Phf21b is a novel histone reader essential for epigenetic silencing of cell cycle genes during neurogenesis. Phf21b is a novel histone reader essential for epigenetic silencing of cell cycle genes during neurogenesis

The role of chromatin-mediated regulation in cell-fate specification during mammalian development is been increasingly appreciated. However, very little is known about epigenetic regulatory networks during embryonic neurogenesis. In search for novel epigenetic regulators of neuronal development, we find that Phf21b is specifically expressed in the cortex during embryonic neurogenesis. Interestingly, knockdown of Phf21b during cortical development leads to a defective neurogenesis. Immunoprecipitation assay combined with mass-spectrometry revealed that Phf21b associates with the repressive chromatin machinery including histone deacetylases as well as Lsd1, suggesting that it likely functions in gene repression. Biochemical analysis revealed that Phf21b has higher affinity for H3K4me1 compared to other modifications states of histone H3 at Lysine 4 (K4). Our data suggests that Phf21b targets the regulatory regions of the cell cycle genes where they recruit Lsd1 and Hdac2 to repress them via loss of H3K4me1 and H3K27ac. Overall, we identify Phf21b as a novel essential regulator of neurogenesis, which mediates epigenetic silencing of cell cycle genes during cortical development. Overall design: (1) mRNA expression profiles of mESC, N2a, and IUE mouse embryonic cortex upon depletion of Phf21b were examined by Illumina Hi-seq 2500 and Next seq 500. (2) ChIP-seq of Phf21b was examined using mESC and N2a day2 differentiated system (3) ATAC-seq of N2a Undifferentiated and Day2 differentiated system upon depletion of Phf21b was profiled

染色质介导的调控在哺乳动物发育过程中细胞命运特化环节的作用正日益受到学界重视。然而，目前对于胚胎神经发生过程中的表观遗传调控网络，我们所知甚少。为探寻神经元发育的新型表观遗传调控因子，本研究发现Phf21b在胚胎神经发生阶段的大脑皮层中特异性表达。有趣的是，在皮层发育过程中敲低Phf21b会导致神经发生缺陷。免疫共沉淀联合质谱检测实验显示，Phf21b可与组蛋白去乙酰化酶及Lsd1等抑制性染色质调控复合物结合，提示其可能参与基因转录抑制。生化分析结果表明，相较于组蛋白H3第4位赖氨酸（K4）的其他修饰状态，Phf21b对H3K4me1具有更高的结合亲和力。本研究数据显示，Phf21b可靶向结合细胞周期基因的调控区域，并通过招募Lsd1与Hdac2，经由去除H3K4me1与H3K27ac修饰实现基因沉默。综上，本研究鉴定Phf21b为神经发生过程中一种全新的必需调控因子，其在皮层发育阶段介导细胞周期基因的表观遗传沉默。实验整体设计如下：1. 采用Illumina HiSeq 2500与NextSeq 500测序平台，检测Phf21b敲低后的小鼠胚胎干细胞（mouse embryonic stem cell, mESC）、N2a细胞以及经宫内电转染（intrauterine electroporation, IUE）处理的小鼠胚胎皮层的mRNA表达谱；2. 利用小鼠胚胎干细胞及分化第2天的N2a细胞系统，开展Phf21b的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）；3. 对未分化及分化第2天的N2a细胞在Phf21b敲低后的样本进行转座酶可及性测序（assay for transposase-accessible chromatin using sequencing, ATAC-seq）分析。