Dataset Information

Mapping of histone-binding sites in histone replacement-completed spermatozoa.

ABSTRACT: The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Here, we find that the swim-up sperm used in many studies contain about 10% population of sperm which have not yet completed the histone-to-protamine replacement. We develop a method to purify histone replacement-completed sperm (HRCS) and to completely solubilize histones from cross-linked HRCS without MNase digestion. Our results indicate that histones are retained at specific promoter regions in HRCS. This method allows the study of epigenetic status in mature sperm.

SUBMITTER: Yoshida K

PROVIDER: S-EPMC6155156 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Mapping of histone-binding sites in histone replacement-completed spermatozoa.

Yoshida Keisuke K Muratani Masafumi M Araki Hiromitsu H Miura Fumihito F Suzuki Takehiro T Dohmae Naoshi N Katou Yuki Y Shirahige Katsuhiko K Ito Takashi T Ishii Shunsuke S

Nature communications 20180924 1

The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Her ...[more]

PMID: 30250204

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Project description:Background Analysis of the 3D structures of protein–ligand binding sites can provide valuable insights for drug discovery. Binding site comparison (BSC) studies can be employed to elucidate the function of orphan proteins or to predict the potential for polypharmacology. Many previous binding site analyses only consider binding sites surrounding an experimentally observed bound ligand. Results To encompass potential protein–ligand binding sites that do not have ligands known to bind, we have incorporated fpocket cavity detection software and assessed the impact of this inclusion on BSC performance. Using fpocket, we generated a database of ligand-independent potential binding sites and applied the BSC tool, SiteHopper, to analyze similarity relationships between protein binding sites. We developed a method for clustering potential binding sites using a curated dataset of structures for six therapeutically relevant proteins from diverse protein classes in the protein data bank. Two clustering methods were explored; hierarchical clustering and a density-based method adept at excluding noise and outliers from a dataset. We introduce circular plots to visualize binding site structure space. From the datasets analyzed in this study, we highlight a structural relationship between binding sites of cationic trypsin and prothrombin, protein targets known to bind structurally similar small molecules, exemplifying the potential utility of objectively and holistically mapping binding site space from the structural proteome. Conclusions We present a workflow for the objective mapping of potential protein–ligand binding sites derived from the currently available structural proteome. We show that ligand-independent binding site detection tools can be introduced without excessive penalty on BSC performance. Clustering combined with intuitive visualization tools can be applied to map relationships between the 3D structures of protein binding sites.Graphical abstract Mapping binding site space. Electronic supplementary material The online version of this article (doi:10.1186/s13321-016-0180-0) contains supplementary material, which is available to authorized users.

Project description:Hypoxia Inducible Factor (HIF) regulates a cascade of transcriptional events in response to decreased oxygenation, acting from the cellular to the physiological level. This response is evolutionarily conserved, allowing the use of zebrafish (Danio rerio) as a model for studying the hypoxic response. Activation of the hypoxic response can be achieved in zebrafish by homozygous null mutation of the von Hippel-Lindau (vhl) tumour suppressor gene. Previous work from our lab has focused on the phenotypic characterisation of this mutant, establishing the links between vhl mutation, the hypoxic response and cancer. To further develop fish as a model for studying hypoxic signalling, we examine the transcriptional profile of the vhl mutant with respect to Hif-1?. As our approach uses embryos consisting of many cell types, it has the potential to uncover additional HIF regulated genes that have escaped detection in analogous mammalian cell culture studies.We performed high-density oligonucleotide microarray analysis of the gene expression changes in von Hippel-Lindau mutant zebrafish, which identified up-regulation of well-known hypoxia response genes and down-regulation of genes primarily involved in lipid processing. To identify the dependency of these transcriptional changes on HIF, we undertook Chromatin Immunoprecipitation linked next generation sequencing (ChIP-seq) for the transcription factor Hypoxia Inducible Factor 1? (HIF-1?). We identified HIF-1? binding sites across the genome, with binding sites showing enrichment for an RCGTG motif, showing conservation with the mammalian hypoxia response element.Transcriptome analysis of vhl mutant embryos detected activation of key hypoxia response genes seen in human cell models of hypoxia, but also suppression of many genes primarily involved in lipid processing. ChIP-seq analysis of Hif-1? binding sites unveiled an unprecedented number of loci, with a high proportion containing a canonical hypoxia response element. Whether these sites are functional remains unknown, nevertheless their frequent location near transcriptional start sites suggests functionality, and will allow for investigation into the potential hypoxic regulation of genes in their vicinity. We expect that our data will be an excellent starting point for analysis of both fish and mammalian gene regulation by HIF.

Dataset Information

Mapping of histone-binding sites in histone replacement-completed spermatozoa.

Publications

Mapping of histone-binding sites in histone replacement-completed spermatozoa.

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