Dynamic enhancer partitioning instructs Zdbf2 activation during epigenetic reprogramming [bisulfite-seq]

Early mammalian development is accompanied by a profound global remodeling of chromatin-based regulation. The Zdbf2 locus provides a valuable model to uncover the effect of dynamic chromatin transition, as it is polycomb silenced in embryonic stem cells (ESCs) and only becomes activated after Long isoform of Zdbf2 (Liz) transcription-dependent de novo DNA methylation during differentiation. Here we show that four enhancers contribute to the Liz-to-Zdbf2 promoter switch, concomitantly with dynamic changes in chromatin architecture. CTCF plays a key role in partitioning the locus in ESCs, when Liz is active and Zdbf2 is silenced. The partition is relieved when Zdbf2 becomes DNA methylated and active. Mutant ESCs that lack the partition fail to properly activate Zdbf2. Notably, the CTCF-based regulation occurs independently of the polycomb and DNA methylation pathways, suggesting a multi-layered regulatory framework that ensures proper epigenetic programming of a developmentally important gene. Overall design: Circular  chromosome conformation capture followed by sequencing (4C-seq) in mouse ESCs and EpiLCs in WT and mutant backgrounds

早期哺乳动物发育过程中，伴随染色质调控系统的全局性深度重编程。Zdbf2基因座是探究动态染色质转变调控效应的理想模型：该基因座在胚胎干细胞（ESCs）中受多梳蛋白复合体介导沉默，仅在分化进程中，通过依赖于Zdbf2长转录本（Liz）转录的从头DNA甲基化过程后才被激活。
本研究证实，4个增强子共同参与Liz至Zdbf2启动子的开关调控，并伴随染色质三维架构的动态变化。在Liz表达活跃而Zdbf2处于沉默状态的胚胎干细胞中，CTCF在该基因座的区域化分隔中发挥关键作用；当Zdbf2发生DNA甲基化并被激活后，这一区域化分隔被解除。缺失该分隔元件的突变胚胎干细胞无法正常激活Zdbf2。值得注意的是，基于CTCF的调控机制独立于多梳蛋白与DNA甲基化通路之外，提示存在多层调控框架以确保发育相关重要基因的表观遗传编程正常进行。
整体实验设计：在野生型（WT）与突变背景的小鼠胚胎干细胞及外胚层样细胞（EpiLCs）中开展环形染色质构象捕获测序（4C-seq）实验。