Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA107359 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The structural integrity of the nucleosome is central to regulation of DNA metabolism and transcription. We describe a library of 486 systematic histone H3 and H4 substitution and deletion mutants in Saccharomyces cerevisiae that probe the contribution of each residue to nucleosome function and can be episomal or genomically integrated. We tagged each mutant histone gene with unique molecular barcodes, facilitating identification of mutant pools through barcode amplification, labeling, and microarray hybridization. We probed fitness contributions of each residue to chemical perturbagens of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for three forms of transcriptional silencing onto the nucleosome surface. Lethal mutants were surprisingly rare and of distinct types; one set of mutations mapped precisely to the DNA interaction surface. The barcode microarrays were useful for scoring complex phenotypes such as competitive fitness in a chemostat, proficiency of DNA repair, and synthetic genetic interactions. Keywords: genetic modification These nine datasets characterize a microarray platform repurposed for profiling a barcoded comprehensive library of synthetic histone mutants. The first two datasets establish the wide dynamic range and high sensitivity and specificity of data from this platform. The other seven datasets demonstrate the usefulness of this technology for scoring subtle and complex phenotypes of the histone H3 and H4 alleles in this library.
2008-09-18 | E-GEOD-10860 | biostudies-arrayexpress
Project description:Chromatin replication requires tight coordination of nucleosome assembly machinery with DNA replication machinery. While significant progress has been made in characterizing histone chaperones in this process, the mechanism of whereby nucleosome assembly couples with DNA replication remains largely unknown. Here we show that replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein that is essential for DNA replication provides a binding platform for H3-H4 deposition by histone chaperons and is required for nucleosome formation on nascent chromatin. RPA binds free histone H3-H4 but not nucleosomal histones, and a RPA coated ssDNA stimulates assembly of H3-H4 onto double strand DNA in vitro. RPA mutant with reduced H3-H4 binding exhibits synthetic genetic interaction with mutations at key factors involved in replication-coupled (RC) nucleosome assembly, and are defective in assembly of replicating DNA into nucleosomes in cells. These results reveal a novel function for RPA in nucleosome assembly and a mechanism whereby nucleosome assembly is coordinated with DNA replication.
2017-01-27 | GSE83648 | GEO
Project description:The structural integrity of the nucleosome is central to regulation of DNA metabolism and transcription. We describe a library of 486 systematic histone H3 and H4 substitution and deletion mutants in Saccharomyces cerevisiae that probe the contribution of each residue to nucleosome function and can be episomal or genomically integrated. We tagged each mutant histone gene with unique molecular barcodes, facilitating identification of mutant pools through barcode amplification, labeling, and microarray hybridization. We probed fitness contributions of each residue to chemical perturbagens of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for three forms of transcriptional silencing onto the nucleosome surface. Lethal mutants were surprisingly rare and of distinct types; one set of mutations mapped precisely to the DNA interaction surface. The barcode microarrays were useful for scoring complex phenotypes such as competitive fitness in a chemostat, proficiency of DNA repair, and synthetic genetic interactions. Keywords: genetic modification
2008-09-19 | GSE10860 | GEO
Project description:Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a non-essential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutations, H3 Q5A, H3 R40A, H3 G44A, H3 R53A, H3 N108A, H3 L109A, H4 K44A, H4 V81A, and H4 Y98A, were common to both screens and exhibited kinetochore biorientation defects. Five of the mutants map near the unstructured nucleosome entry site and their genetic interaction with decreased Ipl1 function can be suppressed by increasing the dosage of the Sgo1 protein. In addition, the composition of purified kinetochores was altered in five of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies of the role of the underlying chromatin structure in segregation.
