BioProject

Chromatin performs numerous functions during cellular differentiation, and therefore it must be capable of adopting a multitude of different structures. More...

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 ‘H2A.Bbd-like’ 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 Overall design: 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 manufacturer’s recommendations (Invitrogen). DNA was either analysed by semi–quantitative 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). Less...