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。人的三倍 21号染色体（HSA 21）导致整个表观基因组和转录组的广泛变化。最近 研究已经确定了与少突胶质细胞（OL）发育，分化， 和髓磷脂的维持。这些转录变化表现在延迟的 髓鞘形成开始，有髓纤维密度降低，其网格状组织破坏，和 有髓神经纤维传导速度降低，这意味着异常胶质细胞生成和白色物质 三体大脑的改变导致DS的智力缺陷。此外，与减少 在DS中报告了OL，即星形胶质细胞的扩增群体。 OL细胞的特化和分化是通过特定阶段获得的定义的细胞因子来严格控制的。 染色质标记。不同的表观遗传特征，包括不同的DNA脱乙酰化模式， 在DS中观察到了甲基化和改变的组蛋白标记。因此，基因的失调导致了 OL的发育和成熟可能是异常的DS相关表观遗传结构的结果， 在扰动OL生产和白色物质赤字中观察到DS。在初步研究中，我们 来自同源三体和整倍体对照诱导的多能干细胞（iPSC）的分化OL，所述诱导的多能干细胞来源于 患有DS和确定的三体性相关的OL承诺，发育和成熟失调的人。 然而，已发表的方案固有的异质性培养限制了下游分析， 鉴定功能和转录上不同的OL亚型以及检查其 在DS的发展轨迹。因此，我们的目标是工程化两个同基因DS衍生的iPSC系，以表达 在内源性血小板衍生生长因子受体α（PDGFRα）基因座的控制下， 有助于DS中白色物质缺乏的分子病因学研究。PDGFRα主要表达于 增殖OPCs和PDGFRα+祖细胞，产生成熟OL以及星形胶质细胞。因此，DS衍生的 PDGFRα报告细胞系将允许检查胶质细胞生成的变化，OL和星形胶质细胞的不平衡， 生产，并监测OL的发展和成熟。使用这些细胞系，我们将进行单细胞测定， 转座酶可接近染色质（ATAC）-seq检查染色质可接近性的变化如何影响OL DS患者来源的细胞中的谱系发育轨迹。我们的研究将在概念上将DS相关 OL分化到表观遗传结构的改变是异常OL的根本原因 导致DS智力缺陷的发展。

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.