Studying epigenetic dysregulation in Down Syndrome using fluorescent reporter cell lines

摘要： 唐氏综合征(DS)智力障碍的确切病因尚不清楚。人类的三倍增长 21号染色体(HSA21)导致整个表观基因组和转录组的广泛变化。近期 研究已经确定了与少突胶质细胞(OL)发育、分化、 以及人类和小鼠脑内髓鞘的维持。这些转录变化表现在延迟的 开始髓鞘形成，有髓纤维密度降低，其格子样组织破坏，以及 有髓纤维传导速度减慢，意味着异常的胶质生成和白质 三体大脑的改变导致了DS患者的智力缺陷。此外，与减少 据报道，在DS中星形胶质细胞的数量增加。 通过特定阶段获取已定义的OL细胞规范和分化受到严格控制 染色质痕迹。不同的表观遗传特征，包括不同的DNA脱乙酰化和 在DS中观察到甲基化和改变的组蛋白标记。因此，驱动基因的失调 OL的发育和成熟可能是DS相关表观遗传结构异常的结果 在紊乱的OL产生和DS中观察到的白质缺陷。在我们的初步研究中，我们 同源三体和整倍体对照诱导来源的多能干细胞分化的成骨细胞 DS患者，并发现三体相关的OL承诺、发育和成熟的失调。 然而，已公布的协议固有的异质文化限制了下游分析和 功能和转录差异的OL亚型的鉴定及其亚型的检测 DS患者的发育轨迹。因此，我们的目标是设计两个同基因DS衍生的ipsc株来表达 受内源血小板衍生生长因子受体α(PDGFRα)基因座控制的番茄 促进DS白质缺陷的分子病因学研究。PDGFRα主要表达于 增殖OPC和PDGFRα+祖细胞，形成成熟的OPC和星形胶质细胞。因此，DS派生 PDGFRα报告系将允许检查胶质形成的变化，OL和星形胶质细胞的失衡 生产，并监督OL的发展和成熟。利用这些品系，我们将进行单细胞检测 转座酶可及染色质(ATAC)-SEQ研究染色质可及性的改变如何影响OL DS患者来源细胞的谱系发育轨迹。我们的研究将从概念上将DS相关 OL分化到表观遗传结构的变化是OL异常的根本原因 发展导致DS的智力缺陷。

Abstract: The exact etiology of the intellectual disability in Down syndrome (DS) is unknown. Triplication of human chromosome 21 (HSA21) results in widespread changes across the entire epigenome and transcriptome. Recent studies have identified dysregulated gene networks related to oligodendrocyte (OL) development, differentiation, and myelin maintenance in human and mouse DS brains. These transcriptional changes manifest in the delayed onset of myelination, reduced density of myelinated fibers, disruption in their lattice-like organization, and decreased conduction velocity across myelinated fibers, implying that aberrant gliogenesis and white matter alterations in trisomic brains contribute to intellectual deficits in DS. Furthermore, in contrast to the reduction of OLs, an expanded population of astrocytes has been reported in DS. OL cell specification and differentiation is tightly controlled through stage-specific acquisition of defined chromatin marks. Distinct epigenetic signatures, including differing patterns of DNA deacetylation and methylation, and altered histone marks have been observed in DS. Thus, the dysregulation of the genes driving OL development and maturation may be a consequence of aberrant DS-related epigenetic architecture resulting in perturbed OL production and the white matter deficit observed in DS. In our preliminary studies, we differentiated OLs from isogenic trisomic and euploid control induced pluripotent stem cells (iPSCs) derived from people with DS and identified trisomy-related dysregulation of OL commitment, development, and maturation. However, the heterogeneous culture inherent to the published protocol limits downstream analysis and identification of functionally and transcriptionally distinct OL subtypes as well as examination of their developmental trajectories in DS. Thus, our goal is to engineer two isogenic DS-derived iPSC lines to express tdTomato under the control of the endogenous platelet-derived growth factor receptor α (PDGFRα) locus to facilitate the study of the molecular etiology of the white matter deficit in DS. PDGFRα is primarily expressed in proliferating OPCs and PDGFRα+ progenitors, giving rise to mature OLs as well as astrocytes. Thus, DS-derived PDGFRα reporter lines will allow to examine changes in gliogenesis, the imbalance in OL and astrocyte production, and monitor OL development and maturation. Using these lines, we will perform single cell assay for transposase-accessible chromatin (ATAC)-seq to examine how changes in chromatin accessibility affect OL lineage developmental trajectory in DS patient-derived cells. Our studies will conceptually link DS-associated alterations in OL differentiation to the epigenetic architecture as the underlying cause for abnormal OL development leading to the intellectual deficits in DS.