NORMAL GENOMIC

• Reo E, Seum C, Spierer P, Bontron S
• Sumoylation of Drosophila SU(VAR)3-7 is required for its heterochromaticfunction.
• Nucleic Acids Res. 2010; 38: 4254-62
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• In Drosophila, SU(VAR)3-7 is an essential heterochromatin component. It isrequired for proper chromatin condensation, and changing its dose modifiesposition-effect variegation. Sumoylation is a post-translationalmodification shown to play a role in diverse biological processes. Here,we demonstrate that sumoylation is essential for proper heterochromatinfunction in Drosophila through modification of SU(VAR)3-7. Indeed,SU(VAR)3-7 is sumoylated at lysine K839; this modification is required forlocalization of SU(VAR)3-7 at pericentric heterochromatin, chromosome 4,and telomeres. In addition, sumoylation of SU(VAR)3-7 is a prerequisitefor its ability to enhance position-effect variegation. Thus, theseresults show that the heterochromatic function of SU(VAR)3-7 depends onits own sumoylation, and unveil a role for sumoylation in Drosophilaheterochromatin.

• El Gazzar M, Yoza BK, Chen X, Hu J, Hawkins GA, McCall CE
• G9a and HP1 couple histone and DNA methylation to TNFalpha transcriptionsilencing during endotoxin tolerance.
• J Biol Chem. 2008; 283: 32198-208
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• TNFalpha gene expression is silenced in the endotoxin tolerant phenotypethat develops in blood leukocytes after the initial activation phase ofsevere systemic inflammation or sepsis. The silencing phase can bemimicked in vitro by LPS stimulation. We reported that the TNFalphatranscription is disrupted in endotoxin tolerant THP-1 human promonocytedue to changes in transcription factor binding and enrichment with histoneH3 dimethylated on lysine 9 (H3K9). Here we show that the TNFalphapromoter is hypermethylated during endotoxin tolerance and that H3K9methylation and DNA methylation interact to silence TNFalpha expression.Chromatin immunoprecipitation and RNA interference analysis demonstratedthat, in tolerant cells, TNFalpha promoter is bound by the H3K9 histonemethyltransferase G9a which dimethylates H3K9 and creates a platform forHP1 binding, leading to the recruitment of the DNA methyltransferaseDnmt3a/b and an increase in promoter CpG methylation. Knockdown of HP1resulted in a decreased Dnmt3a/b binding, sustained G9a binding, and amodest increase in TNFalpha transcription, but had no effect on H3K9dimethylation. In contrast, G9a knockdown-disrupted promoter silencing andrestored TNFalpha transcription in tolerant cells. This correlated with anear loss of H3K9 dimethylation, a significant decrease in HP1 andDnmt3a/b binding and promoter CpG methylation. Our results demonstrate acentral role for G9a in this process and suggest that histone methylationand DNA methylation cooperatively interact via HP1 to silence TNFalphaexpression during endotoxin tolerance and may have implication forproinflammatory gene silencing associated with severe systemicinflammation.

• Maier D, Chen AX, Preiss A, Ketelhut M
• The tiny Hairless protein from Apis mellifera: a potent antagonist ofNotch signaling in Drosophila melanogaster.
• BMC Evol Biol. 2008; 8: 175-175
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• BACKGROUND: The Notch signaling pathway is fundamental to the regulationof many cell fate decisions in eumetazoans. Not surprisingly, members ofthis pathway are highly conserved even between vertebrates andinvertebrates. There is one notable exception, Hairless, which acts as ageneral Notch antagonist in Drosophila. Hairless silences Notch targetgenes by assembling a repressor complex together with Suppressor ofHairless [Su(H)] and the co-repressors Groucho (Gro) and C-terminalbinding protein (CtBP). Now with the availability of genomic databases,presumptive Hairless homologues are predicted, however only in insectspecies. To further our understanding of Hairless structure and function,we have cloned the Hairless gene from Apis mellifera (A.m.H) andcharacterized its functional conservation in Drosophila. RESULTS: The ApisHairless protein is only one third of the size of the Drosophilaorthologue. Interestingly, the defined Suppressor of Hairless bindingdomain is interrupted by a nonconserved spacer sequence and the N-terminalmotif is sufficient for binding. In contrast to Apis Hairless, theDrosophila orthologue contains a large acidic domain and we provideexperimental evidence that this acidic domain is necessary to silenceHairless activity in vivo. Despite the dramatic size differences, ApisHairless binds to the Drosophila Hairless interactors Su(H), Gro, CtBP andPros26.4. Hence, Apis Hairless assembles a repressor complex withDrosophila components that may have a different topology. Nevertheless,Apis Hairless is sufficient to repress the Notch target gene vestigial inDrosophila. Moreover, it is able to rescue Hairless mutant phenotypes,providing in vivo evidence for its function as a bona fide Notchantagonist. CONCLUSION: This is the first interspecies-complementationanalysis of the Hairless gene. Guided by evolutionary comparisons, we hopeto eventually identify all the relevant structural domains and cofactorsof Hairless, thereby opening an avenue for further insights into therepressor-complexes that down-regulate Notch signaling also in other,higher eukaryotes.

• Isaac CE et al.
• The retinoblastoma protein regulates pericentric heterochromatin.
• Mol Cell Biol. 2006; 26: 3659-71
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• The retinoblastoma protein (pRb) has been proposed to regulate cell cycleprogression in part through its ability to interact with enzymes thatmodify histone tails and create a repressed chromatin structure. Wecreated a mutation in the murine Rb1 gene that disrupted pRb's ability tointeract with these enzymes to determine if it affected cell cyclecontrol. Here, we show that loss of this interaction slows progressionthrough mitosis and causes aneuploidy. Our experiments reveal that whilethe LXCXE binding site mutation does not disrupt pRb's interaction withthe Suv4-20h histone methyltransferases, it dramatically reduces H4-K20trimethylation in pericentric heterochromatin. Disruption ofheterochromatin structure in this chromosomal region leads to centromerefusions, chromosome missegregation, and genomic instability. These resultsdemonstrate the surprising finding that pRb uses the LXCXE binding cleftto control chromatin structure for the regulation of events beyond theG(1)-to-S-phase transition.

• Fuks F, Hurd PJ, Deplus R, Kouzarides T
• The DNA methyltransferases associate with HP1 and the SUV39H1 histonemethyltransferase.
• Nucleic Acids Res. 2003; 31: 2305-12
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• The DNA methyltransferases, Dnmts, are the enzymes responsible formethylating DNA in mammals, which leads to gene silencing. Repression byDNA methylation is mediated partly by recruitment of themethyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associateand facilitate histone methylation at Lys9 of H3, which is a keyepigenetic modification involved in gene silencing. Here, we show thatendogenous Dnmt3a associates primarily with histone H3-K9methyltransferase activity as well as, to a lesser extent, with H3-K4enzymatic activity. The association with enzymatic activity is mediated bythe conserved PHD-like motif of Dnmt3a. The H3-K9 histonemethyltransferase that binds Dnmt3a is likely the H3-K9 specific SUV39H1enzyme since we find that it interacts both in vitro and in vivo withDnmt3a, using its PHD-like motif. We find that SUV39H1 also binds to Dnmt1and, consistent with these interactions, SUV39H1 can purify DNAmethyltransferase activity from nuclear extracts. In addition, we showthat HP1beta, a SUV39H1-interacting partner, binds directly to Dnmt1 andDnmt3a and that native HP1beta associates with DNA methyltransferaseactivity. Our data show a direct connection between the enzymesresponsible for DNA methylation and histone methylation. These resultsfurther substantiate the notion of a self-reinforcing repressive chromatinstate through the interplay between these two global epigeneticmodifications.

