Heterochromatin-mediated Gene Silencing Research Articles

Mitotic phosphorylation of HP1α regulates its cell cycle-dependent chromatin binding.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that plays a crucial role in heterochromatin-mediated gene silencing. We previously showed that mammalian HP1α is constitutively phosphorylated at its N-terminal serine residues by casein kinase II (CK2), and that this phosphorylation enhances HP1α's binding specificity for nucleosomes containing lysine 9-methylated histone H3 (H3K9me). Although the presence of additional HP1α phosphorylation during mitosis was reported more than a decade ago, its biological significance remains largely elusive. Here we found that mitosis-specific HP1α phosphorylation affected HP1α's ability to bind chromatin. Using biochemical and mutational analyses, we showed that HP1α's mitotic phosphorylation was located in its hinge region and was reversibly regulated by Aurora B kinase and serine/threonine phosphatases. In addition, chromatin fractionation and electrophoretic mobility shift assays revealed that hinge region-phosphorylated HP1α was preferentially dissociated from mitotic chromatin and exhibited a reduced DNA-binding activity. Although HP1's mitotic behaviour was previously linked to H3 serine 10 phosphorylation, which blocks the binding of HP1's chromodomain to H3K9me3, our findings suggest that mitotic phosphorylation in HP1α's hinge region also contributes to changes in HP1α's association with mitotic chromatin.

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part from the ability of HP1 proteins to spread across large regions of the genome, compact the underlying chromatin and recruit repressive activities1–3. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation driven phase-separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets but other molecules such as the transcription factor TFIIB show no preference. Using single-molecule DNA curtains we find that unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, though with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands based on nuclear context.

Role of Inner Nuclear Membrane Protein Complex Lem2-Nur1 in Heterochromatic Gene Silencing

Heterochromatin in the fission yeast Schizosaccharomyces pombe is clustered at the nuclear periphery and interacts with a number of nuclear membrane proteins. However, the significance and the factors that sequester heterochromatin at the nuclear periphery are not fully known. Here, we report that an inner nuclear membrane protein complex Lem2-Nur1 is essential for heterochromatin-mediated gene silencing. We found that Lem2 is physically associated with another inner nuclear membrane protein, Nur1, and deletion of either lem2 or nur1 causes silencing defect at centromeres, telomeres, and rDNA loci. We analyzed the genome-wide association of Lem2 using ChIP sequencing and we found that it binds to the central core region of centromeres, in striking contrast to Chp1, a component of pericentromeric heterochromatin, which binds H3K9me-rich chromatin in neighboring sequences. The recruitment of Lem2 and Nur1 to silent regions of the genome is dependent on H3K9 methyltransferase, Clr4. Finally, we show that the Lem2-Nur1 complex regulates the local balance between the underln]Snf2/HDAC-containing repressor complex (SHREC) histone deacetylase complex and the anti-silencing protein Epe1. These findings uncover a novel role for Lem2-Nur1 as a key functional link between localization at the nuclear periphery and heterochromatin-mediated gene silencing.

Mechanisms of functional promiscuity by HP1 proteins

Heterochromatin protein 1 (HP1) proteins were originally identified as critical components in heterochromatin-mediated gene silencing and are now recognized to play essential roles in several other processes including gene activation. Several eukaryotes possess more than one HP1 paralog. Despite high sequence conservation, the HP1 paralogs achieve diverse functions. Further, in many cases, the same HP1 paralog is implicated in multiple functions. Recent biochemical studies have revealed interesting paralog-specific biophysical differences and unanticipated conformational versatility in HP1 proteins that may account for this functional promiscuity. Here we review these findings and describe a molecular framework that aims to link the conformational flexibility of HP1 proteins observed in vitro with their functional promiscuity observed in vivo.

