NORMAL GENOMIC

• Khan A et al.
• BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase.
• Proc Natl Acad Sci U S A. 2015; 112: 8338-43
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• Ribosome biogenesis dictates the translational capacity of cells. Several mechanisms establish and maintain transcriptional output from eukaryotic ribosomal DNA (rDNA) loci. rDNA silencing is one such mechanism that ensures the inactivity and hence the maintenance of a silenced state of a subset of rRNA gene copies. Whereas oncogenic agents stimulate rRNA gene transcription, tumor suppressors decrease rRNA gene transcription. We demonstrate in mammalian cells that BANP, E5R, and Nac1 (BEN) domain 3 (BEND3), a quadruple BEN domain-containing protein, localizes in nucleoli and binds to ribosomal RNA gene promoters to help repress rRNA genes. Loss of BEND3 increases histone H3K4 trimethylation and, correspondingly, decreases rDNA promoter DNA methylation, consistent with a role for BEND3 in rDNA silencing. BEND3 associates with the nucleolar-remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TTF-1-interacting protein 5 via association with ubiquitin specific protease 21 (USP21) debiquitinase. Our results provide mechanistic insights into how the novel rDNA transcription repressor BEND3 acts together with NoRC to actively coordinate the establishment of rDNA silencing.

• Yutin N, Faure G, Koonin EV, Mushegian AR
• Chordopoxvirus protein F12 implicated in enveloped virion morphogenesis is an inactivated DNA polymerase.
• Biol Direct. 2014; 9: 22-22
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• Through the course of their evolution, viruses with large genomes have acquired numerous host genes, most of which perform function in virus reproduction in a manner that is related to their original activities in the cells, but some are exapted for new roles. Here we report the unexpected finding that protein F12, which is conserved among the chordopoxviruses and is implicated in the morphogenesis of enveloped intracellular virions, is a derived DNA polymerase, possibly of bacteriophage origin, in which the polymerase domain and probably the exonuclease domain have been inactivated. Thus, F12 appears to present a rare example of a drastic, exaptive functional change in virus evolution. REVIEWERS: This article was reviewed by Frank Eisenhaber and Juergen Brosius.

• Branco AT, Hartl DL, Lemos B
• Chromatin-associated proteins HP1 and Mod(mdg4) modify Y-linked regulatory variation in the drosophila testis.
• Genetics. 2013; 194: 609-18
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• Chromatin remodeling is crucial for gene regulation. Remodeling is often mediated through chemical modifications of the DNA template, DNA-associated proteins, and RNA-mediated processes. Y-linked regulatory variation (YRV) refers to the quantitative effects that polymorphic tracts of Y-linked chromatin exert on gene expression of X-linked and autosomal genes. Here we show that naturally occurring polymorphisms in the Drosophila melanogaster Y chromosome contribute disproportionally to gene expression variation in the testis. The variation is dependent on wild-type expression levels of mod(mdg4) as well as Su(var)205; the latter gene codes for heterochromatin protein 1 (HP1) in Drosophila. Testis-specific YRV is abolished in genotypes with heterozygous loss-of-function mutations for mod(mdg4) and Su(var)205 but not in similar experiments with JIL-1. Furthermore, the Y chromosome differentially regulates several ubiquitously expressed genes. The results highlight the requirement for wild-type dosage of Su(var)205 and mod(mdg4) in enabling naturally occurring Y-linked regulatory variation in the testis. The phenotypes that emerge in the context of wild-type levels of the HP1 and Mod(mdg4) proteins might be part of an adaptive response to the environment.

• Foltman M et al.
• Eukaryotic replisome components cooperate to process histones during chromosome replication.
• Cell Rep. 2013; 3: 892-904
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• DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.

