Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, 815 Mercer St,, Seattle, WA 98109, USA.
Epigenetics Chromatin. 2011 Aug 3;4:14. doi: 10.1186/1756-8935-4-14.
Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (IGF2), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the IGF2 gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.
Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed IGF2BP1 gene. In vitro binding assays using the CTCF target motif at the IGF2BP1 gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic IGF2BP1 expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express IGF2BP1 primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of IGF2BP1 gene expression.
Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.
随机单等位基因表达导致细胞和生物体的表型变异。然而，对于个体等位基因被随机选择表达的表观遗传机制尚不清楚。受胰岛素样生长因子 2 基因 2（IGF2）等印迹基因座染色质特征的启发，我们评估了锌指蛋白 CTCF 的作用，该蛋白对于 IGF2 基因的亲本来源特异性表达是必需的，以及其与 DNA 甲基化、组蛋白修饰和 RNA 聚合酶 II 的等位基因特异性关联的作用。
使用基于阵列的染色质免疫沉淀，我们鉴定了 293 个与 CTCF 和组蛋白 H3 赖氨酸 9 三甲基化（H3K9me3）都相关的基因组位点。将它们的基因组位置与先前发表的单等位基因表达基因进行比较，发现单等位基因表达基因与共定位的 CTCF/H3K9me3 之间没有显著重叠。为了更详细地分析 CTCF 和 H3K9me3 对基因调控的贡献，我们专注于单等位基因表达的 IGF2BP1 基因。使用 IGF2BP1 基因上的 CTCF 靶基序的体外结合测定以及对胞嘧啶甲基化和 CTCF 结合的等位基因特异性分析表明，CTCF 不调节单或双等位基因 IGF2BP1 的表达。令人惊讶的是，我们发现在表达 IGF2BP1 的 B 淋巴母细胞中，RNA 聚合酶 II 检测到母本和父本等位基因上，而 IGF2BP1 主要从一个等位基因表达。因此，RNA 聚合酶 II 延伸的等位基因特异性控制调节 IGF2BP1 基因表达的等位基因偏向性。
CTCF 和 H3K9me3 的共定位并不代表单等位基因表达的可靠染色质特征。此外，个体等位基因与活性（H3K4me3）和沉默（H3K27me3）染色质修饰（等位基因双价染色质）或与 RNA 聚合酶 II 的关联也不能识别单等位基因表达的基因座。个体等位基因的表达选择部分发生在转录延伸过程中。
