Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis. Mus musculus

The histone variant H2A.Z has been implicated in the regulation of gene expression, and in plants antagonizes DNA methylation. Here we ask whether a similar relationship exists in mammals, using a mouse lymphoma model, where chromatin states can be monitored during tumorigenesis. Using native ChIP-on-chip, we found a progressive depletion of H2A.Z around transcriptional start sites (TSSs) during cell state transformations. In addition, we found that H2A.Z and DNA methylation are anti-correlated around TSSs in wild-type and Myc-transformed cells. Surprisingly, approximately 30% of genes show a redistribution of H2A.Z from around TSSs to bodies of active genes during oncogenesis but not before, and these changes are accompanied by DNA methylation changes in the opposite direction. Our results suggest that antagonism between H2A.Z deposition and DNA methylation is a conserved feature of eukaryotic genes, and that transcription-coupled H2A.Z changes may play a role in cancer initiation and progression. Keywords: Chromatin affinity-purification on microarray Overall design: Two types of experiments were performed on wild type pre-B cells, Myc induced pre-B cells, and B cell tumors: H2AZ profiling was performed by comparing DNA isolated by Chromatin immunoprecipitation (ChIP) using antibody against H2AZ, to total MNase fragmented genomic DNA isolated from each of the three cell types. Methylation profiling was performed by comparing DNA isolated using immunoprecipitation with an antibody against single stranded DNA (MeDIP) to total sonicated genomic DNA isolated from each of the three cell types. For each sample, the input and IP material was differentially labeled and cohybridized to a promoter microarray chip. Expression analysis was done using single channel microarrays. In brief, total RNA was isolated from each of the three cell types, followed by poly-AAA CDNA synthesis and labeling.

组蛋白变体H2A.Z已被证实参与基因表达调控，且在植物中可拮抗DNA甲基化。本研究旨在探讨哺乳动物中是否存在类似的关联，采用小鼠淋巴瘤模型开展实验，该模型可在肿瘤发生过程中实时监测染色质状态。通过原生染色质免疫沉淀-芯片（native ChIP-on-chip）技术，我们发现细胞状态转化过程中，转录起始位点（Transcription Start Sites, TSSs）周围的H2A.Z呈现渐进性耗竭。此外，我们发现在野生型及Myc转化细胞中，转录起始位点周围的H2A.Z与DNA甲基化呈负相关。令人意外的是，约30%的基因在肿瘤发生过程中（而非发生前）出现H2A.Z从转录起始位点周围向活性基因编码区重新分布的现象，且此类变化伴随方向相反的DNA甲基化改变。本研究结果表明，H2A.Z沉积与DNA甲基化之间的拮抗作用是真核生物基因的保守特征，而转录偶联的H2A.Z变化可能在癌症发生与进展中发挥重要作用。

关键词：微阵列染色质亲和纯化（Chromatin affinity-purification on microarray）

整体实验设计：我们对野生型前B细胞、Myc诱导的前B细胞以及B细胞肿瘤开展两类实验：H2A.Z分布谱分析通过将使用抗H2A.Z抗体通过染色质免疫沉淀（ChIP）获取的DNA，与从三种细胞类型分别分离的微球菌核酸酶（MNase）酶切全基因组DNA进行比对分析。甲基化谱分析则通过将使用抗单链DNA抗体进行免疫沉淀（MeDIP）获取的DNA，与从三种细胞类型分别分离的超声破碎全基因组DNA进行比对分析。对于每个样本，输入组与免疫沉淀组分采用不同标记方式，并共同杂交至启动子微阵列芯片。表达分析采用单通道微阵列技术：简要而言，我们从三种细胞类型中分别分离总RNA，随后进行poly(A)尾cDNA合成与标记。