• Hammonds AS et al.
• Spatial expression of transcription factors in Drosophila embryonic organ development.
• Genome Biol. 2013; 14: 140-140
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• BACKGROUND: Site-specific transcription factors (TFs) bind DNA regulatory elements to control expression of target genes, forming the core of gene regulatory networks. Despite decades of research, most studies focus on only a small number of TFs and the roles of many remain unknown. RESULTS: We present a systematic characterization of spatiotemporal gene expression patterns for all known or predicted Drosophila TFs throughout embryogenesis, the first such comprehensive study for any metazoan animal. We generated RNA expression patterns for all 708 TFs by in situ hybridization, annotated the patterns using an anatomical controlled vocabulary, and analyzed TF expression in the context of organ system development. Nearly all TFs are expressed during embryogenesis and more than half are specifically expressed in the central nervous system. Compared to other genes, TFs are enriched early in the development of most organ systems, and throughout the development of the nervous system. Of the 535 TFs with spatially restricted expression, 79% are dynamically expressed in multiple organ systems while 21% show single-organ specificity. Of those expressed in multiple organ systems, 77 TFs are restricted to a single organ system either early or late in development. Expression patterns for 354 TFs are characterized for the first time in this study. CONCLUSIONS: We produced a reference TF dataset for the investigation of gene regulatory networks in embryogenesis, and gained insight into the expression dynamics of the full complement of TFs controlling the development of each organ system.

• Duan H et al.
• Insensitive is a corepressor for Suppressor of Hairless and regulates Notch signalling during neural development.
• EMBO J. 2011; 30: 3120-33
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• The Notch intracellular domain functions as a co-activator for the DNA-binding protein Suppressor of Hairless (Su(H)) to mediate myriad cell fate decisions. Notch pathway activity is balanced by transcriptional repression, mediated by Su(H) in concert with its Drosophila corepressor Hairless. We demonstrate that the Drosophila neural BEN-solo protein Insensitive (Insv) is a nuclear factor that inhibits Notch signalling during multiple peripheral nervous system cell fate decisions. Endogenous Insv was particularly critical when repressor activity of Su(H) was compromised. Reciprocally, ectopic Insv generated several Notch loss-of-function phenotypes, repressed most Notch targets in the E(spl)-C, and opposed Notch-mediated activation of an E(spl)m3-luc reporter. A direct role for Insv in transcriptional repression was indicated by binding of Insv to Su(H), and by strong chromatin immunoprecipitation of endogenous Insv to most E(spl)-C loci. Strikingly, ectopic Insv fully rescued sensory organ precursors in Hairless null clones, indicating that Insv can antagonize Notch independently of Hairless. These data shed first light on the in vivo function for a BEN-solo protein as an Su(H) corepressor in the Notch pathway regulating neural development.

• Sathyan KM, Shen Z, Tripathi V, Prasanth KV, Prasanth SG
• A BEN-domain-containing protein associates with heterochromatin and represses transcription.
• J Cell Sci. 2011; 124: 3149-63
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• In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression.

• Bergner LM et al.
• A novel predicted bromodomain-related protein affects coordination between meiosis and spermiogenesis in Drosophila and is required for male meiotic cytokinesis.
• DNA Cell Biol. 2010; 29: 487-98
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• Temporal coordination of meiosis with spermatid morphogenesis is crucial for successful generation of mature sperm cells. We identified a recessive male sterile Drosophila melanogaster mutant, mitoshell, in which events of spermatid morphogenesis are initiated too early, before meiotic onset. Premature mitochondrial aggregation and fusion lead to an aberrant mitochondrial shell around premeiotic nuclei. Despite successful meiotic karyokinesis, improper mitochondrial localization in mitoshell testes is associated with defective astral central spindles and a lack of contractile rings, leading to meiotic cytokinesis failure. We mapped and cloned the mitoshell gene and found that it encodes a novel protein with a bromodomain-related region. It is conserved in some insect lineages. Bromodomains typically bind to histone acetyl-lysine residues and therefore are often associated with chromatin. The Mitoshell bromodomain-related region is predicted to have an alpha helical structure similar to that of bromodomains, but not all the crucial residues in the ligand-binding loops are conserved. We speculate that Mitoshell may participate in transcriptional regulation of spermatogenesis-specific genes, though perhaps with different ligand specificity compared to traditional bromodomains.

