Effects of sodium valproate on the chromatin of Triatoma infestans (Klug, 1834) (Hemiptera,Reduviidae) under in vitro culture conditions

Sodium valproate (VPA) is a classic anticonvulsive, a histone deacetylase inhibitor, and a chromatin remodeling inducer. When injected into specimens of Triatoma infestans, a vector of Chagas disease, VPA affects the chromatin supraorganization of chromocenter heterochromatin in only a few cells of the Malpighian tubules. To test whether this result was explained by the inaccessibility of all of the organ’s cells to the drug, we investigated the nuclear phenotypes and global acetylation of lysine 9 in histone H3 (H3K9ac) in Malpighian tubules cultivated in vitro for 1–24 h in the presence of 0.05 mM–1 mM VPA. The present results revealed that the chromatin decondensation event in the chromocenter body, which was detected only under low VPA concentrations up to a 4-h treatment, was not frequent during organ culture, similar to the results for injected insects. Cultivation of T. infestans Malpighian tubules in vitro for 24 h revealed inadequate for cell preservation even in the absence of the drug. Immunofluorescence signals for H3K9ac following VPA treatment showed a slightly increased intensity in the euchromatin, but were never detected in the chromocenter bodies, except with great intensity at their periphery, where the 18S rDNA is located. In conclusion, when VPA affects the chromocenter heterochromatin in this animal cell model, it occurs through a pathway that excludes a classic global H3K9ac mark. Investigation of nonhistone proteins associated with histone methylation marks is still required to further explain the differential response of T. infestans chromatin to VPA.     

The Polycomb Group Protein SUZ12 regulates histone H3 lysine 9 methylation and HP1α distribution

Regulation of histone methylation is critical for proper gene expression and chromosome function. Suppressor of Zeste 12 (SUZ12) is a requisite member of the EED/EZH2 histone methyltransferase complexes, and is required for full activity of these complexes in vitro. In mammals and flies, SUZ12/Su(z)12 is necessary for trimethylation of histone H3 on lysine 27 (H3K27me3) on facultative heterochromatin. However, Su(z)12 is unique among Polycomb Group Proteins in that Su(z)12 mutant flies exhibit gross defects in position effect variegation, suggesting a role for Su(z)12 in constitutive heterochromatin formation. We investigated the role of Suz12 in constitutive heterochromatin and discovered that Suz12 is required for histone H3 lysine 9 tri-methylation (H3K9me3) in differentiated but not undifferentiated mouse embryonic stem cells. Knockdown of SUZ12 in human cells caused a reduction in H3K27me3 and H3K9me3, and altered the distribution of HP1α. In contrast, EZH2 knockdown caused loss of H3K27me3 but not H3K9me3, indicating that SUZ12 regulates H3-K9 methylation in an EZH2-independent fashion. This work uncovers a role for SUZ12 in H3-K9 methylation.     

The proper connection between shelterin components is required for telomeric heterochromatin assembly

Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of shelterin. Through affinity purification, we found that shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by shelterin to chromosome ends and the proper connection of shelterin components, which allows CLRC to skip telomeric repeats to internal regions.     

Enhancer-associated H3K4 monomethylation by Trithorax-related,the Drosophila homolog of mammalian Mll3/Mll4

Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers.     

Histone Acetylation Level and Histone Acetyltransferase/Deacetylase Activity in Ejaculated Sperm from Normozoospermic Men

Purpose

The aim of this work was to evaluate nuclear histone acetylation level and total histone acetyltransferase (HAT) and deacetylase (HDAC) activity in ejaculated sperm and their relevance to conventional sperm parameters.

Materials and Methods

Thirty-three normozoospermic men were included in this study. Semen samples were processed by swim-up and then immunostained by six acetylation antibodies (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac). Our preliminary study verified the expression of HAT/HDAC1 in mature human sperm. From vitrified-warmed sperm samples, total HAT/HDAC activity was measured by commercially available kits. Nuclear DNA integrity was also measured by TUNEL assay.

Results

The levels of six acetylation marks were not related with conventional sperm parameters including sperm DNA fragmentation index (DFI) as well as HAT/HDAC activity. However, sperm DFI was positively correlated with HAT activity (r=0.038 after adjustment, p<0.02). HAT activity showed a negative relationship with HDAC activity (r=-0.51, p<0.01). Strict morphology was negatively correlated with acetylation enzyme index (=HAT activity/HDAC activity) (r=-0.53, p<0.01).

