કોળી પટેલ સ્ત્રીઓમાં સામાજિક-કાયદાકીય અને રાજકીય જાગૃતિ - એક અભ્યાસ (અમદાવાદ જિલ્લાના સાણંદ તાલુકાના સંદર્ભમાં)

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The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9

<p>Abstract</p> <p>Background</p> <p>Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in <it>Neurospora crassa</it>, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized.</p> <p>Results</p> <p>The selectable marker, <it>hph</it>, was inserted at the subtelomere of Linkage Group VR in an <it>nst-1 </it>(n<it>eurospora </it>s<it>ir </it>t<it>wo</it>-1) mutant and was silenced when <it>nst-1 </it>function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, <it>bar</it>, tested at two other subtelomeres, was similarly sensitive to <it>nst-1 </it>function. Mutation of three additional SIR2 homologues, <it>nst-2</it>, <it>nst-3 </it>and <it>nst-5</it>, partially relieved silencing. Two genes showed stronger effects: <it>dim-5</it>, which encodes a histone H3 K9 methyltransferase and <it>hpo</it>, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased.</p> <p>Conclusion</p> <p>We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in <it>de novo </it>DNA methylation.</p

Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans

Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans

Substitutions in the Amino-Terminal Tail of Neurospora Histone H3 Have Varied Effects on DNA Methylation

Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3K9L or hH3K9R) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3WT). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9

DNA Methylation and Normal Chromosome Behavior in Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC

Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora

Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation

Understanding the function of histone modifications across inducible genes in mammalian cells requires quantitative, comparative analysis of their fate during gene activation and identification of enzymes responsible. We produced high-resolution comparative maps of the distribution and dynamics of H3K4me3, H3K36me3, H3K79me2 and H3K9ac across c-fos and c-jun upon gene induction in murine fibroblasts. In unstimulated cells, continuous turnover of H3K9 acetylation occurs on all K4-trimethylated histone H3 tails; distribution of both modifications coincides across promoter and 5′ part of the coding region. In contrast, K36- and K79-methylated H3 tails, which are not dynamically acetylated, are restricted to the coding regions of these genes. Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5′ and 3′ ends, respectively. Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells. These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus

The ASH1 HOMOLOG 2 (ASHH2) Histone H3 Methyltransferase Is Required for Ovule and Anther Development in Arabidopsis

BACKGROUND:SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction. METHODOLOGY/PRINCIPAL FINDINGS:A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2. CONCLUSIONS/SIGNIFICANCE:The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences