Puri Deepika, Maaßen Catharina, Varona Baranda Monica, Zeevaert Kira, Hahnfeld Lena, Hauser Annika, Fornero Giulia, Elsafi Mabrouk Mohamed H, Wagner Wolfgang
Institute for Stem Cell Biology, RWTH Aachen University Medical School, Aachen, Germany.
Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany.
Front Cell Dev Biol. 2023 Nov 30;11:1302448. doi: 10.3389/fcell.2023.1302448. eCollection 2023.
Pluripotent stem cells are characterized by their differentiation potential toward endoderm, mesoderm, and ectoderm. However, it is still largely unclear how these cell-fate decisions are mediated by epigenetic mechanisms. In this study, we explored the relevance of CCCTC-binding factor (CTCF), a zinc finger-containing DNA-binding protein, which mediates long-range chromatin organization, for directed cell-fate determination. We generated human induced pluripotent stem cell (iPSC) lines with deletions in the protein-coding region in exon 3 of CTCF, resulting in shorter transcripts and overall reduced protein expression. Chromatin immunoprecipitation showed a considerable loss of CTCF binding to target sites. The CTCF deletions resulted in slower growth and modest global changes in gene expression, with downregulation of a subset of pluripotency-associated genes and neuroectodermal genes. CTCF deletion also evoked DNA methylation changes, which were moderately associated with differential gene expression. Notably, CTCF-deletions lead to upregulation of endo-mesodermal associated marker genes and epigenetic signatures, whereas ectodermal differentiation was defective. These results indicate that CTCF plays an important role in the maintenance of pluripotency and differentiation, especially towards ectodermal lineages.
多能干细胞的特征在于它们向内胚层、中胚层和外胚层分化的潜力。然而，这些细胞命运决定如何由表观遗传机制介导在很大程度上仍不清楚。在本研究中，我们探讨了CCCTC结合因子（CTCF）——一种含锌指的DNA结合蛋白，其介导长程染色质组织——与定向细胞命运决定的相关性。我们生成了在CTCF第3外显子的蛋白质编码区域存在缺失的人诱导多能干细胞（iPSC）系，导致转录本缩短且蛋白质表达总体降低。染色质免疫沉淀显示CTCF与靶位点的结合显著减少。CTCF缺失导致生长缓慢和基因表达的适度全局变化，多能性相关基因和神经外胚层基因的一个子集下调。CTCF缺失还引发了DNA甲基化变化，这些变化与差异基因表达适度相关。值得注意的是，CTCF缺失导致内中胚层相关标记基因和表观遗传特征上调，而外胚层分化存在缺陷。这些结果表明CTCF在维持多能性和分化中起重要作用，尤其是对外胚层谱系的分化。
