Bidirectional changes in postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics

The functional and transcriptional maturation of neurons is a prolonged process that extends well beyond mitotic exit and terminal fate commitment of progenitors. The differentiation of cerebellar granule neurons (CGNs) in the postnatal mouse cerebellum provides a useful model to identify chromatin mechanisms that orchestrate temporal changes in transcription as postmitotic CGNs mature. Here we report that CGN maturation is associated with dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), a modification best known for its role in repressing alternative fates during cell specification. H3K27me3 is gained rapidly in newly postmitotic CGNs at progenitor-expressed genes that are repressed in neurons. H3K27me3 is lost more gradually at the promoters of a subset of neuronal genes that are transcriptionally induced upon CGN maturation. The loss of H3K27me3 is facilitated by the lysine demethylase KDM6B, and genes that are induced in maturing CGNs show impaired expression in the cerebellum of conditional KDM6B knockout mice. Genes that lose H3K27me3 gain H3K27 acetylation and binding of the pro-maturation ZIC1/2 transcription factors, suggesting that developmental loss of H3K27me3 may be required to permit the onset of transcriptional maturation. Interestingly, pharmacological inhibition of the H3K27 methyltransferase EZH2 in early postmitotic CGNs not only blocked the repression of progenitor genes but also impaired the induction of mature CGN genes. These data show that regulation of H3K27me3 functions in developing postmitotic neurons beyond the period of cell fate commitment to regulate the dynamics of gene expression programs that underlie functional neuronal maturation. Overall design: H3K27me3 CUT&RUN-seq for 1) cultured cerebellar granule neurons (CGN) at 1, 3, 5 and 7 days in vitro, 2) CGNs treated with DMSO or 5 uM Ezh2 inhibitor GSK-126 at DIV1 and harvested at DIV5, 3) CGNs treated with DMSO or 1 uM Kdm6b inhibitor GSK-J4 at DIV1 and harvested at DIV5. (n=3 biological replicates each)

神经元的功能与转录成熟是一个持续漫长的过程，其进程远早于祖细胞的有丝分裂退出与终末命运决定。出生后小鼠小脑中的小脑颗粒神经元（cerebellar granule neurons, CGNs）分化，可为探究调控有丝分裂后CGN成熟过程中转录组时序变化的染色质机制，提供优质的研究模型。
本研究发现，CGN成熟过程与组蛋白H3赖氨酸27三甲基化（histone H3 lysine 27 trimethylation, H3K27me3）的基因组分布动态变化密切相关；该修饰最广为人知的功能是在细胞命运特化阶段抑制替代性细胞命运。在新生有丝分裂后CGN中，神经元中被沉默的祖细胞表达基因位点会快速获得H3K27me3修饰；而在CGN成熟时被转录激活的部分神经元基因的启动子区域，H3K27me3则会逐渐丢失。
赖氨酸去甲基化酶（lysine demethylase）KDM6B可促进H3K27me3的去除；在条件性敲除KDM6B的小鼠小脑中，成熟CGN中被诱导表达的基因其表达会出现显著缺陷。
丢失H3K27me3的基因会同时获得H3K27乙酰化修饰，并结合促成熟转录因子ZIC1/2，这提示发育过程中H3K27me3的去除或许是开启转录成熟程序的必要条件。
有趣的是，在早期有丝分裂后CGN中对H3K27甲基转移酶（H3K27 methyltransferase）EZH2进行药物抑制，不仅会阻断祖细胞基因的沉默过程，还会损伤成熟CGN基因的诱导表达。
本研究数据表明，在发育中的有丝分裂后神经元中，H3K27me3的调控功能不仅限于细胞命运决定阶段，还可调控介导神经元功能成熟的基因表达程序的动态变化。
实验整体设计如下：对以下样本进行H3K27me3 CUT&RUN测序（CUT&RUN-seq）：1) 体外培养1、3、5、7天的CGN；2) 在体外培养第1天（DIV1）分别用二甲基亚砜（dimethyl sulfoxide, DMSO）或5 μM Ezh2抑制剂GSK-126处理，于体外培养第5天（DIV5）收获的CGN；3) 在DIV1分别用DMSO或1 μM Kdm6b抑制剂GSK-J4处理，于DIV5收获的CGN。每组设置3次生物学重复。