PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations [EM-seq]

The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned – ensuring precise gene expression and developmental fidelity – remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA demethylation and developmental gene expression. Surprisingly, we find that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element de-repression. Our findings identify PROSER1 as a key factor which both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment. Overall design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for endogenous PROSER1 in mESCs using PROSER1 KO as background control, and endogenous TET2 in wildtype and PROSER1 KO mESCs. ChIP-seq in wildtype and PROSER1 KO mESCs for the following histone modifications: H3K27ac, H3K4me1. Enzymatic methyl-seq (EM-seq) in wildtype, PROSER1 KO and PROSER1 KO+Rescue mESCs. RNA sequencing (RNA-seq) in wildtype, PROSER1 KO and PROSER1 KO+Rescue mESCs and wildtype, PROSER1 KO and PROSER1 KO+Rescue mESCs after 2 days differentiation in N2B27 medium ("day2" samples).

DNA甲基化与神经发育障碍之间的关联已得到充分证实。然而，DNA甲基化如何被精准调控——以确保基因表达的精确性与发育保真性——目前仍知之甚少。PROSER1作为一种已知的TET2互作蛋白，近期被发现与一种重型神经发育障碍相关。本研究证实，PROSER1可与所有TET家族酶相互作用，并稳定染色质结合的TET-OGT-PROSER1-DBHS（TOPD）复合物，该复合物可调控DNA去甲基化与发育相关基因的表达。令人意外的是，本研究还发现PROSER1能够螯合TET酶，从而抑制全基因组范围的DNA去甲基化与转座元件的去抑制。本研究结果表明，PROSER1是同时正向与负向调控DNA去甲基化的关键因子，而这一调控过程对于哺乳动物神经发育至关重要。

实验整体设计如下：
1. 以PROSER1敲除（PROSER1 KO）小鼠胚胎干细胞（mESCs）作为背景对照，对内源PROSER1进行染色质免疫沉淀测序（ChIP-seq）；同时在野生型与PROSER1敲除小鼠胚胎干细胞中对内源TET2进行染色质免疫沉淀测序。
2. 针对组蛋白修饰H3K27ac、H3K4me1，在野生型与PROSER1敲除小鼠胚胎干细胞中开展染色质免疫沉淀测序。
3. 在野生型、PROSER1敲除以及PROSER1敲除+挽救（PROSER1 KO+Rescue）小鼠胚胎干细胞中进行酶促甲基化测序（EM-seq）。
4. 分别在常规培养的野生型、PROSER1敲除、PROSER1敲除+挽救小鼠胚胎干细胞，以及在N2B27培养基中分化2天的上述三类细胞（即"day2"样本）中开展RNA测序（RNA-seq）。