PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations [RNA-seq]. PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations [RNA-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甲基化的精细调控机制——即如何保障精准的基因表达与发育保真性——仍不甚明晰。PROSER1作为一种已知的TET2（Ten-eleven translocation 2）互作蛋白，近期被发现与重度神经发育障碍存在关联。本研究证实，PROSER1可与所有TET酶（Ten-eleven translocation enzymes）相互作用，并稳定染色质结合型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，我们还发现PROSER1能够隔离TET酶，阻止广泛的DNA去甲基化与转座元件的去抑制。本研究结果确定PROSER1是同时正向和负向调控哺乳动物神经发育所必需的DNA去甲基化的关键因子。整体实验设计：在小鼠胚胎干细胞（mESCs）中，以PROSER1基因敲除（KO）细胞作为背景对照，对内源PROSER1进行染色质免疫沉淀测序（ChIP-seq），并在野生型和PROSER1 KO mESCs中对内源TET2开展ChIP-seq实验。针对组蛋白修饰H3K27ac、H3K4me1，分别在野生型和PROSER1 KO mESCs中进行ChIP-seq检测。在野生型、PROSER1 KO以及PROSER1 KO+Rescue mESCs中开展酶促甲基化测序（EM-seq）。在野生型、PROSER1 KO、PROSER1 KO+Rescue mESCs，以及上述细胞系在N2B27培养基中分化2天后的样本（即"day2"样本）中进行RNA测序（RNA-seq）。