkCP enhancers are more likely than dCP enhancers to associate with multiple TSSs, which promotes a higher transcriptional output. Finally, hkCP enhancers preferentially associate with topologically associating domain (TAD) borders, whereas dCP enhancers are enriched at chromatin loop anchors present inside TADs. Interestingly, enhancers activated by both core promoters exhibit more hkCP enhancer like characteristics, indicating that the both CP enhancers may represent an intermediate among the distinctive hkCP and dCP enhancers. Altogether, these results provide strong correlative evidence, supporting a model suggesting that architectural proteins are critical regulators of enhancer-promoter interaction specificity and that the interactions between enhancers and promoters significantly contribute to the generation of 3D chromatin architecture (Cubenas-Potts, 2017).

The importance of architectural proteins in regulating enhancer-promoter interactions in Drosophila is supported by the observation that the vast majority of architectural protein sites present in the genome correspond to enhancers and promoters. Historically, architectural proteins were identified as insulators, which were functionally demonstrated to block enhancer-promoter interactions. The insulator function of architectural proteins correlates with their enrichment at TAD borders. However, several lines of evidence, including ChIA-PET analysis of CTCF- and cohesin-mediated interactions in mammals, suggest that these architectural proteins help mediate long range contacts among regulatory sequences. In Drosophila this study observed that nearly all of the Group 1 and Group 2 architectural protein sites are associated with enhancers or promoters defined by STARR-seq, TSSs or CBP peaks, suggesting that architectural proteins help mediate enhancer-promoter interactions. Notably, Group 3 architectural proteins include the classic insulator proteins CTCF, CP190, Mod(mdg4) and SuHw, and at least 25% of their peaks cannot be explained by enhancers or promoters. It is interesting to speculate that the non-enhancer-promoter sites may be involved in more classical insulator functions or contributing to the chromatin architecture of inactive regions of the genome (Cubenas-Potts, 2017).

The conclusion that architectural proteins are critical regulators of the specificity between enhancers and promoters is supported by two main lines of evidence. First, the current results demonstrate a strong correlation between each enhancer class and distinct architectural protein subcomplexes. Functional evidence supporting this conclusion comes from mutational analyses of the DRE motif in the distinct enhancer classes, which likely recruits DREF and the other hkCP enhancer associated architectural proteins. Zabidi (2015) demonstrated that the tandem DRE motif alone was sufficient to enhance expression of the housekeeping core promoter and that mutation of DRE motifs within an hkCP enhancer reduced its promoter interactions in a luciferase assay. Furthermore, addition of a DRE motif to a dCP enhancer changed its promoter specificity. Because DREF and potentially BEAF-32 bind to the DRE motif, these results strongly support a model suggesting that the differential occupancy of Cap-H2, Chromator, DREF and Z4 in the two enhancer classes is a critical regulator of their specific interactions with the core promoter types. However, the data cannot discount the notion that unique transcription factor binding at proximal TSSs also contribute to the specificity of enhancer-promoter interactions. Although hkCP enhancer identity is most highly correlated with CAP-H2, Chromator, DREF and Z4 localization, these four architectural proteins are not found in isolation within hkCP enhancers. BEAF-32 and CP190 are also strongly enriched in hkCP enhancers, which are also associated with high occupancy APBSs and TAD borders. Thus, the full architectural protein complement at hkCP enhancers is far more complex than the four hkCP-specific architectural proteins. In addition, architectural proteins that are truly unique to dCP enhancers were not detected. Because dCP enhancers exhibit higher cell type specificity, it cannot be discounted that there are additional dCP enhancers present in the Drosophila genome that were not identified by STARR-seq and thus, excluded from this analysis. From these studies, it is unclear if the enrichment of Fs(1)h-L and Rad21, particularly because Fs(1)h-L and Rad21 are present in hkCP enhancers at lower levels, or the absence of BEAF-32, CAP-H2, Chromator, CP190, DREF and Z4 truly distinguishes the architectural protein complexes found at dCP enhancers. In the future, careful biochemical analyses will be required to gain a comprehensive understanding of the complete organization of architectural protein subcomplexes associated with each enhancer class (Cubenas-Potts, 2017).

hkCP enhancers are associated with multi-TSS chromatin interactions and TAD borders. The promoter-clustering by hkCP enhancers results in a dose-dependent increase in transcriptional output for the interacting genes. Thus, one likely molecular mechanism by which hkCP enhancers promote robust transcriptional activation is by increasing the local concentration of RNA Polymerase II and general transcription factors (GTFs) by bringing multiple TSSs into close proximity. It is interesting that the hkCP enhancers, which form promoter clusters, are associated with TAD borders. It is speculated that the hkCP enhancer interactions involve inter-TAD contacts within the A-type compartment, indicative of the formation of transcription factories (70). From this analysis, it is unclear if the hkCP enhancers alone are sufficient for the formation of the 3D interactions or the neighboring TSSs and their associated transcription factors are also contributing to these contacts. It is hypothesized that the genes recruited to the factories contain the housekeeping promoter motifs (DRE, Ohler 1, Ohler 6 and TCT) and that the hkCP enhancer residents Cap-H2, Chromator, DREF and Z4, are critical to the formation of these 3D contacts (Cubenas-Potts, 2017).

dCP enhancers are more likely to be present within TADs and are enriched on the subTAD-like chromatin loop anchors. dCP enhancers do not form promoter clusters, but are more likely to interact with individual TSSs. One possible explanation for this observation is that the genes interacting with dCP enhancers require the binding of sequence-specific transcription factors, and increasing the concentration of GTFs and RNA polymerase II is not an effective mechanism to promote transcriptional output. The chromatin loop association is consistent with dCP enhancers forming a strong contact with a single TSS. However, it is acknowledged that dCP enhancers are likely one of multiple molecular mechanisms contributing to chromatin loop formation. Surprisingly, the chromatin loops that were observed in Drosophila are distinct from the chromatin loops described in humans. A recent study reported approximately 10,000 chromatin loops in the genome of GM12878 lymphoblastoid cells, but this study detected only 458 chromatin loops in Drosophila utilizing a similar method. The reason why there are so few chromatin loops in Drosophila compared to humans is unclear. It is possible that chromatin loops represent a more precise level of architecture within TADs between specific enhancers and promoters in mammals, but because TADs are significantly smaller in flies (median size 32.5 kb compared to 880 kb in mice, the chromatin loops are not as prominent or easily detected in the Drosophila genome. Notably, it appears that the chromatin loops are generated by different architectural proteins in the two species. The chromatin loops in humans are anchored by convergent CTCF motifs, while the results presented in this study demonstrate that the chromatin loop anchors in Drosophila are depleted of CTCF. Because the chromatin loops in Drosophila show a strong enrichment for Fs(1)h-L, a Brd4 homolog, and the architectural proteins Rad21, Nup98, TFIIIC and Mod(mdg4), it is possible that a combination of transcription and architectural proteins is required for chromatin loop formation in flies, which may be different from mammals . Altogether, it is clear that dCP enhancers are involved in individual contacts with TSSs and are likely one mechanism by which chromatin loops form in Drosophila (Cubenas-Potts, 2017).

Surprisingly, only ~20% and ~12.5% of all hkCP enhancer and ~7.5% and ~8.5% of dCP enhancer interactions involve a TSS or enhancer on the opposite anchor, respectively. The biological significance of the enhancer to non-TSS association is unclear. One possible explanation is that current methods for identifying statistically significant interactions are not sufficiently robust and that many of the enhancer to non-TSS interactions are not representative of biologically significant contacts. However, it cannot be discounted that the non-TSS interactions mediated by enhancers are real and the biological significance of these contacts remains to be determined. Throughout this analysis, the patterns of TSS interactions were compared with each enhancer class instead of drawing conclusions about the absolute number of TSSs bound per enhancer, minimizing the impact of any non-specific interactions within the data. Additional molecular studies for the various type of enhancer interactions (enhancer to promoter, enhancer to non-TSS, etc.) will be required to evaluate the various biological contributions of each (Cubenas-Potts, 2017).

This study found that the functional differences between enhancers that activate housekeeping versus developmental genes are reflected in their chromatin and architectural protein composition, and in the type of interactions they mediate. hkCP enhancers are marked by H3K4me3, associate with TAD borders, and mediate large TSS-clustered interactions to promote robust transcription. This class of enhancers contain the architectural proteins CAP-H2, Chromator, DREF and Z4. In contrast, dCP enhancers are marked by H3K4me1, associate with chromatin loop anchors and are more commonly associated with single TSS-contacts. dCP enhancers are depleted of the hkCP-specific architectural proteins and show an enrichment for Fs(1)h-L and Rad21. The results support a model suggesting that the unique occupancy of architectural proteins in the distinct enhancer classes are key contributors to the types of interactions that enhancers can mediate genome-wide, ultimately affecting enhancer-promoter specificity and 3D chromatin organization. In the future, further characterization of the broadly defined housekeeping and developmental enhancers into smaller subclasses may yield additional levels of regulation and formation of unique architectural protein and transcription factor protein complexes as key mediators of long range chromatin contacts (Cubenas-Potts, 2017).

Transcriptional cofactors display specificity for distinct types of core promoters

Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression. Although some COFs have been shown to prefer certain promoter types over others, the extent to which different COFs display intrinsic specificities for distinct promoters is unclear. This study used a high-throughput promoter-activity assay in Drosophila melanogaster S2 cells to screen 23 COFs for their ability to activate 72,000 candidate core promoters (CPs). Differential activation of CPs was observed, indicating distinct regulatory preferences or 'compatibilities' between COFs and specific types of CPs. These functionally distinct CP types are differentially enriched for known sequence elements, such as the TATA box, downstream promoter element (DPE) or TCT motif, and display distinct chromatin properties at endogenous loci. Notably, the CP types differ in their relative abundance of H3K4me3 and H3K4me1 marks, suggesting that these histone modifications might distinguish trans-regulatory factors rather than promoter- versus enhancer-type cis-regulatory elements. The existence was confirmed of distinct COF-CP compatibilities in two additional Drosophila cell lines and in human cells, for which COFs were found that prefer TATA-box or CpG-island promoters, respectively. Distinct compatibilities between COFs and promoters can explain how different enhancers specifically activate distinct sets of genes, alternative promoters within the same genes, and distinct transcription start sites within the same promoter. Thus, COF-promoter compatibilities may underlie distinct transcriptional programs in species as divergent as flies and humans (Haberle, 2019).

To systematically test intrinsic COF-CP preferences for many CPs in a standardized setup, a plasmid-based high-throughput promoter-activity assay and self-transcribing active core promoter-sequencing (STAP-seq) were combined with the specific GAL4 DNA-binding-domain (GAL4-DBD)-mediated recruitment of individual COFs. Using this assay in S2 cells, is this study tested whether 13 different individually tethered D. melanogaster COFs, representing different functional classes and enzymatic activities (two acetyltransferases (P300/CBP and Mof), three H3K4-methyltransferase-complex components (Lpt, Trr and Trx), two chromo and chromo-shadow-domain COFs (Chro and Mof) and three bromodomain COFs (Brd4, Brd8 and Brd9), the mediator complex subunits MED15 and MED25, and two less well-characterized COFs (EMSY and Gfzf) could activate transcription from any of 72,000 CP candidates, 133 base pair (bp) long DNA fragments around a comprehensive genome-wide set of transcription start sites (TSSs) and negative controls. If a tethered COF activates a candidate CP, this generates reporter RNAs with a short 5' sequence tag, derived from the 3' end of the corresponding CP. These reporter transcripts were captured with a 5' RNA linker that includes a 10 nucleotide (nt) long unique molecular identifier (UMI), enabling counting of individual reporter RNA molecules and quantifying of productive transcription initiation events at single-base-pair resolution for all candidate CPs in the library (Haberle, 2019).

Three independent COF-STAP-seq screens for each of the 13 COFs and positive (P65) and negative (GFP) controls in S2 cells were highly similar (all pairwise Pearson's correlation coefficients (PCCs) ≥ 0.89) and showed more initiation events for P65 and the 13 COFs than for GFP, as expected. Initiation mainly occurred at CPs corresponding to annotated gene starts, whereas random negative controls showed the least initiation, corroborating previous findings that gene CPs are specialized sequences, able to strongly respond to activating enhancers (Haberle, 2019).

Each COF showed differential activation of CPs and activated a unique set of CPs. For example, within a representative genomic locus, MED25 and Lpt most strongly activated the CP of CG9782, Chro and Gfzf most strongly activated the CP of RpS19a, and Mof most strongly activated the CPs of mbt and SmG. Indeed, the activation profiles of the COFs across all CPs were characteristically different, as revealed by hierarchical clustering. The differential CP activation by luciferase reporter assays were validated with MED25, Lpt, Mof and Chro for 50 CPs. The two assays agreed well (PCCs ≥ 0.72 except Mof, with PCC = 0.58), and it was confirmed that COFs activate some CPs more strongly than others (MED25, for example, preferentially activates the CPs on the left, Mof preferentially activates those in the middle, and Chro preferentially activates those on the right), which are refered to as distinct preferences, specificities, or 'compatibilities' towards different CPs (Haberle, 2019).

To test whether the COF-CP compatibilities generalize beyond S2 cells, three independent COF-STAP-seq screens were performed for six COFs (MED25, P300, Lpt, Gfzf, Chro and Mof) in two additional D. melanogaster cell lines, one derived from embryos (Kc167) and one from adult ovaries (ovarian somatic cells (OSCs)). For each of the six COFs, the screens were highly similar across all three cell lines (all PCCs ≥ 0.69), validating the distinct CP preferences of the COFs and the observed COF-CP compatibilities. These results establish the observed COF-CP compatibilities as a cell-type-independent, COF- and CP-sequence-intrinsic regulatory principle (Haberle, 2019).

To test whether the COF-CP preferences reflect endogenous gene regulation, the binding was assessed of each COF to genomic CPs of genes expressed in S2 cells. Published chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for P300, Brd4, Trx18, Trr18, Lpt19 and Mof20 from S2 cells, and Chro from D. melanogaster embryos showed stronger COF binding at CPs that were strongly activated in STAP-seq by the respective COF (top 25%) and weaker binding at CPs that were more weakly activated (bottom 25%). Next, the COF-CP preferences were compared with the impact of COF inhibition or depletion on endogenous gene expression. Analyses of published gene expression data upon COF inhibition with small molecules (P300) or RNA interference (RNAi; Brd4 and Trx18) revealed that genes associated with the top 25% of CPs preferentially activated by P300, Brd4 or Trx displayed stronger downregulation upon inhibition of the respective COF, compared to genes associated with the bottom 25% of CPs. Conversely, the CPs of all genes that are downregulated upon inhibition of P300, Brd4 or Trx showed stronger activation by the respective COF in STAP-seq than the CPs of genes not affected by COF inhibition. Together, these results suggest that the distinct COF-CP preferences that were observed are employed during endogenous gene regulation in vivo (Haberle, 2019).

The observed COF-CP compatibilities suggest the existence of distinct CP classes that differentially respond to specific COFs. To address this, K-means clustering was used to define groups of CPs with similar responses. Around 75% of the variance can be explained by five CP groups, which were activated preferentially by: (1) MED25, P300, and strongly by P65; (2) MED25, P300, and weakly by P65; (3) Mof, and weakly by Lpt and Chro; (4) Chro and Gfzf; and (5) Gfzf. Although additional types of CPs are likely to exist in more specialized cell types such as germline cells, screening ten additional COFs—including subunits of prominent COF complexes with diverse enzymatic activities (for example, SAGA, ATAC, NuA4/Tip60 and Enok) and general transcription factors (GTFs; for example, TBP, Trf2 and Taf4)—did not reveal additional CP types in S2 cells, presumably because each of the additional COFs was highly similar to at least one of the original 13 COFs (Haberle, 2019).

Given that COF-STAP-seq measures COF-CP compatibility in an otherwise constant reporter setup, distinct compatibilities are likely to arise from differences in CP sequences. Indeed, the five groups of CPs displayed marked differences in the occurrence of known CP motifs. Group 1 is strongly enriched for the TATA box and a variant of the DPE, whereas group 2 is enriched for a different DPE variant. By contrast, groups 1 and 2 are depleted in motifs Ohler 1, 6 and 7, and in the DNA replication-related element (DRE), all of which are enriched in group 3 and to a lesser extent in group 4. Group 4 is the only group with a strong enrichment for the TCT motif that is known to occur in the promoters of genes encoding ribosomal proteins and other proteins involved in translation, which are indeed among the top 10% of CPs preferentially activated by Chro. In accordance with the differential occurrence of CP motifs, published datasets reveal differential binding of GTFs to these CPs in their endogenous genomic contexts. For instance: the TATA-binding protein (TBP) bound more strongly to group 1 CPs, which are enriched for the TATA box; TAF1 bound more strongly to group 1 and 2 CPs, which are enriched for the Inr motif; and motif 1-binding protein (M1BP) and DRE factor (DREF) bound more strongly to group 3 CPs, which are enriched for motif 1 and DRE. Last, the TBP paralogue TRF2 bound more strongly to group 3, 4 and 5 CPs, consistent with reports that TRF2 regulates ribosomal protein genes. The differences in motif occurrence and GTF binding between the CP groups suggest that COF compatibility might relate to GTF composition at the CP, which is determined by the CP sequence (Haberle, 2019).

The CP groups defined by their COF responsiveness are reminiscent of groups previously defined on the basis of motif content and transcription initiation patterns that differ in chromatin properties, gene function and expression, and enhancer responsiveness. The dataset used in this study might provide a functional link between these observations and the activation of distinct CP types by specific COFs. Indeed, group 1 and 2 CPs are associated with genes that are expressed highly variably across cells in Drosophila embryos and have cell-type-specific or developmental functions, whereas group 3 and 4 CPs are associated with genes that are expressed more uniformly and have housekeeping functions. Furthermore, both the upstream sequences and the nearest enhancers of these CPs were enriched for transcription-factor binding motifs known to occur preferentially in developmental versus housekeeping enhancers, and developmental and housekeeping enhancers indeed preferentially activated group 1 and 2 versus group 3 and 4 CPs, respectively, when tested by STAP-seq. Together, these results directly link enhancer-CP specificity to COF-CP compatibility (Haberle, 2019).

Because COFs can modify nucleosomes and alter the chromatin structure, the endogenous genomic contexts of the five CP groups were tested in S2 cells (only considering CPs of active genes). Nucleosome positioning, DNA accessibility and histone modifications all differed between the CP groups: group 1 and 2 CPs have broader DNA accessible regions around the TSSs and lower nucleosome occupancy and nucleosome phasing downstream of the TSS, compared with group 3 and 4 CPs, which have more narrow nucleosome-depleted regions around the TSS and strongly phased downstream nucleosomes (Haberle, 2019).

Unexpectedly, the CP groups also differed in the methylation status of histone 3 lysine 4 (H3K4). H3K4me3 is thought to be universally associated with active promoters, and indeed strongly marks CPs of groups 3, 4 and 5. By contrast, group 1 and 2 CPs have lower levels of H3K4me3 but higher levels of H3K4me1 compared with group 3, 4 and 5 CPs, a modification that is typically considered an enhancer mark. This difference is consistent with the differential binding of Trr and Set1, which deposit H3K4me1 and H3K4me3, respectively, and does not seem to stem from higher levels of Pol II binding or transcription at group 3, 4 or 5 CPs. Consistent with reports that developmental promoters lack H3K4me3, these results suggest that high levels of H3K4me3 versus H3K4me1 might not be a universal feature of promoters that distinguishes them from enhancers as previously suggested, and instead might depend on the COFs that regulate the respective promoters. Indeed, ranking all active CPs in S2 cells by their H3K4me1:H3K4me3 ratio revealed that those with the highest ratio are preferentially activated by P300 and MED25, and those with the lowest ratio are preferentially activated by Mof or Chro (Haberle, 2019).

To test whether regulatory compatibilities between COFs and CPs exist in other species, proof-of-principle screens were performed in human HCT116 cells for five human COFs (BRD4, MED15, EP300, MLL3 and EMSY) and P65, using a focused library containing 12,000 human CP candidates selected to cover the diversity of human CPs. These screens reveal that CPs also respond differently to different COFs in human cells: whereas the TATA-box-containing CP of REN is, for example, only activated by MED15 and P65, the CpG-island CP of IRAK1 responds most strongly to MLL3; and the tested COFs consistently displayed distinct CP-preferences across the entire CP library. Overall, the CPs most strongly activated by MED15 are enriched for TATA boxes, whereas CPs preferentially activated by MLL3 exhibit a higher GC and CpG content, suggesting that MLL3--but not MED15--preferentially activates CpG-island promoters. Together, this establishes that sequence-encoded COF-CP compatibilities exist in species as divergent as fly and human, suggesting that they constitute a general principle with important implications for transcriptional regulation (Haberle, 2019).

The regulatory compatibilities between COFs and CPs that were observed enable separate transcriptional programs to independently regulate not only different genes, but also alternative promoters and thus different isoforms of the same gene. Notably, composite promoters with differentially activated closely spaced TSSs exist and enable regulation by different COFs and programs, potentially in different developmental contexts. As the CP types differ in sequence elements, these might instruct the assembly of functionally distinct pre-initiation complexes (PICs) that differ in GTF composition or create distinct rate-limiting steps that require activation by different COFs, enabling specific and synergistic regulation. The existence of regulatory COF-CP compatibilities impacts promoter activation and gene expression in endogenous contexts and biotechnological applications and, together with other mechanisms that determine enhancer-promoter targeting in the context of the three-dimensional chromatinized genome, helps to explain how different genes or alternative promoters can be distinctly regulated in species as divergent as flies and humans (Haberle, 2019).

The Chriz-Z4 complex recruits JIL-1 to polytene chromosomes, a requirement for interband-specific phosphorylation of H3S10

The conserved band-interband pattern is thought to reflect the looped-domain organization of insect polytene chromosomes. Previously, it has been shown that the chromodomain protein Chriz (Chromator) and the zinc-finger protein Z4 (Putzig) are essentially required for the maintenance of polytene chromosome structure. This study shows that both proteins form a complex that recruits the JIL-1 kinase to polytene chromosomes, enabling local H3S10 phosphorylation of interband nucleosomal histones. Interband targeting domains were identified at the N-terminal regions of Chriz and Z4, and the data suggest partial cooperation of the complex with the BEAF boundary element protein in polytene and diploid cells. Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific histone H3S10 phosphorylation (Gan, 2011).

The chromatin proteins Chriz and Z4 both are ubiquitous and essential proteins that are required for the maintenance of interphase chromosome structure. Chriz and Z4 directly interact by their central and N-terminal domains, respectively. The Z4 N-terminal domain is required for Z4 interband targeting, mediated by Chriz interaction. When overexpressed, it competes the chromosomal binding of the endogenous Z4 protein. The Chriz N-terminal domain is required for interband targeting of the Chriz protein by an as-yet unknown mechanism. Destabilization of the complex results in strong down-regulation of H3S10 phosphorylation on polytene chromosomes (Gan, 2011).

Surprisingly, neither the Chriz chromodomain nor the Z4 zinc-finger region is needed for chromosomal targeting and no interaction partners for these signature protein domains are yet identified. However, both domains provide important functions. Point mutations in the Chriz chromodomain are unable to complement Chriz mutations. Point mutations within the Z4 zinc-finger region are not yet available, but overexpression of the N-terminal Z4 fragment aa 1-516 including the zinc-finger region results in a 'shrunken' interband phenotype, although neither overexpression of the Z4 full-length protein nor of N-Z4 aa 1-240 affect chromosomal structure. This indicates a dominant negative effect for the zinc-finger region in the aa 1-516 construct (Gan, 2011).

Data from coimmunoprecipitation and colocalization in situ suggest that Chriz and Z4 interact with the boundary element binding protein BEAF and the H3S10-specific histone kinase JIL-1. Chriz and JIL-1 directly interact in vitro (Rath, 2006), but it remains to be shown if the JIL-1-Z4 interaction is also direct or mediated by Chriz protein. Also, direct interaction between Chriz–Z4 and BEAF still has to be demonstrated. Unlike the Chriz-Z4 interaction (Gortchakov, 2005), neither the BEAF-Z4 nor the JIL-1-Z4 colocalization is 100%. All four proteins are located in many interbands, but in each case there are sites that are occupied by one or the other protein exclusively. The mechanism explaining this promiscuity in interaction is not yet fully understood. However, it has been demonstrated that interband proteins can be shared between different chromatin protein complexes. For instance, CP190 is present in complexes with BEAF or Su(Hw) but not both. Furthermore, CP190 binds to CTCF with ~50% of CP190-CTCF complexes also bound by BEAF (Gan, 2011).

Mutual interactions could be mediated both by cooperation or competition between different factors for local binding. It is worth mentioning that Chriz also shows a significant overlap with CP190 in polytene chromosome binding in situ and in ChIP on chip experiments on Drosophila cell lines. The overlap with both BEAF and CP190, both well known for their activity as boundary element factors, suggests that Chriz and Z4 may be involved in the control of boundaries, a hypothesis that is currently being tested. The qualitative impression of colocalization gained from polytene chromosome staining is confirmed at higher resolution by ChIP on chip data from diploid S2 cells. Although these data still await a more systematic analysis, it is evident that in some regions the match in BEAF/Chriz binding sites is rather convincing but elsewhere it is more moderate. JIL-1 also colocalizes with Chriz but often shows a broader distribution overlapping several Chriz peaks or extending from a Chriz peak into an adjacent region. Strikingly, for the two cases of well-studied interband regions at 3C6-7 and 61C7-8, binding of BEAF, Chriz and JIL-1 is also observed in S2 diploid cells, suggesting that this stretch of open chromatin is conserved in structure between cell lineages. In the case of 3C6-7, the binding may be correlated with Notch transcription in S2 cells. However, there is no Notch transcription in salivary glands. The transcriptional activity at interband 61C7-8 is not known (Gan, 2011).