• Karetsou Z et al.
• Identification of distinct SET/TAF-Ibeta domains required for core histone binding and quantitative characterisation of the interaction.
• BMC Biochem. 2009; 10: 10-10
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• BACKGROUND: The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Ibeta belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Ibeta, we designed several SET/TAF-Ibeta truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. RESULTS: Wild type SET/TAF-Ibeta binds to histones H2B and H3 with Kd values of 2.87 and 0.15 microM, respectively. The preferential binding of SET/TAF-Ibeta to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Ibeta, as well as the H3 amino-terminal tail, are dispensable for this interaction. CONCLUSION: This type of analysis allowed us to assess the relative affinities of SET/TAF-Ibeta for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Ibeta and can be valuable to understand the role of SET/TAF-Ibeta in chromatin function.

• Guo L et al.
• Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea.
• Nucleic Acids Res. 2008; 36: 1129-37
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• Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

• Korutla L, Degnan R, Wang P, Mackler SA
• NAC1, a cocaine-regulated POZ/BTB protein interacts with CoREST.
• J Neurochem. 2007; 101: 611-8
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• In this report, CoREST was identified as a protein that interacts with NAC1. NAC1 is a cocaine-regulated Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex (POZ/BTB) repressor protein, which mediates interactions among several other transcriptional regulators. In the present study, an interaction between NAC1 and CoREST was detected in neuro-2A cells and HEK293T cells. We found that the POZ/BTB domain is necessary and sufficient for interaction with CoREST. Surprisingly, only one of five mutations in the POZ/BTB domain that disrupts homodimer assembly interfered with NAC1 and CoREST interactions. These results indicate that POZ/BTB homodimer formation is not required for NAC1-CoREST interaction. CoREST demonstrated protein-protein interactions with both isoforms of NAC1, sNAC1, and lNAC1. Coimmunoprecipitation studies show that NAC1 and CoREST are physically bound together. To further support the results, a direct interaction was demonstrated in glutathione-S-transferase pull down assays. siRNA directed against NAC1 mRNA significantly reduced NAC1 protein expression and resulted in reversal of CoREST-mediated repression in cells. This interaction between NAC1 and CoREST was not found for other POZ/BTB proteins tested. Endogenous interaction was demonstrated in lysates from rat brain samples. This is the first report to demonstrate that a POZ/BTB protein interacts with CoREST. Taken together, the results indicate that CoREST may be part of the NAC1 repressor mechanism.

• Iyer LM, Balaji S, Koonin EV, Aravind L
• Evolutionary genomics of nucleo-cytoplasmic large DNA viruses.
• Virus Res. 2006; 117: 156-84
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• A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses [Iyer, L.M., Aravind, L., Koonin, E.V., 2001. Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria (primarily, endosymbionts and parasites). Amongst the expansions, there are multiple families of predicted virus-specific signaling and regulatory domains. Most NCLDVs have also acquired large arrays of genes related to ubiquitin signaling, and the animal viruses in particular have independently evolved several defenses against apoptosis and immune response, including growth factors and potential inhibitors of cytokine signaling. The mimivirus displays an enormous array of genes of bacterial provenance, including a representative of a new class of predicted papain-like peptidases. It is further demonstrated that a significant number of genes found in NCLDVs also have homologs in bacteriophages, although a vertical relationship between the NCLDVs and a particular bacteriophage group could not be established. On the basis of these observations, two alternative scenarios for the origin of the NCLDVs and other groups of large DNA viruses of eukaryotes are considered. One of these scenarios posits an early assembly of an already large DNA virus precursor from which various large DNA viruses diverged through an ongoing process of displacement of the original genes by xenologous or non-orthologous genes from various sources. The second scenario posits convergent emergence, on multiple occasions, of large DNA viruses from small plasmid-like precursors through independent accretion of similar sets of genes due to strong selective pressures imposed by their life cycles and hosts.

• Zhang D, Martyniuk CJ, Trudeau VL
• SANTA domain: a novel conserved protein module in Eukaryota with potential involvement in chromatin regulation.
• Bioinformatics. 2006; 22: 2459-62
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• Since packaging of DNA in the chromatin structure restricts the accessibility for regulatory factors, chromatin remodeling is required to facilitate nuclear processes such as gene transcription, replication, and genome recombination. Many conserved non-enzymatic protein domains have been identified that contribute to the activities of multiprotein remodeling complexes. Here we identified a novel conserved protein domain in Eukaryota whose putative function may be in regulating chromatin remodeling. Since this domain is associated with a known SANT domain in several vertebrate proteins, we named it the SANTA (SANT Associated) domain. Sequence analysis showed that the SANTA domain is approximately a 90 amino acid module and likely composed of four central beta-sheets and three flanking alpha-helices. Many hydrophobic residues exhibited high conservation along the domain, implying a possible function in protein-protein interactions. The SANTA domain was identified in mammals, chicken, frog, fish, sea squirt, sea urchin, worms and plants. Furthermore, a phylogenetic tree of SANTA domains showed that one plant-specific duplication event happened in the Viridiplantae lineage.

