Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Functional studies of a H2A.Bbd-like histone variant in mouse spermatogenesis

ABSTRACT: This SuperSeries is composed of the following subset Series: GSE29781: Expression data from 30do mouse spermatids [Affymetrix] GSE29913: Genome-wide distribution maps of histone variants H2A.Lap1 and H2A.Z in 30do mouse spermatids[ChIP_seq] Refer to individual Series

ORGANISM(S): Mus musculus

SUBMITTER: Rohan Williams

PROVIDER: E-GEOD-29916 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A unique H2A histone variant occupies the transcriptional start site of active genes.

Soboleva Tatiana A TA Nekrasov Maxim M Pahwa Anuj A Williams Rohan R Huttley Gavin A GA Tremethick David J DJ

Nature structural & molecular biology 20111204 1

Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogene ...[more]

PMID: 22139013

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Project description:Chromatin performs numerous functions during cellular differentiation, and therefore it must be capable of adopting a multitude of different structures. How these various structures are established is poorly understood, but we propose that specific histone H2A variants will have a key role in remodelling chromatin during differentiation. Structurally, we show here that the gain of just a single acidic amino acid residue has generated a new mouse M-bM-^@M-^XH2A.Bbd-likeM-bM-^@M-^Y histone variant, H2A.Lap1, and that when incorporated into nucleosomal arrays imparts on them unique biophysical properties that are distinct from arrays containing either H2A or human H2A.Bbd. Functionally, we identify H2A.Lap1 as a novel chromatin component of active genes that are expressed during spermatogenesis, and in combination with H2A.Z create a unique chromatin landscape at the start site of transcription. During round spermatid differentiation, H2A.Lap1 dramatically loads onto the inactive X chromosome enabling a subset of its genes to be transcriptionally activated. We used microarrays together with ChIP-seq data in order to see distribution of Lap1 and expression level of the Lap1 associated genes Total RNA was isolated using TRIzol (Invitrogen) and the RNeasy Mini Kit (Qiagen). Samples were DNase I treated (Roche) and reverse transcribed using Superscript II reverse transcriptase following the manufacturerM-bM-^@M-^Ys recommendations (Invitrogen). DNA was either analysed by semiM-bM-^@M-^Squantitative PCR, real-time qPCR using SYBR Green PCR master mix (Applied Biosystems), or subjected to DNA microarray analysis (in triplicate) using the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Robust Multichip Average (RMA) correction and probe set summaries were obtained using Affymetrix Power Tools software annotation (Affymetrix) based on the mm9 mouse genome. Unless otherwise stated, all subsequent expression analyses (see Supplementary Methods) were performed in the R statistical computing environment (version 2.11.1).

Project description:Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes and spermatids suggests the existence of precise programs of gene expression in these cells as well as in their neighboring somatic Sertoli cells. The objective of this study was to elucidate genes encoding the proteins that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV of the cycle were isolated by microdissection and Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes and spermatids were purified from enzymatically-dispersed testes. Microarray analysis using Rat Genome 230 2.0 arrays identified a total of 16,971 probe sets that recognized transcripts. A comparison with the transcriptome of other tissues identified 398 testis-specific probe sets, which therefore are potential targets for the development of new contraceptives. Sequential waves of cell and stage-specific gene expression are associated with progression of germ cells through the stages of the cycle of the seminiferous epithelium and 1612 probe sets recognized transcripts whose expressions varied at least 4-fold across the stages of the cycle. Pathway analyses reveal that entire biological processes are regulated cyclically in testicular cells. Important among these are cell cycle and DNA repair. Thus, stage-specific gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells. Experiment Overall Design: Seminiferous tubules at the following stages or groups of stages were isolated by transillumination-assisted microdissection: I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV. Twenty-five segments of tubules, 2 m in length, were collected at each stage or group of stages from each rat. Tubules from two separate animals were pooled to form one sample from each stage or groups of stages. A total of 5 independent samples per stage or groups of stages were analyzed. Mature Sertoli cells were isolated to about 85% purity (n=4). Three groups of germ cells were isolated by centrifugal elutriation from enzyme-dispersed testes: round spermatids (n=4), pachytene spermatocytes (n=4) and spermatogonia plus early spermatocytes (n=3) . Purity of these cells has been shown to vary between 85% to 95%

Dataset Information

Functional studies of a H2A.Bbd-like histone variant in mouse spermatogenesis

Publications

A unique H2A histone variant occupies the transcriptional start site of active genes.

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