The effect of reducing the function of Chriz, Z4 and JIL-1 on polytene chromosome structure was studied by the use of hypomorphic alleles and tissue-specific RNAi knockdown. In general, it results in a phenotype of progressive loss of distinct band interband structure, sometimes concomitant with partial condensation or torsional distortion of polytene chromosomes. Deng (2008) reported that targeting of active JIL-1 kinase resulted in ectopic histone H3S10 phosphorylation and local chromosome decondensation that was dependent on JIL-1 kinase activity. It was reasoned that a major role of the Chriz-Z4 complex might be the recruitment of JIL-1 to the chromosome that would result in local phosphorylation of nearby nucleosomes, allowing the ≥30 nm chromatin fibre to unfold and form a less condensed interband chromatin. In support of this hypothesis, gland-specific Chriz-RNAi resulted in decreased levels of chromosomally bound Z4 and JIL-1 and in a significant loss of interband specific H3S10 phosphorylation. However, not all chromosomes exhibited the loss of structure phenotype mentioned above. Conceivably, RNAi induction might have been too mild and there was still enough Chriz protein left to provide for the maintenance of chromosome structure. The experiments were done at 22-24°C, a temperature at which the GAL4 inducer is still not at its maximal activity. Raising the temperature to 29°C early on made the inducer more effective but resulted in a tiny salivary gland phenotype that was not amenable to cytological analysis. Using the Sgs4 enhancer active in late 3rd instar at 29°C was not effective since the Chriz protein with its long half-life masked the RNAi effect. A different explanation for the lack of chromosomal phenotype may be that 10%-20% of the JIL-1 binding sites that do not depend on Chriz/Z4 are not affected by Chriz-RNAi and are sufficient to sustain the chromosome structure (Gan, 2011).

In conclusion, this study provides evidence for a chromatin complex with the chromodomain protein Chriz at its core. The complex, which, besides the Z4 protein, may contain the sequence-specific DNA-binding protein BEAF, is required for H3S10 phosphorylation of interphase chromosomes, presumably by local recruitment of the tandem kinase JIL-1. Local binding and activity of the complex on polytene chromosomes may result in decondensation of interband regions and related sites on nonpolytene diploid chromosomes (Gan, 2011).

Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome

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 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).

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).

The Drosophila Enhancer of Split gene complex: architecture and coordinate regulation by Notch, Cohesin and Polycomb group proteins

The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains twelve genes that inhibit neural development. In a cell line derived from central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E(spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. This study shows that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment (NICD) to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture this study found that the E(spl) C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. It is posited that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain (Schaaf, 2013).

These studies investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the Chromator-Putzig (Chro-Z4/Pzg; Z4 is an alternative name for Putzig) protein complex in CNS derived BG3 cells, in which the E(spl)-C has a rare restrained state with a cohesin-H3K27me3 overlap. The E(spl)-C has a highly self-interactive structure that is unexpectedly independent of these protein complexes and the level of gene expression. Depletion of any of these three protein complexes, however, significantly increases E(spl)-C transcription. The effects of these three protein complexes on E(spl)-C expression likely reflect changes in expression of Notch ligands, and in the cases of cohesin and PRC1, potentially direct effects on activator and Pol II activity at the E(spl)-C genes (Schaaf, 2013).

Chromosome conformation capture (3C) analysis revealed that the E(spl)-C has a structure in which all positions within the complex interact with each other at a high frequency, but not with flanking regions. Surprisingly, this study found that this architecture is independent of cohesin, the PcG complexes, the Chro-Pzg/Z4 complex, transcription, and stage of the cell cycle. Thus it is not known which factors establish this striking architecture, which defines the E(spl)-C as a structurally independent domain. It is also not yet known which factors control recruitment of cohesin and PcG complexes to the locus. It is speculated, however, that this architecture coordinates transcriptional control of the entire E(spl)-C, based on the finding that in BG3 cells, cohesin, PRC1, and the ubiquityl-Histone H2 (H2Aub) and H3K27me3 histone modifications made by the PRC1 and PRC2 complexes are co-extensive within this architectural domain. Although no known insulators or boundary elements flank the E(spl)-C, and depletion of the CP190 protein required for activity of all known Drosophila insulators does not alter E(spl)-C expression, it is likely that the unknown factors that form this structure limit the spread of these protein complexes and histone marks. The E(spl)-C architectural domain may be evolutionarily significant, because Notch-regulated Enhancer of split complexes with similar structures are conserved in insects and crustaceans over 420 million years (Schaaf, 2013).

Possible clues to the identities of the factors that control the E(spl)-C architecture and/or the recruitment of cohesin and PcG complexes may arise in genetic screens for factors that alter E(spl)-C sensitive phenotypes, such as the Nspl-1 rough eye and bristle phenotypes. These phenotypes are sensitive to mutations in the E(spl)-C and cohesin genes in a highly dosage-sensitive manner, and modest changes in the E(spl)-C architecture or recruitment of cohesin or PcG proteins may have similar effects (Schaaf, 2013).

There is coordinate regulation of gene complexes by cohesin in mammalian cells. The Protocadherin beta (Pchdb) gene complex is downregulated in the embryonic fibroblasts and brains of mice heterozygous mutant for the Nipbl cohesin loading factor, and brains of mice that are homozygous mutant for the SA1 cohesin subunit, and cohesin is involved in enhancer-promoter looping in the Protocadherin alpha (Pchda) complex, helping determine which genes in the complex are active. While this is a positive role for cohesin, as opposed to the repressive role that occurs in the E(spl)-C, it is possible that the protocadherin gene clusters also have a higher order architecture that dictates how cohesin functions within the gene complex. Recent genome-wide analysis also indicates that there are constitutive higher order looping architectures that may organize cell-type specific interactions on a shorter scale, and that cohesin contributes to both types of structures (Schaaf, 2013).

Prior studies showed that depletion of cohesin or PRC1 increases expression of the Serrate Notch ligand gene. This likely explains part of the increase in E(spl)-C transcription upon cohesin and PRC1 depletion, because the E(spl)-C genes are directly activated by Notch. Consistent with this idea, this study detected increases in NICD association with the HLHmβ and HLHm3 genes upon cohesin or PRC1 depletion. EDTA treatment confirms that increasing Notch activation increases NICD binding to the E(spl)-C genes (Schaaf, 2013).

Because cohesin and PRC1, unlike the Chro-Pzg/Z4 complex, bind directly to the E(spl)-C, it is also possible that they also directly control association of NICD with the Su(H) protein bound upstream of the active genes. For example, they could potentially interact with NICD or the Su(H) complex, and interfere with NICD association, or somehow facilitate ubiquitination and degradation of NICD. The lack of an effect of cohesin or PRC1 depletion on NICD association with E(spl)-C genes after EDTA treatment does not rule out this possibility, because under these conditions, the amount of NICD is no longer limiting (Schaaf, 2013).

It remains to be determined if the multiple effects of cohesin on Notch function seen in Drosophila, including regulation of Notch ligand and target genes, also occur in mammals. If so, this could underlie many of the development deficits seen in Cornelia de Lange syndrome, caused by mutations in NIPBL and cohesin subunit genes. Mutations in Notch receptor and ligand genes cause Alagille and other syndromes that affect many of the same tissues as CdLS (Schaaf, 2013).

The possibility cannot be ruled out that cohesin and PRC1 directly repress E(spl)-C transcription independently of any effects on Notch ligand expression or NICD association with the E(spl)-C genes. This is because both bind throughout the complex, and the PRC1-generated H2Aub repressive histone mark is co-extensive with the E(spl)-C architectural domain. Importantly, all genes in BG3 cells that show rare overlap of cohesin and the PRC2-generated H3K27me3 modification, such as the invected and engrailed gene complex, show substantial increases in transcription upon cohesin or PRC1 depletion, even though they are not Notch activated. It is highly unlikely that cohesin or PRC1 depletion increases the expression of all the diverse activators that control these genes, and more likely that cohesin and PRC1 directly repress their transcription (Schaaf, 2013).

At all genes examined that are strongly repressed by cohesin, cohesin restricts the transition of paused RNA Pol II into elongation, irrespective of whether or not they have the H3K27me3 mark (Fay, 2011). PRC1 restricts entry of paused Pol II into elongation at active genes that bind cohesin and PRC1, but lack PRC2 and the H3K27me3 modification (Schaaf, 2013).