• Novatchkova M, Bachmair A, Eisenhaber B, Eisenhaber F
• Proteins with two SUMO-like domains in chromatin-associated complexes: the RENi (Rad60-Esc2-NIP45) family.
• BMC Bioinformatics. 2005; 6: 22-22
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• BACKGROUND: Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation. RESULTS: We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast Rad60, Esc2 and mouse NIP45 http://mendel.imp.univie.ac.at/SEQUENCES/reni/. Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants. CONCLUSIONS: Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line - namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs (in analogy to the way that ubiquitin-like domains mimic ubiquitin). This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity.

• Ichimura T, Watanabe S, Sakamoto Y, Aoto T, Fujita N, Nakao M
• Transcriptional repression and heterochromatin formation by MBD1 and MCAF/AM family proteins.
• J Biol Chem. 2005; 280: 13928-35
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• DNA methylation cooperates with methylation at lysine 9 of histone H3 (H3-K9), a modified histone molecule that is targeted by heterochromatin protein 1, to form a transcriptionally silent chromatin. Methyl CpG-binding protein MBD1 recognizes methylated CpG dinucleotide and recruits H3-K9 methyltransferases such as SETDB1 to genomic regions. Here we show that MBD1-containing chromatin-associated factor (MCAF) 1, also known as the human homologue of murine ATFa-associated modulator (AM), is required for transcriptional repression and heterochromatin formation by MBD1, together with the involvement of SETDB1. Moreover, the amino acid sequence of MCAF1 shows similarity to a number of sequences of the MCAF/AM-related proteins, resulting in the identification of a new member of the protein family, termed MCAF2. Immunoprecipitation and in vitro binding analyses reveal that both MCAF proteins interact with MBD1, SETDB1, and Sp1 via two evolutionarily conserved distinct domains. Furthermore, MCAF1 enhances transcriptional repression by MBD1 together with SETDB1, and exogenous expression of MCAF2 partly compensates for the repressive activity in MCAF1 knockdown HeLa cells. The expression of MBD1 mutant, which lacks interaction with MCAF proteins, perturbs heterochromatin protein 1-enriched heterochromatin formation at the MBD1-containing chromosomal loci. These data suggest that MBD1.MCAF1.SETDB1 complex facilitates the formation of heterochromatic domains, emphasizing the role of MCAF/AM family proteins in epigenetic control.

• Krauss V, Dorn R
• Evolution of the trans-splicing Drosophila locus mod(mdg4) in several species of Diptera and Lepidoptera.
• Gene. 2004; 331: 165-76
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• The modifier of mdg4 (mod[mdg4]) locus of Drosophila melanogaster (Dme) encodes chromatin proteins which are involved in position effect variegation, establishment of chromatin boundaries, nerve pathfinding, meiotic chromosome pairing and apoptosis. It was recently shown that mRNA trans-splicing is involved in the generation of at least 26 different mod(mdg4) transcripts. Here, we show that a similar complex mod(mdg4) locus exists in Drosophila pseudoobscura (Dps), Drosophila virilis (Dvi), Anopheles gambiae (Aga) and Bombyx mori (Bmo). As in D. melanogaster, most isoforms of these species contain a strongly conserved BTB/POZ domain (hereafter referred to as BTB domain) within the common N-terminal part and a Cys(2)His(2) motif containing FLYWCH domain within the isoform-specific C-terminal parts. By sequence comparison, we identified six novel isoforms in D. melanogaster and show that altogether 31 isoforms are perfectly conserved by sequence and position in the mod(mdg4) locus of the Drosophila species analyzed. We found significant differences in evolutionary speed of synonymous/nonsynonymous divergence between the various isoform specific exons. These results were extended by tree reconstruction analysis based on the evolved FLYWCH domains of predicted Mod(mdg4) proteins in Drosophila and Anopheles. Comparative analysis of mod(mdg4) gene structure in species of dipterans implicates that several internal inversions occurred making the mRNA trans-splicing mechanism indispensable for mod(mdg4) expression. Finally, we propose a model for the evolution of trans-splicing implementing effective regulation of many alternative gene products in a composite gene structure.