Modeling of Friedreich ataxia-related iron overloading cardiomyopathy using patient-specific-induced pluripotent stem cells

Friedreich ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is due to GAA repeat expansions within the first intron of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. The triplet codon repeats lead to heterochromatin-mediated gene silencing and loss of frataxin. Nevertheless, inadequacy of existing FRDA-cardiac cellular models limited cardiomyopathy studies. We tested the hypothesis that iron homeostasis deregulation accelerates reduction in energy synthesis dynamics which contributes to impaired cardiac calcium homeostasis and contractile force. Silencing of FXN expressions occurred both in somatic FRDA-skin fibroblasts and two of the induced pluripotent stem cells (iPSC) clones; a sign of stress condition was shown in FRDA-iPSC cardiomyocytes with disorganized mitochondrial network and mitochondrial DNA (mtDNA) depletion; hypertrophic cardiac stress responses were observed by an increase in α-actinin-positive cell sizes revealed by FACS analysis as well as elevation in brain natriuretic peptide (BNP) gene expression; the intracellular iron accumulated in FRDA cardiomyocytes might be due to attenuated negative feedback response of transferring receptor (TSFR) expression and positive feedback response of ferritin (FTH1); energy synthesis dynamics, in terms of ATP production rate, was impaired in FRDA-iPSC cardiomyocytes, which were prone to iron overload condition. Energetic insufficiency determined slower Ca(2+) transients by retarding calcium reuptake to sarcoplasmic reticulum (SR) and impaired the positive inotropic and chronotropic responses to adrenergic stimulation. Our data showed for the first time that FRDA-iPSCs cardiac derivatives represent promising models to study cardiac stress response due to impaired iron homeostasis condition and mitochondrial damages. The cardiomyopathy phenotype was accelerated in an iron-overloaded condition early in calcium homeostasis aspect.

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.

Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model

Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder caused by GAA repeat expansion within the FXN gene, leading to epigenetic changes and heterochromatin-mediated gene silencing that result in a frataxin protein deficit. Histone deacetylase (HDAC) inhibitors, including pimelic o-aminobenzamide compounds 106, 109 and 136, have previously been shown to reverse FXN gene silencing in short-term studies of FRDA patient cells and a knock-in mouse model, but the functional consequences of such therapeutic intervention have thus far not been described. We have now investigated the long-term therapeutic effects of 106, 109 and 136 in our GAA repeat expansion mutation-containing YG8R FRDA mouse model. We show that there is no overt toxicity up to 5months of treatment and there is amelioration of the FRDA-like disease phenotype. Thus, while the neurological deficits of this model are mild, 109 and 106 both produced an improvement of motor coordination, whereas 109 and 136 produced increased locomotor activity. All three compounds increased global histone H3 and H4 acetylation of brain tissue, but only 109 significantly increased acetylation of specific histone residues at the FXN locus. Effects on FXN mRNA expression in CNS tissues were modest, but 109 significantly increased frataxin protein expression in brain tissue. 109 also produced significant increases in brain aconitase enzyme activity, together with reduction of neuronal pathology of the dorsal root ganglia (DRG). Overall, these results support further assessment of HDAC inhibitors for treatment of Friedreich ataxia.

Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms

BackgroundMethylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome.ResultsTargeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1.ConclusionsTargeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.

A Common Regulatory Signature Associated with Barrier Insulators in Human Primary Erythroid Cells