It is thus posited that cohesin and PRC1 together restrict transition of the paused Pol II present at the active E(spl)-C genes into elongation. Because co-depletion of cohesin and PRC1 does not synergistically increase transcription, it is thought likely that they function together at the same step. Cohesin and PRC1 directly interact with each other, and cohesin facilitates binding of PRC1 to active genes that lack the H3K27me3 mark. Cohesin depletion, however, does not significantly alter PRC1 association with the E(spl)-C, likely because PRC1 binding is stabilized by the known interaction of PRC1 with H2K27me3. PRC1 is thus not sufficient to repress E(spl)-C transcription in the absence of cohesin, indicating that cohesin has roles that extend beyond its interaction with PRC1 (Schaaf, 2013).

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. This study shows that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro⁷¹ and Chro⁶¹² have severe microtubule spindle and chromosome segregation defects that are associated with a reduction in brain size. The microtubule spindles formed are incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes are lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggests that the lagging and scattered chromosome phenotypes are caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restores both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects are directly attributable to lack of Chromator function (Ding, 2009).

The co-localization and interactions of Chromator with the spindle matrix complex during mitosis (reviewed in Johansen, 2007) suggests that Chromator may be involved in spindle matrix function (Rath, 2004). A spindle matrix has been hypothesized to provide a stationary or elastic molecular matrix that can provide a substrate for motor molecules to interact with during microtubule sliding and which can stabilize the spindle during force production. Thus a prediction of the spindle matrix hypothesis is that if such a scaffold were interfered with, it would affect the assembly and/or dynamic behavior of the microtubule associated spindle apparatus and lead to abnormal chromosome segregation. This study has examined the phenotypic consequences of loss-of-function Chromator mutations on cell division in third instar larval brains. Mitotic neuroblasts from Chro⁷¹/Chro⁶¹² brain squash preparations have severe tubulin spindle and chromosome segregation defects that are associated with a reduction in brain size. The microtubule spindles at metaphase are incomplete, unfocused, and/or without clear spindle poles. At anaphase chromosomes are lagging and scattered indicating impaired spindle function, a phenotype similar to that previously obtained by Chromator RNAi depletion in S2 cells (Rath, 2004). Expression of full-length Chromator transgenes under endogenous promoter control partly or completely restores both viability, microtubule spindle morphology, as well as brain size strongly indicating that the observed mutant defects are directly attributable to lack of Chromator function. Thus, these data provide evidence that Chromator is a nuclear derived protein that plays a role in proper spindle dynamics leading to chromosome separation during mitosis (Ding, 2009).

In previous studies RNAi depletion of the spindle matrix proteins Skeletor, Megator, and EAST in S2 cells did not reveal any obvious microtubule spindle or chromosome segregation defects (Rath, 2004 and Qi, 2004). However, recently using live imaging of Megator RNAi-depleted S2 cells, Lince-Faria (2009) showed that Megator and its human ortholog Tpr function as spatial regulators of the spindle-assembly checkpoint that ensures the efficient recruitment of Mad2 and Mps1 to unattached kinetochores at the onset of mitosis for proper spindle maturation. This study provides evidence that Mad2 localization is similarly affected after Chromator depletion by RNAi and that Chromator-depleted cells have a lower mitotic index, suggesting that Chromator, like Megator, is required for proper SAC response (Ding, 2009).

The observed Chromator mutant phenotypes could be arrived at in a number of ways. For example, they could be caused by incomplete microtubule spindle formation and failure of the chromosomes to congress. However, time-lapse analysis of mitosis in Chromator RNAi-depleted S2 cells suggested that bipolar microtubule spindle formation was relatively normal and that chromosomes congressed to the metaphase plate. However, in most cases their alignment was incomplete and as anaphase commenced the microtubule spindle often frayed and became unstable resulting in lagging and scattered chromosomes. These observations are compatible with the hypothesis that Chromator may constitute a functional component of a spindle matrix molecular complex that serves to stabilize the microtubule spindle apparatus during anaphase and is necessary for proper chromosome segregation (Ding, 2009).

A potential caveat to the interpretation of loss-of-function Chromator phenotypes is that Chromator is known to participate in at least two different molecular complexes, one of which is associated with nuclear and chromatin structure during interphase (Rath, 2004 and Rath, 2006). Thus, loss-of-function mutations in the Chromator gene in addition to directly affecting mitosis may also influence the expression of other genes due to perturbations of their chromatin environment thereby potentially indirectly affecting the function of the microtubule based spindle apparatus. Using immunoblot analysis this study found that the level of at least one such protein Ncd is reduced by 80% in Chromator loss-of-function mutants. qRT-PCR determination showed that this reduction was likely to have been caused by repressed transcription rather than by increased protein turnover. However, the finding that tubulin levels in the Chromator mutant background as well as Mad2 levels in Chromator RNAi-depleted S2 cell are relatively unaffected suggests that lack of Chromator does not cause a general repression of all genes. Interestingly, in mutant larvae lacking the histone H3S10 kinase JIL-1 which has a similar chromatin phenotype to that of Chro⁷¹/Chro⁶¹² mutants ncd mRNA levels are also reduced. However, in JIL-1 null mutant brains mitosis was close to normal with only a moderate increase in the percentage of spindle and chromosome segregation defects compared to wild type in spite of the perturbed chromosome morphology. While reduced levels of the kinesin 14 family motor protein Ncd by RNAi treatment in S2 cells result in clear spindle defects this phenotype is very different from the Drosophila ncd null mutant, in which meiotic spindle defects are observed, but where spindle formation defects in mitotic cells are very subtle or absent. Thus, the downregulation of Ncd in the Chromator mutant background is unlikely to account for the majority of the mitotic defects observed in the larval neuroblasts. However, it remains a possibility that other unknown genes involved in mitosis may be repressed as well. To determine the genes whose expression is affected by the chromatin perturbation induced by the lack of Chromator will require a future genome-wide survey. Consequently, at present it is not possible to unequivocally link the phenotypes observed in this study with Chromator's function as a spindle matrix member. Nonetheless, that Chromator function, whether directly or indirectly, is required for proper microtubule spindle formation and mitosis was demonstrated by the rescue of mutant phenotypes by transgenic expression of Chromator-GFP (Ding, 2009).

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila Titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In Western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which was identified by mass spectrometry analysis as being titin. The spindle matrix proteins Skeletor, Megator and Chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein (Fabian, 2007).

Although no functional data is available on the role of spindle D-titin, several aspects of spindle physiology are congruent with known roles of D-titin. For one, a putative spindle matrix might provide flexibility and elasticity as an underlying component of spindles, and the matrix might even be involved in force production. The term 'putative' was used because there has been no definitive molecular or morphological description of a matrix, except that the Drosophila proteins Skeletor, Megator and Chromator would seem to be components of (or at least markers of) such a matrix. Skeletor, Megator and Chromator staining reveals the expected morphology, and Skeletor and Megator distribution maintain a spindle shape even after spindle microtubules have been depolymerized. Since titin has a major role in the elasticity of skeletal and cardiac muscle, and since it is closely associated with skeletor in the spindle, it is reasonable to think that Skeletor and D-titin function physiologically as part of a spindle matrix. Spindle actin and myosin are closely associated with D-titin and Skeletor, so they, too, might be part of such a matrix. The data suggest that the interactions among these proteins (D-titin, Skeletor, myosin and actin) and between these proteins and the spindle microtubules change during the course of cell division. Skeletor, for example, is present along the spindle fibers and in between them (the 'matrix') throughout the course of division, but it accumulates progressively in the kinetochore microtubule bundles as division proceeds towards anaphase. D-titin in chromosomes is present in high concentration in prophase but by metaphase and anaphase, the staining of the chromosomes is somewhat reduced, as determined by comparing their staining intensities with that of muscle fibers in the same preparations. In prophase, D-titin is present in the cytoplasmic asters. After nuclear membrane breakdown, as the development of the spindle proceeds towards anaphase, D-titin becomes increasingly concentrated at the poles and along kinetochore fibers with the staining along the kinetochore fibers eventually being as high as 80%-85% the levels of staining observed in muscle fibers. Thus, it can be speculated that the interactions among these proteins develop and continue throughout prometaphase to culminate in a 'mature' matrix by metaphase (Fabian, 2007).

Another potential role for D-titin in spindles may be as part of the elastic 'tethers' extending between arms of separating half-bivalents in crane-fly spermatocytes. Evidence for these tethers comes from experiments showing that laser-severed chromosome arms or entire chromosomes move backwards across the equator. D-titin extends between the arms of separating half-bivalents, so it is reasonable to suggest that the tethers contain D-titin, and that D-titin is responsible for tether elasticity, at least in part (Fabian, 2007).