• Mohrmann L, Langenberg K, Krijgsveld J, Kal AJ, Heck AJ, Verrijzer CP
• Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes.
• Mol Cell Biol. 2004; 24: 3077-88
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• The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. Here, we report the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (i) the BAP complex, defined by the presence of OSA and the absence of Polybromo and BAP170, and (ii) the PBAP complex, containing Polybromo and BAP170 but lacking OSA. Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. We conclude that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. Our results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes.

• Kaul-Ghanekar R, Jalota A, Pavithra L, Tucker P, Chattopadhyay S
• SMAR1 and Cux/CDP modulate chromatin and act as negative regulators of the TCRbeta enhancer (Ebeta).
• Nucleic Acids Res. 2004; 32: 4862-75
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• Chromatin modulation at various cis-acting elements is critical for V(D)J recombination during T and B cell development. MARbeta, a matrix-associated region (MAR) located upstream of the T cell receptor beta (TCRbeta) enhancer (Ebeta), serves a crucial role in silencing Ebeta-mediated TCR activation. By DNaseI hypersensitivity assays, we show here that overexpression of the MAR binding proteins SMAR1 and Cux/CDP modulate the chromatin structure at MARbeta. We further demonstrate that the silencer function of MARbeta is mediated independently by SMAR1 and Cux/CDP as judged by their ability to repress Ebeta-dependent reporter gene expression. Moreover, the repressor activity of SMAR1 is strongly enhanced in the presence of Cux/CDP. These two proteins physically interact with each other and colocalize within the perinuclear region through a SMAR1 domain required for repression. The repression domain of SMAR1 is separate from the MARbeta binding domain and contains a nuclear localization signal and an arginine-serine (RS)-rich domain, characteristic of pre-mRNA splicing regulators. Our data suggest that at the double positive stage of T cell development, cis-acting MARbeta elements recruit the strong negative regulators Cux and SMAR1 to control Ebeta-mediated recombination and transcription.

• Pai CY, Lei EP, Ghosh D, Corces VG
• The centrosomal protein CP190 is a component of the gypsy chromatin insulator.
• Mol Cell. 2004; 16: 737-48
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• Chromatin insulators, or boundary elements, affect promoter-enhancer interactions and buffer transgenes from position effects. The gypsy insulator of Drosophila is bound by a protein complex with two characterized components, the zinc finger protein Suppressor of Hairy-wing [Su(Hw)] and Mod(mdg4)2.2, which is one of the multiple spliced variants encoded by the modifier of mdg4 [mod(mdg4)] gene. A genetic screen for dominant enhancers of the mod(mdg4) phenotype identified the Centrosomal Protein 190 (CP190) as an essential constituent of the gypsy insulator. The function of the centrosome is not affected in CP190 mutants whereas gypsy insulator activity is impaired. CP190 associates physically with both Su(Hw) and Mod(mdg4)2.2 and colocalizes with both proteins on polytene chromosomes. CP190 does not interact directly with insulator sequences present in the gypsy retrotransposon but binds to a previously characterized endogenous insulator, and it is necessary for the formation of insulator bodies. The results suggest that endogenous gypsy insulators contain binding sites for CP190, which is essential for insulator function, and may or may not contain binding sites for Su(Hw) and Mod(mdg4)2.2.

