Hu Rong, Zhao Jin, Lai Kuan Chen, Wang Shikun, Zheng Jianqing, Stoddard Christopher, Lai Laijun
Department of Allied Health Sciences, University of Connecticut, 1390 Storrs Road, Storrs, 06269, CT, USA.
Department of Department of Genetics and Genome Sciences, University of Connecticut Health, Farmington, CT, USA.
Stem Cell Res Ther. 2025 Jun 17;16(1):311. doi: 10.1186/s13287-025-04437-9.
CHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders, including CHARGE (Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital hypoplasia, and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus, liver, pancreas, and intestine.
In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7), heterozygous mutant (CHD7), and control wild-type (CHD7) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers, mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).
We found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.
CHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.
CHD7编码一种ATP依赖的染色质结构域解旋酶DNA结合蛋白；该基因的突变会导致多种发育障碍，包括CHARGE（眼裂、心脏缺陷、后鼻孔闭锁、生长发育迟缓、生殖器发育不全和耳部异常）综合征。这些突变如何导致多种缺陷在很大程度上仍不清楚。胚胎确定性内胚层（DE）产生重要器官的上皮部分，如胸腺、肝脏、胰腺和肠道。
在本研究中，我们使用成簇规律间隔短回文重复序列（CRISPR）/Cas9技术在人类胚胎干细胞（hESCs）中删除CHD7基因，以产生CHD7纯合突变体（CHD7）、杂合突变体（CHD7）和对照野生型（CHD7）细胞。然后，我们研究了hESCs在体外发育成DE以及另外两个胚层（中胚层和外胚层）的能力。我们还通过RNA测序（RNA-seq）和转座酶可及染色质测序分析（ATAC-seq）比较了hESC-DE细胞之间的全局基因表达和染色质可及性。
我们发现CHD7的缺失导致发育成DE和中胚层的能力以剂量依赖的方式降低。CHD7的缺失导致与多个途径相关的基因的表达和染色质可及性发生显著变化。我们鉴定出40个在CHD7缺失的hESC-DE细胞中表达和染色质可及性均高度下调的基因。
CHD7对hESCs的DE和中胚层发育至关重要。我们的结果为CHD7突变导致多种先天性异常的机制提供了新的见解。
