Dataset Information

Dynamic of chromosome-binding protein complexes to meiotic prophase I of mouse spermatocyte

ABSTRACT: Meiotic prophase I (MPI) is the most important event in mammalian meiosis. Even though MPI-related gene expression has been extensively investigated, the status of the chromosome binding proteins (CBPs) and the corresponding complexes in this process has been not scrutinized yet. A quantitative proteomics focused onto the MPI-related CBPs was accomplished in this study, in which the mouse primary spermatocytes in 4 different subphases of MPI were collected and the chromosome-enriched proteins (ChEPs) within the cells were extracted and identified in quantitative mode. According to a stringent criterion, in total of 1136 CBPs in the MPI subphases were identified and quantified. Looking at the dynamic patterns of CBP abundance in response to MPI progression, the patterns were broadly divided to two groups, high abundance in leptotene (L) and zygotene (Z) or that in pachytene (P) and diplotene (D). Most importantly, 26 CBP complexes were found, while some MPI-dependent features of these complexes were perceived for the first time. The functions of these complexes were categorized to DNA replication, pre-initial complex of transcription, transcriptional regulation and synapsis. In agreement with the current theory, the dynamic changes of the CBP complex abundance appeared declined DNA replication and activated transcription and synapsis activities in P and D, and Z and P phase; in converse to the traditional prediction, the abundance response of CBP complexes to MPI emerged condensin activity weakened in P and D phase. Profiling of protein complexes thus offered convincing evidence how important the CBP complexes partake in MPI regulation.

ORGANISM(S): Mus Musculus

SUBMITTER: Siqi Liu

PROVIDER: PXD035062 | iProX | Thu Jun 08 00:00:00 BST 2023

REPOSITORIES: iProX

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Publications

Dynamic Responses of Chromosome-Binding Protein Complexes to Meiotic Prophase I of Mouse Spermatocyte.

Zhang Yuxing Y Yang Lingling L Fang Kailun K Li Qidan Q Xu Hongkai H Ren Yan Y Zi Jin J Chen Charlie Degui CD Liu Siqi S

Journal of proteome research 20221012 11

Meiotic prophase I (MPI) is the most important event in mammalian meiosis. The status of the chromosome-binding proteins (CBPs) and the corresponding complexes and their functions in MPI have not yet been well scrutinized. Quantitative proteomics focused on MPI-related CBPs was accomplished, in which mouse primary spermatocytes in four different subphases of MPI were collected, and chromosome-enriched proteins were extracted and quantitatively identified. According to a stringent criterion, 1136 ...[more]

PMID: 36223561

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Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Ino80 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 in random culture Distribution of Ino80 in G1 phase Distribution of Ino80 in early S phase • Experimental design ChIP analyses: W303 background cells expressing Myc-tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.

Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Ino80 and Arp5 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 and Arp5 in wild type in random culture Distribution of Ino80 in G1 cells Distribution of Ino80 in early S phase cells • Experimental design ChIP analyses: W303 background cells expressing Myc tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP analyses: W303 background cells expressing Myc tagged Ino80 were used for the ChIP using anti-Arp5 polyclonal antibody. ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.

Dataset Information

Dynamic of chromosome-binding protein complexes to meiotic prophase I of mouse spermatocyte

Publications

Dynamic Responses of Chromosome-Binding Protein Complexes to Meiotic Prophase I of Mouse Spermatocyte.

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