• Delattre M, Spierer A, Hulo N, Spierer P
• A new gene in Drosophila melanogaster, Ravus, the phantom of the modifierof position-effect variegation Su(var)3-7.
• Int J Dev Biol. 2002; 46: 167-71
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• In a search for homologues of the dominant modifier of position-effectvariegation Su(var)3-7, we have identified one ORF in Drosophilamelanogaster. The 359 amino acid deduced protein is much shorter than the1169 amino acid protein Su(var)3-7. Surprisingly, the two genes are veryclose to each other at 87E on the polytene chromosome map, and aretranscribed divergently. The triplet coding for the N-terminus amino acidof the new gene lies only 368 base pairs from the start of transcriptionof Su(var)3-7. This opposite orientation of the homologue has led us toname it Ravus. The N-terminus of the Ravus protein contains only one ofthe seven unusual zinc fingers of Su(var)3-7. A second region ofsimilarity encodes an acidic domain. Finally, there is a block of highsimilarity near the C-terminus of the two proteins. It corresponds to anew conserved protein domain, BESS, found also in the BEAF and StonewallDrosophila proteins. We have constructed a tagged Ravus protein, and haveexpressed it as a heat-shock inducible transgene. Ravus associates in vivowith polytene chromosomes but, in contrast to theheterochromatin-associated protein Su(var)3-7, does not show specificityfor the chromocenter. Ravus does not seem either to modify the genomicsilencing of position-effect variegation, as over-expression of thetransgene does not affect the variegated phenotype of a number ofrearrangements tested.

• Donaldson KM, Lui A, Karpen GH
• Modifiers of terminal deficiency-associated position effect variegation inDrosophila.
• Genetics. 2002; 160: 995-1009
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• Terminal deletions of a Drosophila minichromosome (Dp(1;f)1187)dramatically increase the position effect variegation (PEV) of a yellow(+)body-color gene located in cis. Such terminal deficiency-associated PEV(TDA-PEV) can be suppressed by the presence of a second minichromosome, aphenomenon termed "trans-suppression." We performed a screen for mutationsthat modify TDA-PEV and trans-suppression. Seventy suppressors andenhancers of TDA-PEV were identified, but no modifiers oftrans-suppression were recovered. Secondary analyses of the effects ofthese mutations on different PEV types identified 10 mutations that modifyonly TDA-PEV and 6 mutations that modify TDA-PEV and only one other typeof PEV. One mutation, a new allele of Su(var)3-9, affects all forms ofPEV, including silencing associated with the insertion of a transgene intotelomeric regions (TPE). This Su(var)3-9 allele is the first modifier ofPEV to affect TPE and provides a unique link between different types ofgene silencing in Drosophila. The remaining mutations affected multiplePEV types, indicating that general PEV modifiers impact TDA-PEV. Modifiersof TDA-PEV may identify proteins that play important roles in generalheterochromatin biology, including proteins involved in telomere structureand function and the organization of chromosomes in the interphasenucleus.

• Zhang CL, McKinsey TA, Olson EN
• Association of class II histone deacetylases with heterochromatin protein1: potential role for histone methylation in control of muscledifferentiation.
• Mol Cell Biol. 2002; 22: 7302-12
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• Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscledifferentiation through associations with the myocyte enhancer factor 2(MEF2) transcription factor. MEF2-interacting transcription repressor(MITR) is an amino-terminal splice variant of HDAC9 that also potentlyinhibits MEF2 transcriptional activity despite lacking a catalytic domain.Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatinprotein 1 (HP1), an adaptor protein that recognizes methylated lysineswithin histone tails and mediates transcriptional repression by recruitinghistone methyltransferase. Promyogenic signals provided bycalcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITRand HDACs with HP1. Since the histone methyl-lysine residues recognized byHP1 also serve as substrates for deacetylation by HDACs, the interactionof MITR and HDACs with HP1 provides an efficient mechanism for silencingMEF2 target genes by coupling histone deacetylation and methylation.Indeed, nucleosomal histones surrounding a MEF2-binding site in themyogenin gene promoter are highly methylated in undifferentiatedmyoblasts, when the gene is silent, and become acetylated during muscledifferentiation, when the myogenin gene is expressed at high levels. Theability of MEF2 to recruit a histone methyltransferase to target genepromoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling todisrupt these interactions provides an efficient mechanism forsignal-dependent regulation of the epigenetic events controlling muscledifferentiation.

