Project description:Chromatin structure and DNA accessibility are partly modulated by the incorporation of histone variants. H2A.Z, encoded by the non-essential HTZ1 gene in S. cerevisiae, is an evolutionarily conserved H2A histone variant that is predominantly incorporated at transcription start sites by the SWR1-complex (SWR1-C). While H2A.Z has often been implicated in transcription regulation, htz1Δ mutants exhibit minimal changes in gene expression compared to wild-type. However, given that growth defects of htz1Δ mutants are alleviated by simultaneous deletion of SWR1-C subunits, previous work examining the role of H2A.Z in gene expression regulation may be confounded by deleterious activity caused by SWR1-C when its missing its H2A.Z substrate (apo-SWR1-C). Furthermore, as H2A.Z mutants only display significant growth defects in genotoxic stress conditions, a more substantive role for H2A.Z in gene expression may only be uncovered after exposure to cellular stress. To explore this possibility, we generated mRNA transcript profiles for wild-type, htz1Δ, swr1Δ, and htz1Δswr1Δ mutants before and after exposure to hydroxyurea (HU), which induces DNA replication stress. Our data showed that H2A.Z played a more prominent role in gene activation than repression during HU exposure, and its incorporation was important for proper upregulation of several HU-induced genes. We also observed that apo-SWR1-C contributed to gene expression defects in the htz1Δ mutant, particularly for genes involved in phosphate homeostasis regulation. Furthermore, mapping H2A.Z incorporation before and after treatment with HU revealed that decreases in H2A.Z enrichment at transcription start sites was correlated with, but generally not required for, the upregulation of genes during HU exposure. Together this study characterized the regulatory effects of H2A.Z incorporation during the transcriptional response to HU.
Project description:Purpose: ATG41 is involved both in autophagy and zinc-deficient growth. The goal of this study is to compare transcriptomic profiles of wild-type and atg41Δ strains to discover autophagy-independent molecular phenotypes for the mutant. The atg1Δ mutant is a control for autophagy activity. Methods: Wild-type and mutant yeast were grown to mid-log phase in replete medium and shifted to zinc-deficient medium for 8 hours, after which, cells were harvested for RNA sequencing to detect differential gene expression. Results: Gene expression data for virtually every gene (~6,000) was obtained with ~12,000,000 reads per sample. Differential gene expression analysis showed that several hundred genes were differentially experessed in the atg41Δ mutant (greater than 2-fold) at an FDR of 0.5. Conclusions: Most strikingly, we found that the atg41Δ mutant transcriptome shows signs that sulfur metabolism is distrupted during zinc-deficinet growth. Expression of Met4 gene targets is increased.
Project description:Sequencing of mononucleosomal DNA during G1 and S phases in Saccharomyces cerevisiae Samples from mononucleosomal DNA from WT and rpd3 mutant strains (W303-1a background) in G1 or in S phase in the presence of 0.2 M HU were sequenced (Illumina Genome Analyzer IIx) using the single-end read protocol
Project description:Chromatin structure and DNA accessibility are partly modulated by the incorporation of histone variants. H2A.Z, encoded by the non-essential HTZ1 gene in S. cerevisiae, is an evolutionarily conserved H2A histone variant that is predominantly incorporated at transcription start sites by the SWR1-complex (SWR1-C). While H2A.Z has often been implicated in transcription regulation, htz1Δ mutants exhibit minimal changes in gene expression compared to wild-type. However, given that growth defects of htz1Δ mutants are alleviated by simultaneous deletion of SWR1-C subunits, previous work examining the role of H2A.Z in gene expression regulation may be confounded by deleterious activity caused by SWR1-C when its missing its H2A.Z substrate (apo-SWR1-C). Furthermore, as H2A.Z mutants only display significant growth defects in genotoxic stress conditions, a more substantive role for H2A.Z in gene expression may only be uncovered after exposure to cellular stress. To explore this possibility, we generated mRNA transcript profiles for wild-type, htz1Δ, swr1Δ, and htz1Δswr1Δ mutants before and after exposure to hydroxyurea (HU), which induces DNA replication stress. Our data showed that H2A.Z played a more prominent role in gene activation than repression during HU exposure, and its incorporation was important for proper upregulation of several HU-induced genes. We also observed that apo-SWR1-C contributed to gene expression defects in the htz1Δ mutant, particularly for genes involved in phosphate homeostasis regulation. Furthermore, mapping H2A.Z incorporation before and after treatment with HU revealed that decreases in H2A.Z enrichment at transcription start sites was correlated with, but generally not required for, the upregulation of genes during HU exposure. Together this study characterized the regulatory effects of H2A.Z incorporation during the transcriptional response to HU.
Project description:Yeast sensor checkpoint kinase Mec1 is phosphorylated at Ser1991 upon HU replication stress. The phospho-accepter mutant (mec1-S1991A) confers HU sensitivity and synthetic sickness with the mutants that aggravate replication and transcription collision. To understand how the entire chromatin proteome (chromatome) responds to replication stress, we first investigated the global chromatin-bound proteins in S-phase yeast cells in the presence and absence of HU. Second, we performed phospho-proteome analysis in wild-type and mec1-S1991A mutant to find the S1991-phospho-dependent targets of Mec1 in the presence and absence of HU. Samples were duplicated in chromatome and triplicated in phospho-proteome analysis.