• Hung MS, Shen CK
• Eukaryotic methyl-CpG-binding domain proteins and chromatin modification.
• Eukaryot Cell. 2003; 2: 841-6

• Meehan RR, Kao CF, Pennings S
• HP1 binding to native chromatin in vitro is determined by the hinge region and not by the chromodomain.
• EMBO J. 2003; 22: 3164-74
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• We have isolated the complete coding sequences for two Xenopus laevis isoforms of heterochromatin protein 1, corresponding to HP1alpha and HP1gamma. The sequence of xHP1alpha shows considerable divergence from its mammalian homologues, whereas xHP1gamma is highly conserved. Functionally, xHP1alpha behaves identically to human HP1alpha. We observe unexpected differences between the two HP1 variants in binding native soluble chromatin, which seem to correlate with their distinct nuclear distributions in vivo. A surprising finding is that the characteristic interaction of HP1 chromodomains with histone H3 at methylated lysine 9 is not detected in preformed chromatin due to its inaccessibility. Instead, we localize a strong chromatin-binding activity to the short hinge region between the chromodomain and the chromoshadow domain of xHP1alpha but not xHP1gamma. This novel chromatin-binding activity has a non-specific DNA-binding component in addition to a linker histone-dependent preference for an altered chromatin structure with a likely heterochromatin organization.

• Shareef MM, King C, Damaj M, Badagu R, Huang DW, Kellum R
• Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing.
• Mol Biol Cell. 2001; 12: 1671-85
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• Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

• Matsumoto K, Nagata K, Miyaji-Yamaguchi M, Kikuchi A, Tsujimoto M
• Sperm chromatin decondensation by template activating factor I through direct interaction with basic proteins.
• Mol Cell Biol. 1999; 19: 6940-52
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• Template activating factor I (TAF-I) was originally identified as a host factor required for DNA replication and transcription of adenovirus genome complexed with viral basic proteins. Purified TAF-I was shown to bind to core histones and stimulate transcription from nucleosomal templates. Human TAF-I consists of two acidic proteins, TAF-Ialpha and TAF-Ibeta, which differ from each other only in their amino-terminal regions. Here, we report that TAF-I decondenses demembraned Xenopus sperm chromatin. Human TAF-Ibeta has a chromatin decondensation activity comparable to that of NAP-I, another histone binding protein, whereas TAF-Ialpha has only a weak activity. Analysis of molecular mechanisms underlying the chromatin decondensation by TAF-I revealed that TAF-I interacts directly with sperm basic proteins. Deletion of the TAF-I carboxyl-terminal acidic region abolishes the decondensation activity. Interestingly, the acidic region itself is not sufficient for decondensation, since an amino acid substitution mutant in the dimerization domain of TAF-I which has the intact acidic region does not support chromatin decondensation. We detected the beta form of TAF-I in Xenopus oocytes and eggs by immunoblotting, and the cloning of its cDNA led us to conclude that Xenopus TAF-Ibeta also decondenses sperm chromatin. These results suggest that TAF-I plays a role in remodeling higher-order chromatin structure as well as nucleosomal structure through direct interaction with chromatin basic proteins.

• Jenuwein T, Laible G, Dorn R, Reuter G
• SET domain proteins modulate chromatin domains in eu- and heterochromatin.
• Cell Mol Life Sci. 1998; 54: 80-93
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• The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals. Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture. These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin--a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain. Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV. Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g. HOM-C).

• Crevel G et al.
• Molecular and cellular characterization of CRP1, a Drosophila chromatin decondensation protein.
• J Struct Biol. 1997; 118: 9-22
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• CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins revealed strong homologies to Xenopus nucleoplasmin and NO38. CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3. CRP1 mRNA is expressed throughout Drosophila development; it is highest during oogenesis and early embryogenesis. mRNA levels correlate closely with levels of protein expression measured previously. Results of chemical crosslinking indicate that CRP1 is either tetrameric or pentameric; similar ambiguity was revealed by direct visualization using scanning transmission electron microscopy. Consistent with previously published results, parallel crosslinking studies of Xenopus nucleoplasmin suggested a pentameric structure. Scanning transmission electron microscopic examination after negative staining revealed that CRP1 and Xenopus nucleoplasmin are morphologically similar. CRP1 is able to substitute for nucleoplasmin in Xenopus egg extract-mediated sperm chromatin decondensation. In vitro, CRP1-induced decondensation is accompanied by direct binding of CRP1 to chromatin.

• Altschul SF et al.
• Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
• Nucleic Acids Res. 1997; 25: 3389-402
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• The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.