Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration [RNA-seq]

De novo germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated heterozygous H3.3G34R/V/W direct knock-in mutant mice and identified strikingly distinct developmental defects for each amino acid substitution. H3.3G34R-mutant mice uniquely exhibited progressive microcephaly and neurodegeneration, associated with abnormal accumulation of damage-associated microglia and astrocytes, and concurrent neuronal depletion in postnatal brains. On the mutant H3.3 tail, G34R severely decreased H3K36me2, impairing recruitment of DNA methyltransferase DNMT3A and promoting its redistribution on chromatin. This results in loss of CH methylation at complement and other innate immune genes promoting their sustained expression, and, aberrant CG methylation at neuronal gene promoters and undue silencing. Persistent complement expression in G34R neurons led to excessive synaptic pruning, neuroinflammation, and neuronal damage accounting for progressive neurodegeneration. Our study reveals H3.3G34-substitutions have differential impact on chromatin, which underlie the diverse phenotypes observed. Notably, we uncover unappreciated roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating pruning and neuroinflammation in post-natal brains. 23 RNA-seq samples of mouse cortex at 7 days or 10 weeks with H3f3a G34R/V/W histone substitutions.生殖系新发组蛋白H3.3氨基酸替换（包括H3.3G34R/V）可引发严重的神经发育综合征。为解析此类突变影响大脑发育的具体机制，我们构建了杂合H3.3G34R/V/W定点敲入突变小鼠，并发现不同氨基酸替换会导致截然不同的发育缺陷。仅H3.3G34R突变小鼠表现出进行性小头畸形与神经退行性变，伴随损伤相关小胶质细胞和星形胶质细胞的异常聚集，以及出生后大脑神经元的持续性丢失。在突变的H3.3尾部区域，G34R突变显著降低了H3K36me2（组蛋白H3赖氨酸36二甲基化）水平，削弱了DNA甲基转移酶DNMT3A的招募，并促进其在染色质上的重新分布。这一变化导致补体及其他先天免疫基因位点的CH甲基化丢失，进而引发这些基因的持续表达；同时还会造成神经元基因启动子区域的异常CG甲基化，导致基因过度沉默。G34R神经元中补体的持续表达会引发过度的突触修剪、神经炎症与神经元损伤，最终导致进行性神经退行性变。本研究揭示，H3.3G34位点的氨基酸替换对染色质具有差异化调控作用，这正是观察到的多样化表型的分子基础。值得注意的是，我们还发现了H3K36me2与DNMT3A依赖的CH甲基化在调控出生后大脑突触修剪与神经炎症中此前未被重视的功能。本数据集包含23份来自7日龄或10周龄小鼠皮层的RNA测序（RNA-seq）样本，对应携带H3f3a G34R/V/W组蛋白替换的模型。