Tiwari Vijay K, McGarvey Kelly M, Licchesi Julien D F, Ohm Joyce E, Herman James G, Schübeler Dirk, Baylin Stephen B
Cancer Biology Division, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University Medical Institutions, Baltimore, Maryland, USA.
PLoS Biol. 2008 Dec 2;6(12):2911-27. doi: 10.1371/journal.pbio.0060306.
Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream ( approximately 30 kb) of and extending approximately 60 kb around one such gene, GATA-4, is organized-in Tera-2 undifferentiated embryonic carcinoma (EC) cells-in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)-mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a approximately 60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells.
在成体癌症中，许多发生DNA高甲基化并在表观遗传上沉默的基因在胚胎干细胞（ES细胞）中被多梳蛋白家族（PcG）标记。我们发现，在一个这样的基因GATA-4周围，其上游大约30 kb且延伸约60 kb的大片区域，在Tera-2未分化胚胎癌细胞中呈现出一种拓扑结构复杂的多环构象，该构象由多个内部长程接触区域形成，这些区域靠近富含EZH2、其他PcG蛋白以及标志性PcG组蛋白标记H3K27me3的区域。在未分化的Tera-2细胞中，通过小干扰RNA（siRNA）介导的EZH2缺失导致GATA-4基因座处长程关联的频率显著降低，这似乎依赖于影响这些染色质区域周围的H3K27me3富集，同时GATA-4转录适度增加。当Tera-2细胞接收到诱导GATA-4表达增加约60倍的分化信号时，染色质环完全溶解，同时PcG蛋白和H3K27me3标记消失。然而，在结肠癌细胞中，当GATA-4没有基础转录且环包含多个异常DNA高甲基化的CpG岛，并且甲基胞嘧啶结合蛋白MBD2定位于这些CpG岛，包括基因启动子附近的CpG岛时，长程相互作用的频率增加。通过对DNA甲基转移酶进行基因敲除（DKO细胞）来去除DNA甲基化，会导致MBD2占据减少以及长程接触频率降低，使得这些细胞现在更类似于未分化的Tera-2细胞。我们的研究结果揭示了干细胞/前体细胞和成体癌症在高阶染色质构象上意想不到的相似性。我们还提供了新的见解，即PcG占据且富含H3K27me3的区域可以形成染色质环，并在哺乳动物细胞中围绕单个基因在顺式方向上发生物理相互作用。这些环在胚胎癌细胞中与一种准备就绪的低转录状态相关联，并且随着DNA甲基化的增加，在成体癌细胞中导致转录完全被抑制。