Another possible role is suggested by recent experiments in which titin modulates the velocity of actin filaments that slide along an HMM-coated surface, titin-actin interactions acting as a `viscous bumper mechanism' to slow the movements. One could envisage a similar effect on the velocity of chromosome movement because titin can interact with microtubules in addition to muscle proteins (Fabian, 2007).

The identification of D-titin in spindles, together with other muscle proteins, actin, myosin and zyxin, suggests that these proteins indeed function during mitosis, perhaps in a spindle matrix and in producing forces for chromosome movement. If the arrangements (and polarities) of these proteins in the spindle were known, their function might be better understood. With respect to D-titin, following several markers could, in theory, give information about how the molecules are arranged; since D-titin is so large, double staining with different antibodies could result in resolvable separation for epitopes reasonably far apart on the molecule. Separate dots were indeed found after staining with the three different antibodies. Because the staining is punctate, and the molecules are arranged in three dimensions and not just the two observed in the image, certainty about the arrangement of the individual D-titin molecules is not possible. Nonetheless, if it is true that two D-titin epitopes are 2 microm apart along the length of the kinetochore microtubules, but only 0.2 microm apart at an oblique angle, as seen in double stained images, this would suggest that the individual D-titin molecules may be arranged obliquely to the kinetochore microtubules rather than along them (Fabian, 2007).

The presence of D-titin in prophase nuclear membranes and in nascent membranes formed around the daughter nuclei in telophase, is consistent with the finding that D-titin is associates with and binds to C. elegans lamins (Fabian, 2007).

EAST and Chromator control the destruction and remodeling of muscles during Drosophila metamorphosis

Metamorphosis involves the destruction of larval, the formation of adult and the transformation of larval into adult tissues. This study demonstrates the role of the Drosophila nuclear proteins EAST and Chromator in tissue destruction and remodeling. To better understand the function of east, a yeast two-hybrid screen was performed, and the euchromatin associated protein Chromator was identified as a candidate interactor. To analyze the functional significance of the two-hybrid data, a set of novel pupal lethal Chro alleles were generated by P-element excision. The pupal lethal Chro mutants resemble lethal east alleles as homozygous mutants develop into pharates with normal looking body parts, but fail to eclose. The eclosion defect of the Chro alleles is rescued in an east heterozygous background, indicating antagonistic genetic interactions between the two genes. Live cell imaging was applied to study muscle development during metamorphosis. Consistent with the eclosion defects, mutant pharates of both genes show loss and abnormal differentiation of adult eclosion muscles. The two genes have opposite effects on the destruction of larval muscles in metamorphosis. While Chro mutants show incomplete histolysis, muscles degenerate prematurely in east mutants. Moreover east mutants affect the remodeling of abdominal larval muscles into adult eclosion muscles. During this process, loss of east interferes with the spatial coordination of thinning of the larval muscles. Overexpression of EAST-GFP can prevent the disintegration of polytene chromosomes during programmed cell death. It is proposed that Chro activates and east inhibits processes and genes involved in tissue destruction and remodeling (Wasser, 2007).

The chromodomain protein, Chromator, interacts with JIL-1 kinase and regulates the structure of Drosophila polytene chromosomes

This study generated two new hypomorphic Chro alleles and analyzed the consequences of reduced Chromator protein function on polytene chromosome structure. In Chro⁷¹/Chro⁶¹² mutants the polytene chromosome arms were coiled and compacted with a disruption and misalignment of band and interband regions and with numerous ectopic contacts connecting non-homologous regions. Furthermore, Chromator co-localizes with the JIL-1 kinase at polytene interband regions and the two proteins interact within the same protein complex. That both proteins are necessary and may function together is supported by the finding that a concomitant reduction in JIL-1 and Chromator function synergistically reduces viability during development. Overlay assays and deletion construct analysis suggest that the interaction between JIL-1 and Chromator is direct and that it is mediated by sequences in the C-terminal domain of Chromator and by the acidic region within the C-terminal domain of JIL-1. Taken together these findings indicate that Chromator and JIL-1 interact in an interband-specific complex that functions to establish or maintain polytene chromosome structure in Drosophila (Rath, 2006).

Chromator was originally identified in a yeast two-hybrid screen as an interaction partner of the putative spindle matrix component, skeletor, and localizes to the spindle and the centrosomes during mitosis (Rath, 2004). Furthermore, functional assays using RNAi-mediated depletion in S2 cells suggest that Chromator directly affects spindle function and chromosome segregation (Rath, 2004). However, localization of Chromator to polytene interbands suggests it also has a functional role in maintaining chromatin structure during interphase. Such a role is supported by the finding (Eggert, 2004) that Chromator (which these workers refer to as Chriz) is found in a protein complex together with the interband-specific zinc-finger protein Z4 (Eggert, 2004; Gortchakov, 2005). That Z4 participates in regulating polytene chromosomal structure is likely because Z4-null mutant chromosomes show a decompaction of chromatin and a loss of a clear band/interband pattern (Eggert, 2004). However, the effect of Chromator on polytene chromosome morphology has been difficult to study because null alleles of Chro die as embryos or first-instar larvae before salivary gland polytene chromosomes can be analyzed (Rath, 2004; Gortchakov, 2005). For this reason an EMS mutagenesis screen was performed that generated two new Chro hypomorphic alleles. The analysis of these alleles shows that impaired Chromator function leads to disorganization and misalignment of band/interband regions resulting in coiling and folding of the polytene chromosomes. In addition, Chromator directly interacts with JIL-1 kinase and the two proteins extensively co-localize at polytene interband regions. Taken together these findings indicate that Chromator and JIL-1 interact in an interband-specific complex that functions to establish or maintain polytene chromosome structure in Drosophila (Rath, 2006).

Although the Drosophila polytene chromosome has served as a widely used model for studying chromatin structure, remarkably little is known about its spatial organization or about the molecular basis for the conjugation of homologous chromatids in the process of polytenization. It has been demonstrated that JIL-1 kinase, which phosphorylates histone H3 Ser10 in interband regions, plays a crucial role in maintaining polytene chromosome structure. In the absence of JIL-1 there is a shortening and folding of the chromosomes with a non-orderly intermixing of euchromatin and the compacted chromatin characteristic of banded regions and there is a striking redistribution of the heterochromatin markers dimethyl H3K9 and HP1 to ectopic chromosome sites. This suggested a model where JIL-1 kinase activity functions to maintain chromosome structure and euchromatic regions by counteracting heterochromatization mediated by histone H3 dimethylation and HP1 recruitment. However, the Chro mutant analysis presented in this study suggests that JIL-1 activity is necessary but not sufficient for maintaining some of these aspects of polytene chromosome morphology and that Chromator function is also required. Nonetheless, it should be noted that although the polytene chromosome phenotypes of JIL-1 and Chro mutants resemble each other with coiled and compacted chromosome arms they are not identical. In contrast to JIL-1 mutant polytene chromosomes, in Chro mutants there is still a clear demarcation between band and interband regions at the ultrastructural level although these bands are severely misaligned. Furthermore, in JIL-1 null mutants the male X chromosome is differentially affected with a 'puffed' appearance whereas in Chro mutants the morphology of the male X chromosome is similar to that of the autosomes. Thus, it is likely that JIL-1 and Chromator control different but related aspects of chromosome morphology within the complex. That both proteins are necessary and may function synergistically is supported by the finding that a concomitant reduction in JIL-1 and Chromator function dramatically reduces viability during development (Rath, 2006).

An important feature of the Chromator protein is the presence of a chromodomain. The function of most chromodomain proteins identified thus far has been related to the establishment or maintenance of a variety of chromatin conformations. For example, HP1 binds to methylated histone H3 and is essential for the assembly of heterochromatin. Thus, it is possible that Chromator through interactions mediated by its chromodomain participates in a complex with JIL-1 that is required for maintaining properly separated and aligned interband regions as well as a more open chromatin configuration. However, loss of JIL-1 or Chromator function also influences the coherence and organization of bands although neither protein is present in these regions. This suggests that JIL-1 and Chromator function may affect the distribution and/or activity of other molecules important for influencing chromatin structure such as boundary elements and/or the molecular machinery regulating heterochromatin formation and spreading. In support of this notion it has recently been demonstrated (Zhang, 2006) that the lethality as well as some of the chromosome morphology defects observed in JIL-1 null or hypomorphic mutant backgrounds may be the result of ectopic histone methyltransferase activity (Rath, 2006).

