The Saccharomyces cerevisiae Histone Chaperone Rtt106 Mediates the Cell Cycle Recruitment of SWI/SNF and RSC to the HIR-Dependent Histone Genes

In Saccharomyces cerevisiae, three out of the four histone gene pairs (HTA1-HTB1, HHT1-HHF1, and HHT2-HHF2) are regulated by the HIR co-repressor complex. The histone chaperone Rtt106 has recently been shown to be present at these histone gene loci throughout the cell cycle in a HIR- and Asf1-dependent manner and involved in their transcriptional repression. The SWI/SNF and RSC chromatin remodeling complexes are both recruited to the HIR-dependent histone genes; SWI/SNF is required for their activation in S phase, whereas RSC is implicated in their repression outside of S phase. Even though their presence at the histone genes is dependent on the HIR complex, their specific recruitment has not been well characterized. In this study we focused on characterizing the role played by the histone chaperone Rtt106 in the cell cycle-dependent recruitment of SWI/SNF and RSC complexes to the histone genes.Using GST pull-down and co-immunoprecipitation assays, we showed that Rtt106 physically interacts with both the SWI/SNF and RSC complexes in vitro and in vivo. We then investigated the function of this interaction with respect to the recruitment of these complexes to HIR-dependent histone genes. Using chromatin immunoprecipitation assays (ChIP), we found that Rtt106 is important for the recruitment of both SWI/SNF and RSC complexes to the HIR-dependent histone genes. Furthermore, using synchronized cell cultures, we showed by ChIP assays that the Rtt106-dependent SWI/SNF recruitment to these histone gene loci is cell cycle regulated and restricted to late G1 phase just before the peak of histone gene expression in S phase.Overall, these data strongly suggest that the interaction between the histone chaperone Rtt106 and both the SWI/SNF and RSC chromatin remodeling complexes is important for the cell cycle regulated recruitment of these two complexes to the HIR-dependent histone genes

Distribution of Introns in Fungal Histone Genes

Saccharomycotina and Taphrinomycotina lack intron in their histone genes, except for an intron in one of histone H4 genes of Yarrowia lipolytica. On the other hand, Basidiomycota and Perizomycotina have introns in their histone genes. We compared the distributions of 81, 47, 79, and 98 introns in the fungal histone H2A, H2B, H3, and H4 genes, respectively. Based on the multiple alignments of the amino acid sequences of histones, we identified 19, 13, 31, and 22 intron insertion sites in the histone H2A, H2B, H3, and H4 genes, respectively. Surprisingly only one hot spot of introns in the histone H2A gene is shared between Basidiomycota and Perizomycotina, suggesting that most of introns of Basidiomycota and Perizomycotina were acquired independently. Our findings suggest that the common ancestor of Ascomycota and Basidiomycota maybe had a few introns in the histone genes. In the course of fungal evolution, Saccharomycotina and Taphrinomycotina lost the histone introns; Basidiomycota and Perizomycotina acquired other introns independently. In addition, most of the introns have sequence similarity among introns of phylogenetically close species, strongly suggesting that horizontal intron transfer events between phylogenetically distant species have not occurred recently in the fungal histone genes

Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

The ability to reactivate ultraviolet (UV) damaged phage ΦCbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage ΦCbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2–2 h at 30°C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5–10 J/m 2 of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec -requiring DNA repair system analogous to W-reactivation in Escherichia coli . In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47557/1/438_2004_Article_BF00329935.pd

Methylated H3K4, a Transcription-Associated Histone Modification, Is Involved in the DNA Damage Response Pathway

Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells

Genome-wide analysis of regions similar to promoters of histone genes

Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes. Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters. Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that participate in regulation of histone and histone-coregulated gene transcription initiation. While these hypotheses still remain to be verified, we believe that these form a useful resource for researchers to further explore regulation of human histone genes and human genome. It is worthwhile to note that the regulatory regions of the human genome remain largely un-annotated even today and this study is an attempt to supplement our understanding of histone regulatory regions.Statistic

