Genome-wide analysis using ChIP to identify isoform-specific gene targets.
Genome-wide analysis using ChIP to identify isoform-specific gene targets.
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DOI：
10.3791/2101
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发表时间：
2010-07-07
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通讯作者：
Benevolenskaya, Elizaveta V
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作者：
Beshiri, Michael L;Islam, Abul;Benevolenskaya, Elizaveta V
将转录和表观遗传因子招募到它们的靶标是它们调控的关键一步。在招募中突出的特征是与特定的组蛋白修饰结合的蛋白质结构域。其中一个结构域是植物同源域(PHD)，它存在于几种染色质结合蛋白中。表观遗传因子RBP2具有多个PHD结构域，但它们具有不同的功能(图4)。特别是，在人类白血病的RBP2致癌融合中发现的C-末端PHD结构域，与组蛋白H3(H3K4me3)中的三甲基化赖氨酸4结合。在原单核细胞、淋巴瘤来源的U937细胞分化为单核细胞的过程中，与含有C-末端PHD的RBP2亚型相对应的转录本积累。与这两组数据一致的是，全基因组分析表明，在分化的U937细胞中，RBP2蛋白定位于H3K4me3高度浓缩的基因组区域。RBP2的靶标定位与RBP2组蛋白去甲基酶活性和转录活性下降导致H3K4me3的下降有关。相比之下，RBP2的另外两个博士无法结合H3K4me3。值得注意的是，在较小的RBP2亚型中，RBP2的C-末端结构域PHD缺失。可以想象，与H3K4me3缺乏相互作用的RBP2的小亚型在基因组位置上不同于大亚型。RBP2亚型基因组位置的差异可能解释了观察到的RBP2功能的多样性。具体地说，RBP2在视网膜母细胞瘤蛋白(PRB)介导的细胞分化中起关键作用。与这些数据一致的是，以前的全基因组分析没有区分异构体，识别出两组不同的RBP2靶基因：1)与RBP2结合的基因以一种独立于分化的方式结合；2)与RBP2结合的基因以依赖分化的方式结合。为了确定异构体之间的定位差异，我们用CHIP-SEQ进行了全基因组定位分析。使用检测两种RBP2亚型的抗体，我们已经定位了所有的RBP2靶点。此外，我们的抗体只结合大的，而不是小的RBP2亚型(图4)。在确定大的异构体靶之后，可以从所有的RBP2靶中减去它们，以揭示小的异构体的靶。这些数据表明染色质相互作用结构域在蛋白质招募中对其在基因组中的结合位置的贡献。
Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3). The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me3. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.