• Netzer C et al.
• SALL1, the gene mutated in Townes-Brocks syndrome, encodes atranscriptional repressor which interacts with TRF1/PIN2 and localizes topericentromeric heterochromatin.
• Hum Mol Genet. 2001; 10: 3017-24
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• The Townes-Brocks syndrome (TBS) is an autosomal dominantly inheritedmalformation syndrome presenting as an association of imperforate anus,triphalangeal and supernumerary thumbs, malformed ears and sensorineuralhearing loss. Mutations in SALL1, a gene mapping to 16q12.1, wereidentified as a cause for TBS. To elucidate how SALL1 mutations lead toTBS, we have performed a series of functional studies with the SALL1protein. Using epifluorescence and confocal microscopy it could be shownthat a GFP-SALL1 fusion protein localizes to chromocenters and smallerheterochromatin foci in transiently transfected NIH-3T3 cells.Chromocenters consist of clustered pericentromeric heterochromatin andcontain telomere sequences. Indirect immunofluorescence revealed a partialcolocalization of GFP-SALL1 with M31, the mouse homolog of the Drosophilaheterochromatic protein HP1. It was further demonstrated that SALL1 actsas a strong transcriptional repressor in mammalian cells. Transcriptionalrepression could not be relieved by the addition of the histonedeacetylase inhibitor Trichostatin-A. In a yeast two-hybrid screen weidentified PIN2, an isoform of telomere-repeat-binding factor 1 (TRF1), asan interaction partner of SALL1, and showed that the N-terminus of SALL1is not necessary for the interaction with PIN2/TRF1. The interaction wasconfirmed in vitro in a GST-pulldown assay. The association of thedevelopmental regulator SALL1 with heterochromatin is striking andunexpected. Our results propose an involvement of SALL1 in the regulationof higher order chromatin structures and indicate that the protein mightbe a component of a distinct heterochromatin-dependent silencing process.We have also provided new evidence that there is a close functional linkbetween the centromeric and telomeric heterochromatin domains not only inDrosophila and yeast, but also in mammalian cells.

• Brasher SV et al.
• The structure of mouse HP1 suggests a unique mode of single peptiderecognition by the shadow chromo domain dimer.
• EMBO J. 2000; 19: 1587-97
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• The heterochromatin protein 1 (HP1) family of proteins is involved in genesilencing via the formation of heterochromatic structures. They arecomposed of two related domains: an N-terminal chromo domain and aC-terminal shadow chromo domain. Present results suggest that chromodomains may function as protein interaction motifs, bringing togetherdifferent proteins in multi-protein complexes and locating them inheterochromatin. We have previously determined the structure of the chromodomain from the mouse HP1beta protein, MOD1. We show here that, incontrast to the chromo domain, the shadow chromo domain is a homodimer.The intact HP1beta protein is also dimeric, where the interaction ismediated by the shadow chromo domain, with the chromo domains movingindependently of each other at the end of flexible linkers. Mappingstudies, with fragments of the CAF1 and TIF1beta proteins, show that anintact, dimeric, shadow chromo domain structure is required for complexformation.

• Sedkov Y et al.
• Molecular genetic analysis of the Drosophila trithorax-related gene whichencodes a novel SET domain protein.
• Mech Dev. 1999; 82: 171-9
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• The products of the trithorax and Polycomb groups genes maintain theactivity and silence, respectively, of many developmental genes includinggenes of the homeotic complexes. This transcriptional regulation is likelyto involve modification of chromatin structure. Here, we report thecloning and characterization of a new gene, trithorax-related (trr), whichshares sequence similarities with members of both the trithorax andPolycomb groups. The trr transcript is 9.6 kb in length and is presentthroughout development. The TRR protein, as predicted from the nucleotidesequence of the open reading frame, is 2431 amino acids in length andcontains a PHD finger-like domain and a SET domain, two highly conservedprotein motifs found in several trithorax and Polycomb group proteins, andin modifiers of position effect variegation. TRR is most similar insequence to the human ALR protein, suggesting that trr is a Drosophilahomologue of the ALR. TRR is also highly homologous to DrosophilaTRITHORAX protein and to its human homologue, ALL-1/HRX. However,preliminary genetic analysis of a trr null allele suggests that TRRprotein may not be involved in regulation of homeotic genes (i.e. not amember of the trithorax or Polycomb groups) or in position effectvariegation.