The Role of Histone H4 Biotinylation in the Structure of Nucleosomes

Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation

Dia2 Controls Transcription by Mediating Assembly of the RSC Complex

Background: Dia2 is an F-box protein found in the budding yeast, S. cerevisiae. Together with Skp1 and Cul1, Dia2 forms the substrate-determining part of an E3 ubiquitin ligase complex, otherwise known as the SCF. Dia2 has previously been implicated in the control of replication and genome stability via its interaction with the replisome progression complex. Principal Findings: We identified components of the RSC chromatin remodelling complex as genetic interactors with Dia2, suggesting an additional role for Dia2 in the regulation of transcription. We show that Dia2 is involved in controlling assembly of the RSC complex. RSC belongs to a group of ATP-dependent nucleosome-remodelling complexes that controls the repositioning of nucleosomes. The RSC complex is expressed abundantly and its 17 subunits are recruited to chromatin in response to both transcription activation and repression. In the absence of Dia2, RSC-mediated transcription regulation was impaired, with concomitant abnormalities in nucleosome positioning. Conclusions: Our findings imply that Dia2 is required for the correct assembly and function of the RSC complex. Dia2, by controlling the RSC chromatin remodeller, fine-tunes transcription by controlling nucleosome positioning during transcriptional activation and repression

H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

<p>Abstract</p> <p>Background</p> <p>The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA.</p> <p>Results</p> <p>Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in <it>Saccharomyces cerevisiae</it>. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an <it>htb-K123R </it>mutation, leads to synthetic lethality.</p> <p>Conclusion</p> <p>These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.</p

An NF-Y-Dependent Switch of Positive and Negative Histone Methyl Marks on CCAAT Promoters

Background: Histone tails have a plethora of different post-translational modifications, which are located differently in ‘‘open’ ’ and ‘‘closed’ ’ parts of genomes. H3K4me3/H3K79me2 and H4K20me3 are among the histone marks associated with the early establishment of active and inactive chromatin, respectively. One of the most widespread promoter elements is the CCAAT box, bound by the NF-Y trimer. Two of NF-Y subunits have an H2A-H2B-like structure. Principal findings: We established the causal relationship between NF-Y binding and positioning of methyl marks, by ChIP analysis of mouse and human cells infected with a dominant negative NF-YA: a parallel decrease in NF-Y binding, H3K4me3, H3K79me2 and transcription was observed in promoters that are dependent upon NF-Y. On the contrary, changes in the levels of H3K9-14ac were more subtle. Components of the H3K4 methylating MLL complex are not recruited in the absence of NF-Y. As for repressed promoters, NF-Y removal leads to a decrease in the H4K20me3 mark and deposition of H3K4me3. Conclusions: Two relevant findings are reported: (i) NF-Y gains access to its genomic locations independently from the presence of methyl histone marks, either positive or negative; (ii) NF-Y binding has profound positive or negative consequences on the deposition of histone methyl marks. Therefore NF-Y is a fundamental switch at the heart of decisio

Genome-Wide uH2A Localization Analysis Highlights Bmi1-Dependent Deposition of the Mark at Repressed Genes

Polycomb group (PcG) proteins control organism development by regulating the expression of developmental genes. Transcriptional regulation by PcG proteins is achieved, at least partly, through the PRC2-mediated methylation on lysine 27 of histone H3 (H3K27) and PRC1-mediated ubiquitylation on lysine 119 of histone H2A (uH2A). As an integral component of PRC1, Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome-wide binding patterns of some of the PRC1 and PRC2 components, as well as the H3K27me3 mark, there have been no reports describing genome-wide localization of uH2A. Using the recently developed ChIP-Seq technology, here, we report genome-wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site (TSS) of well-annotated genes. This peak is enriched at promoters containing the H3K27me3 mark and represents the least expressed genes in WT MEF cells. In addition, peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower-level expression when compared to genes that do not contain promoter peaks of uH2A. Moreover, we demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout. Importantly, local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. We describe the enrichment of H2A ubiquitylation at high-density CpG promoters and provide evidence to suggest that DNA methylation may be linked to uH2A at these regions. Thus, our work not only reveals Bmi1-dependent H2A ubiquitylation, but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells