Chromatin Chromatin remodeling protein CHD4 regulates axon guidance of spiral ganglion neurons in developing inner ear

The chromodomain helicase binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler—De-novo pathogenic variants of CHD4 cause Sifrim-Hitz-Weiss syndrome (SIHIWES). Patients with SIHIWES show delayed development, intellectual disability, facial dysmorphism, and hearing loss. Many cochlear cell types, including spiral ganglion neurons (SGNs), express CHD4. SGNs are the primary afferent neurons that convey sound information from the cochlea, but the function of CHD4 in SGNs is unknown. We employed the Neurog1(Ngn1) CreERT2 Chd4 conditional knockout animals to delete Chd4 in SGNs. SGNs are classified as type I and type II neurons. SGNs lacking CHD4 showed abnormal fasciculation of type I neurons along with improper pathfinding of type II fibers. CHD4 binding to chromatin from immortalized multipotent otic progenitor-derived neurons was used to identify candidate target genes in SGNs. Gene ontology analysis of CHD4 target genes revealed cellular processes involved in axon guidance, axonal fasciculation, and ephrin receptor signaling pathway. We validated increased Epha4 transcripts in SGNs from Chd4 conditional knockout cochleae. The results suggest that CHD4 attenuates the transcription of axon guidance genes to form the stereotypic pattern of SGN peripheral projections. The results implicate how epigenetic changes affect circuit wiring by modulating axon guidance molecule expression and provide insights into neurodevelopmental diseases. Overall design: CUT&Tag for CHD4, H3K4me3, p300 and IgG control from proliferating iMOPs (immortablized mutipotent otic progenitors) and iMOP-derived neurons.

染色质域解旋酶结合蛋白4（chromodomain helicase binding protein 4，CHD4）是一种ATP依赖型染色质重塑因子。CHD4的新发致病变异可导致西夫里姆-希茨-魏斯综合征（Sifrim-Hitz-Weiss syndrome，SIHIWES）。该综合征患者表现为发育迟缓、智力障碍、面部畸形及听力损失。诸多耳蜗细胞类型均可表达CHD4，其中包括螺旋神经节神经元（spiral ganglion neurons，SGNs）。SGNs是负责从耳蜗传递声音信息的初级传入神经元，但CHD4在SGNs中的功能尚不明确。我们借助Neurog1（Ngn1）CreERT2 Chd4条件性敲除动物模型，在SGNs中敲除Chd4。SGNs可分为I型与II型神经元。缺失CHD4的SGNs可出现I型神经元束化异常，同时II型神经纤维的路径选择出现异常。我们通过分析永生化多能耳蜗祖细胞衍生神经元的染色质与CHD4的结合情况，鉴定出SGNs中的候选靶基因。对CHD4靶基因进行基因本体（Gene Ontology，GO）富集分析后发现，其参与的细胞过程包括轴突导向、轴突束化及Ephrin受体信号通路。我们验证了Chd4条件性敲除耳蜗的SGNs中Epha4转录本水平升高。研究结果表明，CHD4可通过抑制轴突导向基因的转录，从而形成SGNs外周投射的典型模式。本研究结果阐明了表观遗传改变如何通过调控轴突导向分子的表达影响神经环路连接，并为神经发育疾病的研究提供了新的见解。实验整体设计：对增殖期永生化多能耳蜗祖细胞（immortalized multipotent otic progenitors，iMOPs）及其衍生神经元开展CHD4、组蛋白H3赖氨酸4三甲基化（H3K4me3）、p300蛋白（p300）及IgG对照的CUT&Tag测序。