In addition to the present studies demonstrating an interaction with JIL-1, Chromator has been shown to interact with the spindle matrix protein skeletor (Walker, 2000; Rath, 2004) and with the zinc-finger protein Z4 (Eggert, 2004; Gortchakov, 2005). The interaction with skeletor was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays (Rath, 2004). Immunocytochemical labeling of Drosophila embryos, S2 cells and polytene chromosomes demonstrated that the two proteins show extensive co-localization during the cell cycle although their distributions are not identical (Rath, 2004). During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of skeletor. During mitosis both Chromator and skeletor detach from the chromosomes and align together in a spindle-like structure with Chromator additionally being localized to centrosomes that are devoid of skeletor-antibody labeling. The extensive co-localization of the two proteins is compatible with a direct physical interaction between skeletor and Chromator. However, at present it is not known whether such an interaction occurs throughout the cell cycle or is present only at certain stages with additional proteins mediating complex assembly at other stages. The interaction of Chromator with Z4 was identified in co-immunoprecipitation experiments and the two proteins colocalize extensively at interband polytene regions (Eggert, 2004). However, Chromator and Z4 do not appear to associate directly and their chromosomal binding is independent of each other (Gorthakov, 2005). Interestingly, the phenotype of loss of Z4 function is somewhat different from that of loss of JIL-1 or Chromator function. Z4 mutant chromosomes decompact and attain a cloudy appearance when losing their band/interband organization (Eggert, 2004) instead of coiling and shortening as in JIL-1 and Chro loss-of-function mutants. This differential effect on polytene chromosome banding patterns and morphology may reflect that these constituents contribute different activities within one complex or may indicate the presence of more than one molecular assembly, each with different functions. Thus, future studies will be necessary to clarify the interactions of Chromator with interband-specific proteins and its functional role in establishing or maintaining polytene chromosome structure (Rath, 2006).

Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes

Polytene interphase chromosomes are compacted into a series of bands and interbands reflecting their organization into independent chromosomal domains. In order to understand chromosomal organization, the role was studied of proteins that are selective for interbands. This work describes the Drosophila melanogaster chromodomain protein Chriz that is coimmunoprecipitated with the zinc finger protein Z4. Both proteins colocalize exclusively to the interbands on Drosophila polytene chromosomes. Like Z4, Chriz is ubiquitously expressed throughout development and is associated with chromatin in all interphase nuclei. Following dissociation from chromatin, early in mitosis Chriz binds to the centrosomes and to the mitotic spindle. Newly induced amorphic Chriz alleles are early lethal, and ubiquitous overexpression of Chriz is lethal as well. Available Chriz hypomorphs which survive until pupal stage have a normal chromosomal phenotype. Reducing Z4 protein does not affect Chriz binding to polytene chromosomes and vice versa. Z4 is still chromosomally bound when Chriz protein is depleted by RNA interference (Gortchakov, 2005).

Identification of the Drosophila interband-specific protein Z4 as a DNA-binding zinc-finger protein determining chromosomal structure

The subdivision of polytene chromosomes into bands and interbands suggests a structural chromatin organization that is related to the formation of functional domains of gene expression. Use was made of the antibody Z4 to gain insight into this level of chromosomal structure, since the Z4 antibody mirrors this patterning by binding to an antigen that is present in most interbands. The Z4 gene encodes a protein with seven zinc fingers, it is essential for fly development and acts in a dose-dependent manner on the development of several tissues. Z4 mutants have a dose-sensitive effect on w^m4 position effect variegation with a haplo-suppressor and triplo-enhancer phenotype, suggesting Z4 is involved in chromatin compaction. This assumption is further supported by the phenotype of Z4 mutant chromosomes, which show a loss of the band/interband pattern and are subject to an overall decompaction of chromosomal material. By co-immunoprecipitations, a novel chromo domain protein, which was named Chriz (Chromo domain protein interacting with Z4), identified in Flybase as Chromator; was identified as an interaction partner of Z4. Chriz localizes to interbands in a pattern that is identical to the Z4 pattern. These findings together with the result that Z4 binds directly to DNA in vitro strongly suggest that Z4 in conjunction with Chriz is intimately involved in the higher-order structuring of chromosomes (Eggert, 2004).

The localization of Z4 to all interbands and the concomitant absence from transcriptional active loci as represented by the puffed regions strengthens the view that Z4 predominantly participates in the formation of particular chromatin structures. Several different chromatin components with an impact on chromatin structure have been identified by their dose-dependent effect on the expression of the variegating w^m4-allele. In particular, the genes Su(var)2-5, Su(var)3-7 and Su(var)3-9 with a haplo-suppressor and triploenhancer phenotype were shown to encode proteins associated with heterochromatin. The localization to heterochromatin is in accordance with the presumed function of these proteins to influence the expression of w^m4 at the euchromatin/heterochromatin border by variably establishing highly compacted repressive chromatin structures. In contrast to these proteins, Z4 does not bind to heterochromatin but is distributed exclusively within the euchromatic part of chromosomes in the interbands. Although the detailed structure of chromatin constituting bands and interbands is unknown, it is generally accepted that DNA contained within an interband is less compacted than DNA contained within a band. Therefore, reducing the dosage of Z4 was expected to favor chromatin compaction, resulting in an enhancement of w^m4 PEV. Conversely, the overexpression of Z4 was expected to favor 'open' chromatin structures and lead to a suppression of w^m4 PEV. Surprisingly, Z4 in contrast to these expectations turned out to have a haplo-suppressor and a triplo-enhancer effect. This result indicates that Z4 structures chromosomes by supporting the condensation of chromatin. This conclusion is further substantiated by the analysis of chromosomes from 3rd instar larvae mutant for a hypomorphic allele of Z4. In these animals chromosomes are evident which have lost the organization into bands and interbands and altogether appear as a less compact mass of chromatin. The loss of chromosomal structure could be the result of an unpairing of the chromosomal fibres that are oriented in parallel bundles in polytene chromosomes. However, it is found to be rather unlikely that Z4 might have a primary function in the pairing of chromatids. A null-allele of Z4 is embryonic lethal, which exhibits an essential function of Z4 in diploid cells unrelated to chromatid pairing. A possible role of Z4 could involve the establishment of chromosomal borders that separate chromatin domains of different compaction levels and determine the extent of interband formation. The exact length of DNA included within interbands is still unclear, but has been estimated to range from a few hundred to a few thousand base pairs of DNA. Furthermore it is unknown whether Z4 proteins cover the whole length of interbands or are present only at the borders of bands and interbands to exert a classical boundary function. The latter is supported by the finding that within the hsp70 heat-shock puffs Z4 localizes exactly at one of the borders of each structural domain. This is very reminiscent to the localization of two proteins involved in insulator function, Zw5 and BEAF; to the proximal and distal edges of the 87A puff, and suggests common functions in the definition of structural chromosomal domains (Eggert, 2004).

In addition to Z4, several different proteins have been shown to localize to the interbands of polytene chromosomes. JIL-1, a protein with two conserved serine/threonine kinase domains is present in hundreds of interbands, with a twofold enrichment on the male X-chromosome compared with autosomes, suggesting an involvement of JIL-1 in the hyperactivation of X-chromosomal genes in the male for dose compensation. Hypomorphic mutants of JIL-1 have decreased levels of histone H3Ser10 phosphorylation and chromosomes are highly condensed due to the loss of the euchromatic interbands. These results provided evidence for a role of JIL-1 in the establishment or maintenance of an open chromatin structure correlated with the interbands to facilitate gene transcription. Quite evident from the chromosomal phenotypes of the corresponding mutants, Z4 and JIL-1 have opposite effects on chromosomal structure, despite the fact that both proteins localize to interbands. This indicates that different activities contribute to the formation of the banding pattern. Although the function of JIL-1 seems to be tightly linked to the modulation of chromatin in interbands to achieve a more decondensed state, the function of Z4 could be primarily associated with the establishment of chromosomal borders influencing the chromatin structure of the chromosomal bands as well (Eggert, 2004).

