ChIP-chip with antibodies for histone 3 lysine 4 trimethylation, histone 3, and PolII in Mll1+/+ and Mll1-/- MEFs. Mus musculus

Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II A common landmark of activated genes is the presence of trimethylation on lysine 4 of histone H3 (H3K4) at promoter regions. The Set1/COMPASS was the founding member and the only H3K4 methylases in S. cerevisiae, however, in mammals at least six H3K4 methylases Set1A/B and MLL1-4 are found in COMPASS-like complexes capable of methylating H3K4. To gain further insight into the different roles and functional targets for the H3K4 methylases, we have undertaken a genome-wide analysis of H3K4 methylation pattern in wild-type Mll1+/+ and Mll1-/- mouse fibroblasts (MEFs). We found that Mll1 is required for the H3K4 trimethylation of less than 5% of promoters carrying this modification. Many of these genes, which include developmental regulators such as Hox genes show decreased levels of RNA polymerase II recruitment and expression concomitant with the loss of H3K4 methylation. Although Mll1 is only required for the methylation of a subset of Hox genes, Menin, a component of the Mll1 and Mll2 complexes, is required for the overwhelming majority of H3K4 methylation at Hox loci. However, the loss of MLL3/4 and/or the Set1 complexes have little to no effect on the Hox loci H3K4 methylation or expression levels in these MEFs. Together these data provide insight into redundancy and specialization of COMPASS-like complexes in mammals and provide evidence on a possible role for Mll1-mediated H3K4 methylation in the regulation of transcriptional initiation. Overall design: Chromatin Immunoprecipitation was performed with antibodies for histone 3 lysine 4 trimethylation, histone 3, and PolII in Mll1+/+ and Mll1-/- mouse embryonic fibroblasts. DNA was hybridized to a custom Agilent tiling array (4x44k format) that covers three of the hox regions (A,B,D) and a collection of other genes.

组蛋白H3赖氨酸4甲基化的全景分析：明确MLL家族成员靶标并揭示MLL1介导的H3K4甲基化在RNA聚合酶II调控转录起始中的作用
激活基因的共同标志性特征之一，是启动子区域存在组蛋白H3赖氨酸4三甲基化（H3K4）修饰。Set1/COMPASS复合物是首个被发现的H3K4甲基转移酶，也是酿酒酵母（S. cerevisiae）中唯一的H3K4甲基化酶；而在哺乳动物中，至少存在6种H3K4甲基化酶，即Set1A、Set1B以及MLL1至MLL4，它们均组装于可介导H3K4甲基化的类COMPASS复合物中。为深入解析不同H3K4甲基化酶的功能角色与靶标基因，我们对野生型Mll1+/+与Mll1敲除（Mll1-/-）小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中的H3K4甲基化图谱开展了全基因组分析。
研究发现，仅不足5%携带该修饰的启动子的H3K4三甲基化依赖于Mll1。这类基因包含Hox家族等发育调控因子，其RNA聚合酶II招募水平与基因表达量均随H3K4甲基化的丢失而降低。尽管Mll1仅参与部分Hox基因的甲基化修饰，但作为Mll1与Mll2复合物的组分之一，Menin蛋白对Hox基因座上绝大多数的H3K4甲基化是必需的。然而，敲除MLL3/4或Set1复合物对这些MEFs中Hox基因座的H3K4甲基化及基因表达水平几乎无影响。
综上，本研究揭示了哺乳动物类COMPASS复合物的功能冗余性与特异性，并为Mll1介导的H3K4甲基化调控转录起始提供了实验证据。
总体实验设计：针对Mll1+/+与Mll1-/-小鼠胚胎成纤维细胞，分别使用针对组蛋白H3赖氨酸4三甲基化、组蛋白H3以及RNA聚合酶II的抗体进行染色质免疫沉淀（Chromatin Immunoprecipitation），将所得DNA与定制化安捷伦平铺芯片（Agilent tiling array，4×44k规格）进行杂交，该芯片覆盖3个Hox基因座（A、B、D）以及一系列其他基因。