Bidirectional changes in postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics. 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: Paired-end RNA-seq for WT and Kdm6b-cKO cerebellar tissue harvested at P14. (n=2 biological replicates)

神经元的功能与转录成熟是一个持续周期较长的过程，其进程远超出祖细胞的有丝分裂退出与终末命运决定阶段。出生后小鼠小脑中的小脑颗粒神经元（cerebellar granule neurons, CGNs）分化模型，可为探究协调有丝分裂后CGNs成熟过程中转录时序变化的染色质调控机制，提供了实用的研究模型。本研究发现，CGN成熟与组蛋白H3赖氨酸27三甲基化（histone H3 lysine 27 trimethylation, H3K27me3）的基因组分布动态改变密切相关，该修饰最广为人知的功能是在细胞特化阶段抑制替代细胞命运的表达。在新生有丝分裂后CGNs中，H3K27me3会快速富集于在神经元中被沉默的祖细胞表达基因区域；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。H3K27me3的丢失由赖氨酸去甲基化酶KDM6B（lysine demethylase KDM6B）介导，且在成熟CGNs中被诱导表达的基因，在条件性KDM6B敲除小鼠的小脑中其表达会受到显著损害。丢失H3K27me3的基因会获得H3K27乙酰化修饰，并结合促成熟的ZIC1/2转录因子，这提示发育过程中H3K27me3的丢失或许是允许转录成熟程序启动的必要条件。值得注意的是，在早期有丝分裂后CGNs中对H3K27甲基转移酶EZH2进行药物抑制，不仅会阻断祖细胞基因的沉默过程，还会损害成熟CGN基因的诱导表达。上述结果表明，在发育中的有丝分裂后神经元中，H3K27me3的调控功能不止局限于细胞命运决定阶段，而是用于调控介导神经元功能成熟的基因表达程序的动态变化。总体实验设计：对出生后第14天（P14）采集的野生型与Kdm6b条件性敲除小鼠小脑组织进行双端RNA测序（Paired-end RNA-seq），共设置2次生物学重复。