• Nielsen AL et al.
• Interaction with members of the heterochromatin protein 1 (HP1) family andhistone deacetylation are differentially involved in transcriptionalsilencing by members of the TIF1 family.
• EMBO J. 1999; 18: 6385-95
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• Mammalian TIF1alpha and TIF1beta (KAP-1/KRIP-1) are relatedtranscriptional intermediary factors that possess intrinsic silencingactivity. TIF1alpha is believed to be a euchromatic target for ligandednuclear receptors, while TIF1beta may serve as a co-repressor for thelarge family of KRAB domain-containing zinc finger proteins. Here, wereport an association of TIF1beta with both heterochromatin andeuchromatin in interphase nuclei. Co-immunoprecipitation of nuclearextracts shows that endogenous TIF1beta, but not TIF1alpha, is associatedwith members of the heterochromatin protein 1 (HP1) family. However, invitro, both TIF1alpha and TIF1beta interact with and phosphorylate the HP1proteins. This interaction involves a conserved amino acid motif, which iscritical for the silencing activity of TIF1beta but not TIF1alpha. Wefurther show that trichostatin A, an inhibitor of histone deacetylases,can interfere with both TIF1 and HP1 silencing. The silencing activity ofTIF1alpha appears to result chiefly from histone deacetylation, whereasthat of TIF1beta may be mediated via both HP1 binding and histonedeacetylation.

• Perrin L et al.
• The Drosophila modifier of variegation modulo gene product binds specificRNA sequences at the nucleolus and interacts with DNA and chromatin in aphosphorylation-dependent manner.
• J Biol Chem. 1999; 274: 6315-23
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• modulo belongs to the modifier of Position Effect Variegation class ofDrosophila genes, suggesting a role for its product in regulatingchromatin structure. Genetics assigned a second function to the gene, inprotein synthesis capacity. Bifunctionality is consistent with proteinlocalization in two distinct subnuclear compartments, chromatin andnucleolus, and with its organization in modules potentially involved inDNA and RNA binding. In this study, we examine nucleic acid interactionsestablished by Modulo at nucleolus and chromatin and the mechanism thatcontrols the distribution and balances the function of the protein in thetwo compartments. Structure/function analysis and oligomerselection/amplification experiments indicate that, in vitro, two basicterminal domains independently contact DNA without sequence specificity,whereas a central RNA Recognition Motif (RRM)-containing domain allowsrecognition of a novel sequence-/motif-specific RNA class. Phosphorylationmoreover is shown to down-regulate DNA binding. Evidence is provided thatin vivo nucleolar Modulo is highly phosphorylated and belongs to aribonucleoprotein particle, whereas chromatin-associated protein is notmodified. A functional scheme is finally proposed in which modification byphosphorylation modulates Mod subnuclear distribution and balances itsfunction at the nucleolus and chromatin.

• Brockdorff N, Duthie SM
• X chromosome inactivation and the Xist gene.
• Cell Mol Life Sci. 1998; 54: 104-12
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• Recent years have seen rapid progress towards understanding the molecularmechanisms involved in X chromosome inactivation (X inactivation). Thisprogress has largely revolved around the discovery of the X inactivespecific transcript (Xist) gene, which is known now to represent themaster switch locus regulating X inactivation. In adult cells Xist istranscribed exclusively from the inactive X chromosome. The transcript hasno apparent protein-coding potential and is retained in the nucleus inclose association with the domain occupied by the inactive X chromosome.It is thus thought to represent a functional RNA molecule which acts asthe primary signal responsible for the propagation of X inactivation.Developmental regulation of Xist correlates with the developmental timingof X inactivation. Recent results have demonstrated that Xist is bothnecessary and sufficient for X inactivation. Goals for the future are tounderstand the mechanism of Xist regulation which underlies theestablishment of appropriate X inactivation patterns and to determine howXist RNA participates in the process of propagating inactivation in cis.