Project description:Numerous nucleosome remodeling enzymes tightly regulate nucleosome positions in eukaryotic cells. Transcription and statistical positioning of nucleosomes may also contribute to proper nucleosome organization. Individual contributions remain controversial due to strong redundancy of processes acting on the nucleosome landscape. By incisive yeast genome engineering we radically decreased their redundancy. We find the transcriptional machinery to be disruptive of evenly spaced nucleosomes, and proper nucleosome density critical for their biogenesis. INO80 spaces nucleosomes in vivo and positions the first nucleosome covering genes. It requires its Arp8 and Ies2 subunits, but unexpectedly not the Nhp10 module, for spacing. Whereas H2A.Z stimulates INO80 in vitro, its presence is dispensable for INO80 +1 positioning function in vivo. DNA damage, recombination and transposon integration assays suggest that evenly spaced nucleosomes protect cells against genotoxic stress. We derive a unifying model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.
Project description:Among the collection of chromatin modifications that influence its function and structure, the substitution of canonical histones by the so-called histone variants is one of the most prominent actions. Since crucial meiotic transactions are modulated by chromatin, here we investigate the functional contribution of the H2A.Z histone variant during both unperturbed meiosis and upon challenging conditions where the meiotic recombination checkpoint is triggered in budding yeast by the absence of the synaptonemal complex component Zip1. We have found that H2A.Z localizes to meiotic chromosomes in an SWR1-dependent manner. Although meiotic recombination is not substantially altered, the htz1 mutant (lacking H2A.Z) shows slower meiotic progression, impaired sporulation and reduced spore viability. These phenotypes are likely accounted for by the misregulation of meiotic gene expression landscape observed in htz1. In the zip1 mutant, the absence of H2A.Z results in a tighter meiotic arrest imposed by the meiotic recombination checkpoint. We have found that Mec1-dependent Hop1-T318 phosphorylation and the ensuing Mek1 activation are not significantly altered in zip1 htz1; however, downstream checkpoint targets, such as the meiosis I-promoting factors Ndt80, Cdc5 and Clb1, are drastically down-regulated. The study of the checkpoint response in zip1 htz1 has also allowed us to reveal the existence of an additional function of the Swe1 kinase, independent of CDK inhibitory phosphorylation, which is relevant to restrain meiotic cell cycle progression. In summary, our study shows that the H2A.Z histone variant impacts various aspects of meiotic development adding further insight into the relevance of chromatin dynamics for accurate gametogenesis.
Project description:The histone variant H2A.Z is a hallmark of nucleosomes flanking the promoters of protein coding genes, and is often found in nucleosomes that also carry lysine 56- acetylated histone H3 (H3-K56Ac), a mark which promotes rapid replication- independent turnover of nucleosomes. Although H2A.Z and H3-K56Ac have been generally implicated in transcriptional activation, their exact contributions have remained elusive. Here we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither H3- K56Ac nor H2A.Z has a significant impact on steady state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and noncoding RNAs (ncRNAs) are only revealed in the absence of the nuclear RNA exosome. H2A.Z is also necessary for expression of divergent, promoter-proximal ncRNAs in mouse embryonic stem cells, suggesting a conserved role for H2A.Z across eukaryotes. Finally, we show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher order chromatin structures. 2 replicates of WT (CY1089), rtt109â (CY2210), rrp6â (CY2071) and one replicate of the W303 input (Sample 7). TableS5.xlsx contains the processed IP/input values for each ORF transcript.