Conclusion

Our works demonstrated a significant relationship of acetylation-associated enzyme activity and strict morphology or sperm DFI.     

A conserved ncRNA-binding protein recruits silencing factors to heterochromatin through an RNAi-independent mechanism

Long noncoding RNAs (lncRNAs) can trigger repressive chromatin, but how they recruit silencing factors remains unclear. In Schizosaccharomyces pombe, heterochromatin assembly on transcribed noncoding pericentromeric repeats requires both RNAi and RNAi-independent mechanisms. In Saccharomyces cerevisiae, which lacks a repressive chromatin mark (H3K9me [methylated Lys9 on histone H3]), unstable ncRNAs are recognized by the RNA-binding protein Nrd1. We show that the S. pombe ortholog Seb1 is associated with pericentromeric lncRNAs. Individual mutation of dcr1⁺ (Dicer) or seb1⁺ results in equivalent partial reductions of pericentromeric H3K9me levels, but a double mutation eliminates this mark. Seb1 functions independently of RNAi by recruiting the NuRD (nucleosome remodeling and deacetylase)-related chromatin-modifying complex SHREC (Snf2–HDAC [histone deacetylase] repressor complex).     

The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa

Transposable elements are common in genomes and must be controlled. Many organisms use DNA methylation to silence such selfish DNA, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. We identified a JmjC domain protein in Neurospora, DNA METHYLATION MODULATOR-1 (DMM-1), that prevents aberrant spreading of DNA and histone H3K9 methylation from inactivated transposons into nearby genes. Mutation of a conserved residue within the JmjC Fe(II)-binding site abolished dmm-1 function, as did mutations in conserved cysteine-rich domains. Mutants defective only in dmm-1 mutants grow poorly, but growth is restored by reduction or elimination of DNA methylation using the drug 5-azacytosine or by mutation of the DNA methyltransferase gene dim-2. DMM-1 relies on an associated protein, DMM-2, which bears a DNA-binding motif, for localization and proper function. HP1 is required to recruit the DMM complex to the edges of methylated regions.     

Defining heterochromatin in C. elegans through genome-wide analysis of the heterochromatin protein 1 homolog HPL-2

Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In most organisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavily studied model C. elegans, which possesses holocentric chromosomes with dispersed centromeres, we analyzed the genome-wide distribution of the heterochromatin protein 1 (HP1) ortholog HPL-2 and compared its distribution to other features commonly associated with heterochromatin. HPL-2 binding highly correlates with histone H3 mono- and dimethylated at lysine 9 (H3K9me1 and H3K9me2) and forms broad domains on autosomal arms. Although HPL-2, like other HP1 orthologs, binds H3K9me peptides in vitro, the distribution of HPL-2 in vivo appears relatively normal in mutant embryos that lack H3K9me, demonstrating that the chromosomal distribution of HPL-2 can be achieved in an H3K9me-independent manner. Consistent with HPL-2 serving roles independent of H3K9me, hpl-2 mutant worms display more severe defects than mutant worms lacking H3K9me. HPL-2 binding is enriched for repetitive sequences, and on chromosome arms is anticorrelated with centromeres. At the genic level, HPL-2 preferentially associates with well-expressed genes, and loss of HPL-2 results in up-regulation of some binding targets and down-regulation of others. Our work defines heterochromatin in an important model organism and uncovers both shared and distinctive properties of heterochromatin relative to other systems.Eukaryotic genomes are packaged into two general types of chromatin: euchromatin and heterochromatin. This packaging is important for the regulation of gene expression and organization of the genome. Initially, heterochromatin was cytologically defined as the condensed, dark-staining chromatin that remains visible throughout the cell cycle (Heitz 1928). Since then, numerous molecular characteristics of heterochromatin have been identified. These include an enrichment of repetitive DNA elements, such as satellite DNA and sequences derived from transposable elements, and enrichment of histone H3 methylated at lysine 9 (H3K9me) (Grewal and Elgin 2002). Another hallmark of heterochromatin is the enrichment of heterochromatin protein 1 (HP1), a highly conserved, small nonhistone protein first identified in Drosophila (James and Elgin 1986). Heterochromatin is typically concentrated at pericentric and subtelomeric regions. How heterochromatin is distributed in an organism with numerous centromeres distributed along the length of each chromosome (i.e., holocentric) is not known. This paper defines the distribution of an HP1 protein and heterochromatin in the nematode C. elegans, which possesses holocentric chromosomes and is a valuable model for genome organization, chromosome segregation, gene expression, and development.It has been shown through a variety of methods that the chromo domain (CD) of metazoan HP1 proteins specifically recognizes H3K9me2 and H3K9me3 (Bannister et al. 2001; Jacobs et al. 2001; Lachner et al. 2001). Evidence that this interaction is important for proper HP1 protein localization comes from the observation that loss or reduction of H3K9me results in loss or reduction of HP1 binding in vivo (Bannister et al. 2001; Lachner et al. 2001; Schotta et al. 2002; Seum et al. 2007; Tzeng et al. 2007). Other interactions besides the CD-H3K9me interaction are involved in HP1 localization as well, as Drosophila SU(VAR)205 (also known as HP1A) is able to associate with promoter regions of genes independently of H3K9me (Figueiredo et al. 2012), and HP1A lacking its CD is able to associate with heterochromatin (Smothers and Henikoff 2001). Furthermore, in vitro studies have shown that mouse CBX1, CBX3, and CBX5 (also known as HP1β, HP1γ, and HP1α, respectively) bind the histone-fold domain of histone H3 (Nielsen et al. 2001) and that fly HP1A binds DNA in a sequence-independent manner (Zhao et al. 2000). Interestingly, studies in fission yeast, flies, and mammals have demonstrated that the RNAi machinery and RNA itself contribute to HP1 protein localization (Pal-Bhadra et al. 2004; Verdel et al. 2004; Maison et al. 2011). Taken together, these studies implicate interactions between HP1 and methylated histone tails, histone cores, DNA, and RNA as contributing to the recruitment and retention of HP1 at particular DNA regions in vivo. In this study, we specifically tested whether H3K9me is required for proper HP1 localization in C. elegans.The nematode C. elegans has two HP1 paralogous proteins: HP1 Like (heterochromatin protein) 1 and 2 (HPL-1 and HPL-2) (Couteau et al. 2002). HPL-2 serves more roles and/or more important roles than HPL-1, as hpl-2 mutants display diverse defects while hpl-1 mutants generally lack observable mutant phenotypes. HPL-2 is an important factor for germline health, as hpl-2 mutants display maternal-effect sterility at elevated temperature (25°C) (Coustham et al. 2006) and a reduced ability to silence exogenous “non-self” sequences in the germline (Couteau et al. 2002; Robert et al. 2005; Ashe et al. 2012; Shirayama et al. 2012). HPL-2 is also important in somatic development, as hpl-2 mutants show larval, somatic gonad, and vulval developmental defects (Schott et al. 2006). Comparisons of hpl-2 hpl-1 double mutants and hpl-2 single mutants suggest that HPL-2 and HPL-1 have some overlapping roles, as double mutant worms display more severe phenotypes than hpl-2 alone (Schott et al. 2006; Shirayama et al. 2012). Because HPL-2 is the more important of the two C. elegans HP1 homologs and is the only HP1 homolog in C. briggsae, a close relative of C. elegans (Vermaak and Malik 2009), we focused our current study on HPL-2.Here, we show that HPL-2 binding to chromatin highly correlates with H3K9me1 and H3K9me2 throughout the genome and that HPL-2-enriched regions form domains that are also enriched for repetitive DNA elements. These observations suggest that HPL-2 indeed has functions associated with heterochromatin and that HPL-2-enriched domains represent the distribution of heterochromatin in C. elegans. Surprisingly, H3K9me is not necessary for the normal distribution of HPL-2, as the genome-wide pattern of HPL-2 is largely unchanged in met-2 set-25 mutant embryos, which Towbin et al. reported and we verified to lack H3K9me (Towbin et al. 2012). Consistent with HPL-2 having roles independent of H3K9me, met-2 set-25 mutants display less sterility at elevated temperature than hpl-2. Interestingly, worm heterochromatin has a unique distribution relative to other organisms: enrichment on the autosomal “arms” and on the leftmost region of the X chromosome. On autosomal arms, elevated HPL-2 levels flank centromeric chromatin, creating regions that resemble pericentric heterochromatin. HPL-2 shows a bias toward association with well-expressed genes, where it seems to repress the expression of some genes it binds and promote the expression of others. Our studies uncover both shared and unique properties of worm heterochromatin compared to other organisms, and reveal how heterochromatin is distributed in an organism with holocentric chromosomes.     

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.     

Histone acetylation and methylation marks in chromatin of Panstrongylus megistus (Hemiptera,Reduviidae)

Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me₂ in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me₃ in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me₂ marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation.     

Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4

Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD_4–6) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4''s nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD_4–6 reduce PHD_4–6''s binding ability and MLL4''s catalytic activity. PHD_4–6''s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s''s interference with PHD_4–6''s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation.     

Elimination of a specific histone H3K14 acetyltransferase complex bypasses the RNAi pathway to regulate pericentric heterochromatin functions

In Schizosaccharomyces pombe, the RNAi pathway is required for the formation of pericentric heterochromatin, proper chromosome segregation, and repression of pericentric meiotic recombination. Here we demonstrate that, when the activity of the histone H3 Lys 14 (H3K14) acetyltransferase Mst2 is eliminated, the RNAi machinery is no longer required for pericentric heterochromatin functions. We further reveal that reducing RNA polymerase II recruitment to pericentric regions is essential for maintaining heterochromatin in the absence of RNAi.     

Systematic screen reveals new functional dynamics of histones H3 and H4 during gametogenesis

Profound epigenetic differences exist between genomes derived from male and female gametes; however, the nature of these changes remains largely unknown. We undertook a systematic investigation of chromatin reorganization during gametogenesis, using the model eukaryote Saccharomyces cerevisiae to examine sporulation, which has strong similarities with higher eukaryotic spermatogenesis. We established a mutational screen of histones H3 and H4 to uncover substitutions that reduce sporulation efficiency. We discovered two patches of residues—one on H3 and a second on H4—that are crucial for sporulation but not critical for mitotic growth, and likely comprise interactive nucleosomal surfaces. Furthermore, we identified novel histone post-translational modifications that mark the chromatin reorganization process during sporulation. First, phosphorylation of H3T11 appears to be a key modification during meiosis, and requires the meiotic-specific kinase Mek1. Second, H4 undergoes amino tail acetylation at Lys 5, Lys 8, and Lys 12, and these are synergistically important for post-meiotic chromatin compaction, occurring subsequent to the post-meiotic transient peak in phosphorylation at H4S1, and crucial for recruitment of Bdf1, a bromodomain protein, to chromatin in mature spores. Strikingly, the presence and temporal succession of the new H3 and H4 modifications are detected during mouse spermatogenesis, indicating that they are conserved through evolution. Thus, our results show that investigation of gametogenesis in yeast provides novel insights into chromatin dynamics, which are potentially relevant to epigenetic modulation of the mammalian process.     

Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomyces cerevisiae

SWR1-Com, which is responsible for depositing H2A.Z into chromatin, shares four subunits with the NuA4 histone acetyltransferase complex. This overlap in composition led us to test whether H2A.Z was a substrate of NuA4 in vitro and in vivo. The N-terminal tail of H2A.Z was acetylated in vivo at multiple sites by a combination of NuA4 and SAGA. H2A.Z acetylation was also dependent on SWR1-Com, causing H2A.Z to be efficiently acetylated only when incorporated in chromatin. Unacetylatable H2A.Z mutants were, like wild-type H2A.Z, enriched at heterochromatin boundaries, but were unable to block spreading of heterochromatin. A mutant version of H2A.Z that could not be acetylated, in combination with a mutation in a nonessential gene in the NuA4 complex, caused a pronounced decrease in growth rate. This H2A.Z mutation was lethal in combination with a mutant version of histone H4 that could not be acetylated by NuA4. Taken together, these results show a role for H2A.Z acetylation in restricting silent chromatin, and reveal that acetylation of H2A.Z and H4 can contribute to a common function essential to life.