AbstractAbstract 3868Insulators are DNA sequences and associated binding proteins that establish and/or maintain boundaries between regions of active and silenced chromatin domains. In higher organisms, there are 2 types of insulators, enhancer-blocking insulators, which establish chromatin domains to separate enhancers and promoters, and barrier insulators, which create a barrier to protect against heterochromatin-mediated gene silencing. Despite their role as critical regulators of tissue-specific gene expression, barrier insulators are poorly understood in mammalian cells, with much of our knowledge from studies of the barrier insulator in the chicken β-globin locus, cHS4. The DNA region of cHS4 that functions as a barrier binds upstream stimulatory factor (USF) proteins that recruit histone methyltransferase (HMT) activity, and histone acetyltransferase (HAT) activity, supporting a model that recruitment of enzymes and other proteins associated with activating histone modifications block the mechanism(s) that lead to spreading of gene-silencing. Our goal is to identify a regulatory signature associated with barrier insulators in erythroid cells. We utilized chromatin immunoprecipitation coupled with ultrahigh throughput Solexa sequencing (ChIP-seq) to generate genome-wide maps of regulatory and chromatin modifying proteins in erythroid cells. To generate cells for ChIP, human CD34+ cells were cultured in serum free media with erythropoietin to yield a population of CD71+/GPA+ erythroid cells (R3/R4 population). First, a genome-wide map of USF1 and USF2 occupancy in primary erythroid cells was created. A total of 19213 USF1 and 20115 USF2 sites of occupancy were identified. USF1 and USF2 frequently heterodimerize; co-localization was present at 15882 sites (83% of USF1 and 79% of USF2 sites). USF heterodimers were commonly located near proximal promoters (within 1KB of TSS, 48% of sites) and enhancers (>1kb from RefSeq gene, 30% of sites). To analyze co-localizing barrier-associated arginine methyltransferases from erythroid cells, ChIP-seq was performed with PRMT1 and PRMT4/CARM1. A total of 7062 PRMT1 sites and 15900 PRMT4 sites were identified. PRMT1 and PRMT4 were commonly found at sites of USF occupancy, with 6120 sites demonstrating occupancy of all four factors, consistent with the hypothesis that the USF proteins frequently recruit HMT's in mammalian cells. Sites of PRMT/USF co-occupancy were more likely to be at proximal promoters (68%) than sites of USF occupancy alone. Genome-wide occupancy of four acetyltransferases commonly found in erythroid cells, CBP, p300, PCAF, and SRC1, was also studied using ChIP-seq. 6804, 46932, 25688, and 25833 sites of occupancy were found for CBP, p300, PCAF, and SRC1 respectively. Co-localization with the p300, PCAF, and SRC1 with the USF/PRMT binding sites was common, occurring in 3825 sites. These sites were most commonly located near proximal promoters (71%) and enhancers (17%). In contrast, CBP co-localized with the USF/PRMT/p300/PCAF/SRC regions in only 10 locations and sites of CBP occupancy were more commonly found at enhancers (64%) and introns (29%) than at promoters (0.4%). Detection of barrier insulators near gene promoters is not surprising. Recent studies have revealed many similarities between barriers and promoters, including binding of specific transcription factors, and have led to the suggestion that barrier insulators have evolved as specialized derivatives of gene promoters, each with specific, yet discrete function. The regulatory protein CTCF mediates enhancer-blocking insulator activity. ChIP-seq was utilized to create a genome-wide map of CTCF binding in erythroid cells. 38503 sites of CTCF occupancy were identified. These sites were located at enhancers (41%), introns, (28%) and proximal promoters (18%). 4459 CTCF sites (12%) co-localized with regions of USF/PRMT/p300/PCAF/SRC binding. These sites most commonly occurred at promoters (65%) and enhancers (19%). The role of CTCF in barrier insulator function is controversial; our data are consistent with recent data demonstrating its mark at chromatin boundaries. The signature composed of USF/PRMT/p300/PCAF/SRC/CTCF was found in the well characterized functional erythroid barrier located in the ankyrin-1 gene proximal promoter region. These data indicate that a common regulatory signature is likely associated with barrier elements in erythroid cells. Disclosures:No relevant conflicts of interest to declare.

Friedreich's Ataxia Induced Pluripotent Stem Cells Model Intergenerational GAA⋅TTC Triplet Repeat Instability

The inherited neurodegenerative disease Friedreich's ataxia (FRDA) is caused by GAA⋅TTC triplet repeat hyperexpansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA⋅TTC repeats cause heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA⋅TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells and occupies FXN intron 1, and shRNA silencing ofMSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.

Drosophila vigilin, DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum

Functional characterisation of vigilin, a highly conserved multi-KH-domain protein that binds RNA and ssDNA, remains elusive and, to some extent, controversial. Studies performed in Saccharomyces cerevisiae and human cells indicate that vigilin localises to the cytoplasm, binds ribosomes, associates to RER and regulates mRNA translation. On the other hand, we and others reported a contribution to heterochromatin-mediated gene silencing (PEV) and chromosome segregation in S. cerevisiae, Drosophila and human cells. Whether this contribution is direct remains, however, unclear. Here, we report that Drosophila vigilin, DDP1, vastly localises to the cytoplasm, being largely excluded from the nucleus. We also show that DDP1 preferentially associates to RER and co-purifies with several ribosomal proteins, suggesting a contribution to mRNA translation. In light of these results, the contribution of DDP1 to PEV was re-examined. Here, we show that a newly generated null ddp1 Δ mutation is only a weak suppressor of PEV, which is in contrast with our own previous results showing dominant suppression in the presence of a strong hypomorphic ddp1 15.1 mutation. Similar results were obtained in the fission yeast Schizosaccharomyces pombe, where vigilin (Vgl1) also associates to RER, having no significant contribution to PEV at centromeres, telomeres and the mating-type locus. Altogether, these results indicate that cytoplasmic localisation and association to RER, but not contribution to heterochromatin organisation, are evolutionarily conserved features of vigilin, favouring a model by which vigilin acts in the cytoplasm, regulating RNA metabolism, and affects nuclear functions only indirectly.

The molecular basis for stability of heterochromatin-mediated silencing in mammals

The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

Heterochromatin-Mediated Gene Silencing Is Not Affected by Drosophila CBP Activity

Cyclic AMP Response Element Binding protein (CREB)-binding protein (CBP) is an acetyltransferase important for modifying histones and chromatin-associated proteins and thus affecting transcription and other DNA metabolic processes. We found that the Drosophila CBP (dCBP) is associated with the NAD(+)-dependent deacetylase, SIR2, which was originally identified as a silencing information regulator in yeast that models silenced and repeated sequence chromatin such as centric heterochromatin, telomeres, and the repeated rDNA sequences. As in yeast, Drosophila sir2 (dsir2) affects the formation and/or function of centric heterochromatin. The fact that we found dCBP in immunecomplexes with dSIR2 in vivo and found that dCBP can interact with dSIR2 directly in vitro suggested that dCBP might affect the packaging of silencing heterochromatin as well. A careful study of the dCBP mutations provides evidence that dCBP does not affect the formation and/or function of centric heterochromatin and thus may affect other dSIR2 functions.

Insulator Elements in Erythrocyte Membrane Genes

Insulators are DNA sequences and associated binding proteins that establish and/or maintain the boundaries between euchromatin and heterochromatin. One type of insulator establishes chromatin domains to separate enhancers and promoters and prevent their interaction (enhancer-blocking insulators) whereas another type creates a barrier to protect against heterochromatin-mediated gene silencing (barrier insulators). In the well-characterized chicken beta-globin LCR 5′HS4 insulator, binding of CTCF mediates enhancer-blocking functions and USF proteins mediate barrier activity. Because varying transcripts of erythrocyte membrane protein genes are expressed in both erythroid and nonerythroid cells, we hypothesized that that insulator elements that bind USF and CTCF participate in their regulation. Advances in technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNA-associated proteins on a genome-wide scale. Coupling chromatin immunoprecipitation to microarrays that contain genomic regions (ChIP-chip) is a high resolution technique available for mapping protein-DNA interactions in vivo. We used ChIP-chip to identify CTCF and USF factor binding sites with potential insulator function that regulate membrane protein gene expression in erythroid cells. ChIP was performed with K562 and HeLa cells using antibodies against CTCF, USF1, and USF2. DNA obtained from these IPs was hybridized to a custom NimbleGen high-density human genomic DNA microarray. Chip probes were ~50bp in length, Tm ≥76°C, tiled ~65bp apart. Regions of repetitive DNA excluded. The chip included 15 erythrocyte membrane protein genes, most encoding complex loci with multiple tissue-, cell-, and developmental stage-specific transcripts, including alpha spectrin, beta spectrin, ankyrin, spectrin, band 3, ƒnalpha adducin, beta adducin, gamma adducin, ICAM4, erythroid associated membrane protein, protein 4.1R, protein 4.2, dematin, beta actin, tropomodulin, and tropomyosin. Each gene plus 50 to 100kb of flanking DNA were included on the chip. Binding sites on the custom DNA array were identified using the Tamalpais peak calling algorithm using L1–L3 level of stringency (Genom Res 16:595, 2006) or Tilescope using a signal cutoff of 0.9 (Genome Biol 8:R81, 2007). In K562 chromatin, 117 sites of CTCF occupancy were identified. There were 49 sites in 5′ flanking DNA, 3 in promoters, 9 in coding sequence, 20 in introns, 1 in 3′ untranslated region, and 35 in 3′ flanking DNA. Sites of USF1 and USF2 occupancy were identified in 7/15 and 9/15 genes, respectively. 15 USF1 sites were identified; 10 in 5′ flanking DNA, 1 in a promoter (<500bp from transcription start site), and 4 in 3′ flanking DNA. 22 sites of USF2 occupancy were identified; 9 in 5′ flanking DNA, 1 in a promoter, 4 in introns, and 8 in 3′ flanking DNA. USF1 and USF2 frequently heterodimerize and 10 sites bound both USF1 and USF2. To validate ChIP-chip results, a site binding USF2 near the ankyrin gene erythroid promoter was further analyzed. This site was chosen because the USF2-associated sequence directs uniform, copy-number dependent expression of a linked reporter gene in transgenic mice, the erythroid ankyrin promoter is positioned between 2 alternate, nonerythroid ankyrin promoters, and DNaseI mapping revealed that this region is contained within an erythroid-specific chromatin domain. Quantitative ChIP confirmed USF2 occupancy, as well as USF1 occupancy, in this region. For functional analysis, we used a gene silencing/position-effect variegation (PEV) assay to determine whether this region possess barrier element function. Using a HS2-beta globin promoter-GFP cassette as negative control and a cHS4-HS2-beta globin promoter-GFP-cHS4 cassette as positive control, the ankyrin promoter region fragment (p<0.001) functioned as barrier elements in this PEV assay. These data demonstrate that there are numerous binding sites for insulator-associated DNA binding proteins in erythrocyte membrane protein genes and indicate that these elements likely play a significant role in the regulation of tissue-specific expression of many genes expressed in erythroid cells.

Characterization of Drosophila melanogaster JmjC+N histone demethylases

In this article, we characterize histone demethylase activity of the entire family of JmjC+N proteins of Drosophila melanogaster. Our results show that Lid (little imaginal discs), which is structurally homologous to JARID1, demethylates H3K4me3. However, contrary to what would be inferred from its demethylase activity, lid contributes to the establishment of transcriptionally competent chromatin states as: (i) is required for histone H3 acetylation; (ii) contributes to expression of the homoeotic gene Ultrabithorax (Ubx); and (iii) antagonizes heterochromatin-mediated gene silencing (PEV). These results, which are consistent with the identification of lid as a trithorax group (trxG) gene, are discussed in the context of current models for the contribution of H3K4me3 to the regulation of gene expression. Here, we also show that the two Drosophila JMJD2 homologues, dJMJD2(1)/CG15835 and dJMJD2(2)/CG33182, are capable of demethylating both H3K9me3 and H3K36me3. dJMJD2(1)/CG15835 regulates heterochromatin organization, as its over-expression induces spreading of HP1, out of heterochromatin, into euchromatin, without affecting the actual pattern of histone modifications of heterochromatin. dJMJD2(1)/CG15835 is excluded from heterochromatin and localizes to multiple euchromatic sites, where it regulates H3K36 methylation. These results indicate that dJMJD2(1)/CG15835 contributes to delimit hetero- and euchromatic territories through the regulation of H3K36 methylation in euchromatin. On the other hand, dJARID2/CG3654 shows no demethylase activity on H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H4K20me3.

Drosophila sticky/citron kinase Is a Regulator of Cell-Cycle Progression, Genetically Interacts With Argonaute 1 and Modulates Epigenetic Gene Silencing

The sticky/citron kinase protein is a conserved regulator of cell-cycle progression from invertebrates to humans. While this kinase is essential for completion of cytokinesis, sticky/citron kinase phenotypes disrupting neurogenesis and cell differentiation suggest additional non-cell-cycle functions. However, it is not known whether these phenotypes are an indirect consequence of sticky mutant cell-cycle defects or whether they define a novel function for this kinase. We have isolated a temperature-sensitive allele of the Drosophila sticky gene and we show that sticky/citron kinase is required for histone H3-K9 methylation, HP1 localization, and heterochromatin-mediated gene silencing. sticky genetically interacts with Argonaute 1 and sticky mutants exhibit context-dependent Su(var) and E(var) activity. These observations indicate that sticky/citron kinase functions to regulate both actin-myosin-mediated cytokinesis and epigenetic gene silencing, possibly linking cell-cycle progression to heterochromatin assembly and inheritance of gene expression states.

HP1α guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

Drosophila longevity is not affected by heterochromatin-mediated gene silencing

Two highly conserved histone deacetylases, Sir2 and Rpd3, have been linked to caloric restriction and the extension of longevity. Because the Drosophila forms of each protein can silence genes in either euchromatin or heterochromatin, we determined whether longevity extension is mediated by silencing in the latter domain. When silencing was increased and decreased using mutations that affect heterochromatin protein 1 (HP1), but have no direct effect upon Sir2 or Rpd3, lifespan was unaffected. Heterochromatin-mediated gene silencing was then modulated without directly influencing HP1 as well as the deacetylases, again yielding no effect on lifespan. Mortality rates were unchanged by all manipulations, indicating that euchromatic targets are likely to be the effectors of deacetylase-mediated longevity extension in Drosophila [corrected]

TIF1δ, a Novel HP1-interacting Member of the Transcriptional Intermediary Factor 1 (TIF1) Family Expressed by Elongating Spermatids

TIF1 (transcriptional intermediary factor 1) proteins are encoded by an expanding family of developmental and physiological control genes that are conserved from flies to man. These proteins are characterized by an N-terminal RING-B box-coiled-coil (RBCC) motif and a C-terminal PHD finger/bromodomain unit, and have been implicated in epigenetic mechanisms of transcriptional repression involving histone modifiers and heterochromatin-binding proteins. We describe here the isolation and functional characterization of a fourth murine TIF1 gene, TIF1delta. The predicted TIF1delta protein displays all the structural hallmarks of a bona fide TIF1 family member and resembles the other TIF1s in that it can exert a deacetylase-dependent silencing effect when tethered to a promoter region. Moreover, like TIF1alpha and TIF1beta, TIF1delta can homodimerize and contains a PXVXL motif necessary and sufficient for HP1 (heterochromatin protein 1) binding. Although TIF1alpha and TIF1beta also bind nuclear receptors and Kruppel-associated boxes specifically and respectively, TIF1delta appears to lack nuclear receptor- and Kruppel-associated box binding activity. Furthermore, TIF1delta is unique among the TIF1 family proteins in that its expression is largely restricted to the testis and confined to haploid elongating spermatids, where it associates preferentially with HP1 isotype gamma (HP1gamma) and forms discrete foci dispersed within the centromeric chromocenter and the surrounding nucleoplasm. Collectively, these data are consistent with specific, nonredundant functions for the TIF1 family members in vivo and suggest a role for TIF1delta in heterochromatin-mediated gene silencing during postmeiotic phases of spermatogenesis.