A correlation of transcription taking place in the interbands is supported by the finding that the elongating form of RNA PolII is found in hundreds of interbands. In addition, transcription factors like Spt5 and Spt6, CHD1 and the chromatin remodeling complex including Brahma localize to the less compacted interband regions. An involvement of Z4 in the promoter-selective transcription and/or chromatin remodeling is suggested by the recent finding that Z4 is a component of a macromolecular complex containing the TBP-related factor TRF2, DREF, ISWI and NURF-55 (Hochheimer, 2002). However, the chromosomal localizations of the factors involved in general or promoter-selective transcription differs from the localization of Z4 in that the latter is present in nearly all the interbands, whereas the former are found at only a subset of interbands at a few hundred sites. Owing to this difference Z4 is assumed to perform a unique function which is fundamental to the repetitive organization of chromatin into bands and interbands, which in a subset of interbands is possibly used by the transcriptional machinery. Whether this function of Z4 is related to the formation of boundaries is currently unknown. Proteins that bind to boundary or insulator sequences and are distributed in a subset of the interbands in Drosophila have been identified with the BEAF-32, Su(Hw) and Mod(mdg4) proteins. Su(Hw) and Mod(mdg4) are involved in the nuclear organization of about 500 insulator sequences into 20 to 30 insulator bodies, organizing the chromatin fibre into looped domains. A similar organizing capacity is not evident for Z4, as Z4 shows a more uniform distribution in Kc cells, lacking a pronounced concentration in a small number of discrete foci. However, owing to the greater number of sites bound by Z4, the number of nuclear foci organized by Z4 could exceed those formed by Su(Hw) and Mod(mdg4) and remain undetected in a low resolution analysis of nuclei stained for Z4 (Eggert, 2004).

Regardless of the precise chromatin composition that differs between a band and an interband, a primary distinction can be expected to act at the level of the DNA sequence. In this respect the interband DNA should contain one or more sequence motifs that are specifically recognized by one or more proteins, and Z4 with the seven zinc finger motifs is a potential candidate to exert this function. In vitro, Z4 bound to the interband sequence derived from the 5' region of Notch without sequence specificity. Possibly, the accumulated general affinity of the seven zinc fingers for DNA obscured the specific interaction of one or a few of the fingers with its target site in vitro. Still, the question remains regarding how the targeting of Z4 to the interbands is achieved in vivo. This question is especially relevant as a comparison of the few cases of DNA sequences that were unambiguously mapped to the interband regions revealed that these sequences did not contain a single characteristic shared sequence motif. A possible explanation could be given by the capability of Z4 to bind to a variety of consensus sequences, each specifically recognized by single zinc fingers and/or different combinations of the fingers, as has been shown for the vertebrate zinc finger protein CTCF (Eggert, 2004).

Alternatively, or in addition to the interaction with DNA, Z4 could bind to a target protein present in interbands. This requires one or a few proteins covering all the chromosomal binding sites of Z4. Until now the novel protein Chriz is the only candidate displaying a chromosomal localization identical to Z4. Significantly, Chriz contains a chromo domain, a motif that has been found in many chromosomal proteins participating in the maintenance of diverse chromatin conformations. Therefore, Z4 and Chriz seem to be central for the modulation of the higher-order chromatin states distinguishing bands from interbands (Eggert, 2004).

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein Skeleto

A yeast two-hybrid interaction assay was used to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis (Rath, 2004).

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Cubenas-Potts, C., Rowley, M. J., Lyu, X., Li, G., Lei, E. P. and Corces, V. G. (2017). Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture. Nucleic Acids Res 45(4): 1714-1730. PubMed ID: 27899590

Deng, H., Bao, X., Cai, W., Blacketer, M. J., Belmont, A. S., Girton, J., Johansen, J. and Johansen, K. M. (2008). Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila. Development 135: 699-705. PubMed ID: 18199578

Ding, Y., Yao, C., Lince-Faria, M., Rath, U., Cai, W., Maiato, H., Girton, J., Johansen, K. M. and Johansen, J. (2009). Chromator is required for proper microtubule spindle formation and mitosis in Drosophila. Dev Biol 334: 253-263. PubMed ID: 19632217

Eggert, H., Gortchakov, A. and Saumweber, H. (2004). Identification of the Drosophila interband-specific protein Z4 as a DNA-binding zinc-finger protein determining chromosomal structure. J Cell Sci 117: 4253-4264. PubMed ID: 15292401

Fabian, L., Xia, X., Venkitaramani, D. V., Johansen, K. M., Johansen, J., Andrew, D. J. and Forer, A. (2007). Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator. J. Cell Sci. 120(Pt 13): 2190-204. PubMed ID: 17591688

Fay, A., Misulovin, Z., Li, J., Schaaf, C. A., Gause, M., Gilmour, D. S. and Dorsett, D. (2011). Cohesin selectively binds and regulates genes with paused RNA polymerase. Curr Biol 21: 1624-1634. PubMed ID: 21962715

Gan, M., Moebus, S., Eggert, H. and Saumweber, H. (2011). The Chriz-Z4 complex recruits JIL-1 to polytene chromosomes, a requirement for interband-specific phosphorylation of H3S10. J Biosci 36: 425-438. PubMed ID: 21799255

Gortchakov, A. A., Eggert, H., Gan, M., Mattow, J., Zhimulev, I. F. and Saumweber, H. (2005). Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes. Chromosoma 114: 54-66. PubMed ID: 15821938

Haberle, V., Arnold, C. D., Pagani, M., Rath, M., Schernhuber, K. and Stark, A. (2019). Transcriptional cofactors display specificity for distinct types of core promoters. Nature 570(7759):122-126. PubMed ID: 31092928

Hochheimer, A., et al. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420(6914): 439-45. PubMed ID: 12459787

Johansen, K. M. and Johansen, J. (2007). Cell and molecular biology of the spindle matrix. Int Rev Cytol 263: 155-206. PubMed ID: 17725967

Johansen, K. M., Forer, A., Yao, C., Girton, J. and Johansen, J. (2011). Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix? Chromosome Res 19: 345-365. PubMed ID: 21274615

Lince-Faria, M., Maffini, S., Orr, B., Ding, Y., Claudia, F., Sunkel, C. E., Tavares, A., Johansen, J., Johansen, K. M. and Maiato, H. (2009). Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator. J Cell Biol 184: 647-657. PubMed ID: 19273613

Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H. and Akhtar, A. (2006). Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila. Mol Cell 21: 811-823. PubMed ID: 16543150

Qi, H., Rath, U., Wang, D., Xu, Y. Z., Ding, Y., Zhang, W., Blacketer, M. J., Paddy, M. R., Girton, J., Johansen, J. and Johansen, K. M. (2004). Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila. Mol Biol Cell 15: 4854-4865. PubMed ID: 15356261

Qi, H., Rath, U., Ding, Y., Ji, Y., Blacketer, M. J., Girton, J., Johansen, J. and Johansen, K. M. (2005). EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila. J Cell Biochem 95: 1284-1291. PubMed ID: 15962301

Rath, U., Wang, D., Ding, Y., Xu, Y.-Z., Qi, H., Blacketer, M. J., Girton, J., Johansen, J. and Johansen, K. M. (2004). Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein Skeletor. J. Cell. Biochem. 93: 1033-1047. 15389869

Rath, U., et al. (2006). The chromodomain protein, Chromator, interacts with JIL-1 kinase and regulates the structure of Drosophila polytene chromosomes. J. Cell Sci. 119(Pt 11): 2332-41. 16723739

Qi, H., Rath, U., Wang, D., Xu, Y. Z., Ding, Y., Zhang, W., Blacketer, M. J., Paddy, M. R., Girton, J., Johansen, J. and Johansen, K. M. (2004). Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila. Mol Biol Cell 15: 4854-4865. PubMed ID: 15356261

Schaaf, C. A., Misulovin, Z., Gause, M., Koenig, A., Gohara, D. W., Watson, A. and Dorsett, D. (2013). Cohesin and polycomb proteins functionally interact to control transcription at silenced and active genes. PLoS Genet 9: e1003560. PubMed ID: 23818863

Vogelmann, J., Le Gall, A., Dejardin, S., Allemand, F., Gamot, A., Labesse, G., Cuvier, O., Negre, N., Cohen-Gonsaud, M., Margeat, E., Nollmann, M. (2014) Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome. PLoS Genet 10: e1004544. PubMed ID: 25165871

Walker, D. L., Wang, D., Jin, Y., Rath, U., Wang, Y., Johansen, J. and Johansen, K. M. (2000). Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix. J Cell Biol 151: 1401-1412. PubMed ID: 11134070

Wasser, M., Bte Osman, Z. and Chia, W. (2007). EAST and Chromator control the destruction and remodeling of muscles during Drosophila metamorphosis. Dev Biol 307: 380-393. PubMed ID: 17540360

Yao, C., Ding, Y., Cai, W., Wang, C., Girton, J., Johansen, K. M. and Johansen, J. (2012). The chromodomain-containing NH(2)-terminus of Chromator interacts with histone H1 and is required for correct targeting to chromatin. Chromosoma 121: 209-220. PubMed ID: 22203189

Zabidi, M. A., Arnold, C. D., Schernhuber, K., Pagani, M., Rath, M., Frank, O. and Stark, A. (2015). Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518(7540): 556-559. PubMed ID: 25517091

date revised: 29 December 2023

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