2013-12-04 | GSE39903 | GEO
Project description:Histone acetylation is important for the activation of gene transcription but little is known about its direct ‘read/write’ mechanisms. Here, we report cryo-electron microscopy structures in which a p300/CBP multidomain monomer recognizes histone H4 N-terminal tail (NT) acetylation (ac) in a nucleosome and acetylates non-H4 histone NTs within the same nucleosome. p300/CBP not only recognized H4NTac via the bromodomain pocket responsible for ‘reading’, but also interacted with the DNA minor grooves via the outside of that pocket. This directed the catalytic center of p300/CBP to one of the non-H4 histone NTs. The primary target that p300 ‘writes’ by ‘reading’ H4NTac was H2BNT, and H2BNTac promoted H2A-H2B dissociation from the nucleosome. We propose a model in which p300/CBP ‘replicates’ histone NT acetylation within the H3-H4 tetramer to inherit epigenetic storage, and ‘transcribes’ it from the H3-H4 tetramer to the H2B-H2A dimers to activate context-dependent gene transcription through local nucleosome destabilization.
2023-05-08 | PXD040835 | Pride
Project description:Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a non-essential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutations, H3 Q5A, H3 R40A, H3 G44A, H3 R53A, H3 N108A, H3 L109A, H4 K44A, H4 V81A, and H4 Y98A, were common to both screens and exhibited kinetochore biorientation defects. Five of the mutants map near the unstructured nucleosome entry site and their genetic interaction with decreased Ipl1 function can be suppressed by increasing the dosage of the Sgo1 protein. In addition, the composition of purified kinetochores was altered in five of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies of the role of the underlying chromatin structure in segregation. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.
2013-12-04 | E-GEOD-39903 | biostudies-arrayexpress
Project description:A library of unmodified and differentially modified human histones H3 and H4 was prepared using native chemical ligation as described previously (Bartke et al., 2010; Nakamura et al., 2019). The modification status of histone H3 and H4 products was confirmed by LC-MS/MS.
2023-12-10 | PXD020773 | Pride
Project description:Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Pol axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging strands of DNA replication forks. Mutating the H3-H4 binding domain of Mcm2, a subunit of the CMG (Cdc45-MCM-GINS) replicative helicase that translocates along leading strands, impairs the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Pol-primase mutants that disrupt the connection of the CMG helicase via Ctf4-Pol to lagging strands. Our results support a model whereby parental (H3-H4)2 displaced from nucleosomes through leading-strand DNA replication are transferred to lagging strands for nucleosome assembly via the CMG-Ctf4-Pol complex.
2018-09-24 | GSE118580 | GEO
Project description:Nucleosome structure directly influences gene transcription. However, the function of each histone residue remains largely unknown. Here we profiled gene expression changes upon the mutation of individual residues of histone H3 and H4. Histone residues grouped by expression change similarity displayed overall structural relevance. This regulatory functional map of the core histones led to novel findings. First, the residues specific to each histone family tend to be more influential than those commonly found among different histones. Second, unlike histone acetylations, H3K4 trimethylation does not appear to be prerequisite for gene activation. Third, H3Q5 has been newly identified for its putative interactions with many chromatin regulators for transcription control. Lastly, the nucleosome lateral surface seems to play a key role through interactions with the surrounding DNA. Remarkably, we discovered a novel role for H3K56 in chromatin dynamics. The deletion of this residue, but not the alteration of acetylation states, caused a genome-wide decrease in nucleosome mobility and stabilized nucleosome positioning near transcription start and end sites. Occupying the DNA entry/exit site, H3K56 is thought to modulate nucleosome sliding along DNA. Taken together, genomics approaches such as microarray and deep sequencing prove valuable for mapping the function of histone residues. Microarray analysis was performed for 123 histone mutants and four wild-types as two reaplications of H3 and H4 of Saccharomyces ceravisiae.
2012-12-05 | E-GEOD-29059 | biostudies-arrayexpress
Project description:Using histone H3 mutants designed so that they can only form a heterodimer, we studied the gene expression in a collection of mutants, comparing mutations to one histone H3 tail of a nucleosome compared to two histone H3 tails of a nucleosome.
2020-05-01 | GSE81531 | GEO