Role of SETD5 in Moyamoya Disease Pathogenesis

摘要 Moyamoya病(MMD)发生在颈内动脉远端进行性狭窄和 最终闭塞，是儿科中风的常见原因。许多致病基因变异都有 已被确定为引起MMD的原因，但共同的发病机制尚未确定。病理来自 受累血管显示闭塞病变由纤维增殖细胞组成，这些细胞染色阳性 平滑肌细胞特异的α-肌动蛋白；因此我们认为，平滑肌细胞的迁移和增殖 可能是这种疾病的驱动因素。参与染色质重塑的多个编码蛋白质的基因 已被发现导致MMD，包括基因SETD5中的杂合性功能丧失(LOF)变体。SETD5 与核受体-辅阻遏物(NCoR)复合体相互作用，调节组蛋白乙酰化。我们以前的 对ACTA2中导致MMD的致病变异的研究表明，这些变异损害了SMC的分化，并且 未完全分化的细胞增加了增殖和迁移，细胞依赖糖酵解。 能源生产。重要的是，促进氧化磷酸化的治疗恢复了分化和 携带导致MMD的Acta2变异体的小鼠SMC迁移减少，提示一种潜在的治疗方法 策略。基于这些结果和已确定的MMD的遗传触发因素列表，我们提出了一个共同的 发病机制：SMC规范过程中异常的染色质重塑导致细胞增殖 并迁移以闭塞血管。在这里，我们将在两个SETD5中带有LOF变体的细胞中测试这一假设 明确的目标。1)我们将评估SETD5的LOF变异是否影响SMC的分化和表型。我们 将使用Crispr/Cas9基因编辑将SETD5 LOF等位基因引入人诱导的多能干细胞 (IPSCS)。我们将在同基因对照的基础上将这些ipscs分化为神经脊祖细胞，然后再分化为 SMC，并将表征所产生的细胞的分化、增殖、迁移和代谢。2) 我们将评估SETD5是否影响SMC分化关键基因的染色质重塑。我们会 利用靶向Crispr/Cas9基因编辑在SETD5蛋白的C末端引入3xFlag标签 并将使用这些细胞来确定SETD5在IPSCs中起作用的基因组位置，神经脊祖细胞， 染色质免疫沉淀测序。我们将评估SETD5中的LOF变异是否会影响 组蛋白乙酰化和已鉴定基因座上的基因转录。完成这些目标将把SETD5- SMC表型依赖的染色质重塑及LOF的分子机制 SETD5基因的变异会导致MMD。这一结果有可能确定治疗策略以治疗或预防 具有SETD5 LOF变异的患者的MMD。最后，这些数据将极大地促进我们对 可能是MMD发病的共同途径。

ABSTRACT Moyamoya disease (MMD) occurs when the distal internal carotid arteries are progressively narrowed and eventually occluded, and is a common cause of pediatric stroke. Numerous pathogenic genetic variants have been identified to cause MMD, but a common mechanism of pathogenesis has yet to be defined. Pathology from affected vessels shows the occlusive lesions are comprised of fibroproliferative cells that stain positive for smooth muscle cell (SMC)-specific α-actin (SMA); we therefore propose that SMC migration and proliferation may be drivers of the disease. Multiple genes encoding proteins that participate in chromatin remodeling have been identified to cause MMD, including heterozygous loss of function (LOF) variants in the gene SETD5. SETD5 interacts with the nuclear receptor-corepressor (Ncor) complex to regulate histone acetylation. Our previous work on MMD-causing pathogenic variants in ACTA2 showed that these variants impair SMC differentiation, and the incompletely differentiated cells have increased proliferation and migration and rely on glycolysis for cellular energy production. Importantly, treatments that boost oxidative phosphorylation restored differentiation and reduced migration in mouse SMCs with MMD-causing Acta2 variants, suggesting a potential therapeutic strategy. Based on these results and the list of identified genetic triggers for MMD, we propose a common pathogenic mechanism: aberrant chromatin remodeling during SMC specification leads to cells that proliferate and migrate to occlude the vessels. Here, we will test this hypothesis in cells with LOF variants in SETD5 in two specific aims. 1) We will assess whether LOF variants in SETD5 impact SMC differentiation and phenotype. We will use Crispr/Cas9 gene editing to introduce SETD5 LOF alleles into human induced pluripotent stem cells (iPSCs). We will differentiate these iPSCs alongside isogenic controls into neural crest progenitors and then into SMCs, and will characterize the differentiation, proliferation, migration, and metabolism of the resulting cells. 2) We will assess whether SETD5 impacts chromatin remodeling at loci critical for SMC differentiation. We will introduce a 3xFlag tag at the C-terminus of the SETD5 protein using targeted Crispr/Cas9 gene editing in human iPSCs and will use these cells to identify genomic loci where SETD5 is acting in iPSCs, neural crest progenitors, and SMCs by chromatin immunoprecipitation sequencing. We will assess whether LOF variants in SETD5 affect histone acetylation and gene transcription at the identified loci. Completion of these aims will link SETD5- dependent chromatin remodeling with SMC phenotype and elucidate the molecular mechanisms by which LOF variants in SETD5 cause MMD. The results have the potential to identify therapeutic strategies to treat or prevent MMD in patients with SETD5 LOF variants. Finally, these data will dramatically advance our understanding of a potential common pathway for MMD pathogenesis.