Dynamic reorganization of extremely long-range promoter-promoter interactions between two states of pluripotency. Mus musculus

Serum-to-2i interconversion of mouse Embryonic Stem Cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary but not sufficient to establish these interactions, as confirmed by Capture Hi-C on Eed-/- serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation. Overall design: To study dynamics in chromatin architecture and to characterize long-range interaction, we performed Hi-C using DpnII as the restriction enzyme, potentially reaching a genome-wide coverage at a less than 1Kb resolution. We subsequently performed enrichment of interaction by a target capture similar to the exome sequencing approach. We enriched for DNaseI hyper-sensitive sites (DHS’s) in chromatin from mESCs. Probes were designed against the union of all DHS’s of Serum and 2i mESCs. Capture Hi-C reveals Extremely Long-Range Interactions (ELRI) in Serum but not in 2i ESCs. We observed H3K27me3 as a prominent characteristic, but not exclusive feature of ELRI loci in Serum mESCs. To further elucidate the involvement of constituents of PRC1 and PRC2 in ELRI, we performed ChIP-seq experiment on Suz12 and Ring1B during serum-to-2i transition. In addition, RNA-seq was performed to compare the expression levels of genes.

小鼠胚胎干细胞（mouse Embryonic Stem Cells，mESCs）的血清态向2i态的互变，是用于早期胚胎发育研究的极具价值的体外模型。为探究该转化过程中三维染色质组织是否发生改变，我们建立了针对DNase I超敏感位点（DNase I hypersensitive sites）靶序列富集的捕获Hi-C（Capture Hi-C）技术。我们在血清培养的细胞中，检测到少量由H3K27me3修饰的二价启动子之间的超长程染色体内及染色体间相互作用，其中涉及Hox基因簇。值得注意的是，这类启动子介导的相互作用在2i态全能性小鼠胚胎干细胞中并未出现，但在进一步发育为类似始发态的血清态小鼠胚胎干细胞时会重新显现。将血清态小鼠胚胎干细胞逆转回2i态全能干细胞时，这类启动子-启动子相互作用会以时空依赖的方式消失。在2i态全能干细胞的二价启动子上基本不存在的H3K27me3，是建立这类相互作用的必要而非充分条件，这一点通过对Eed敲除（Eed-/-）的血清态小鼠胚胎干细胞进行捕获Hi-C实验得到了验证。我们的研究结果表明，H3K27me3与多梳抑制复合体2（Polycomb Repressive Complex 2，PRC2）在小鼠胚胎干细胞向分化始发态转变过程中的染色质重塑中发挥关键作用。

实验整体设计：为研究染色质构象的动态变化并解析长程相互作用特征，我们使用限制性内切酶DpnII进行Hi-C实验，理论上可实现分辨率低于1kb的全基因组覆盖。随后，我们采用类似外显子组测序的靶标捕获策略，对染色质相互作用进行富集。我们针对小鼠胚胎干细胞染色质中的DNase I超敏感位点（DNase I hypersensitive sites，DHSs）进行富集。探针的设计靶点覆盖血清态与2i态小鼠胚胎干细胞的全部超敏感位点的并集。捕获Hi-C实验结果显示，血清态小鼠胚胎干细胞中存在超长程相互作用（Extremely Long-Range Interactions，ELRI），而2i态小鼠胚胎干细胞中则无此类相互作用。我们发现，H3K27me3是血清态小鼠胚胎干细胞中ELRI位点的显著特征，但并非唯一特征。为进一步阐明多梳抑制复合体1（Polycomb Repressive Complex 1，PRC1）与多梳抑制复合体2（PRC2）的组分在超长程相互作用中的作用，我们在血清态向2i态转化的过程中，针对Suz12与Ring1B进行了染色质免疫共沉淀测序（ChIP-seq）实验。此外，我们还通过RNA测序（RNA-seq）对基因的表达水平进行了比较分析。