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Insights into novel regulators of human somatic cell reprogramming.

机译：深入了解人类体细胞重编程的新型调节剂。

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摘要

Reprogramming of somatic cells generates induced pluripotent stem cells (iPSCs) that are invaluable resources in biomedical research. Transcriptional and epigenetic changes have been investigated to improve our understanding of reprogramming processes. Here, we extend the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface makers. We found that transcriptome changes during early induction of reprogramming are independent of the opening of compact chromatin by Yamanaka factors OCT4, SOX2, KLF4 and MYC. Our data also show that multiple cellular signaling pathways display a bi-phasic change: repression at the early stage and induction at the final stage of reprogramming. RNA-seq allowed us to uncover a switch in allelic specific expression during reprogramming, and to identify multiple spliced forms of genes uniquely expressed at progressive stages of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that significantly enhances reprogramming. Our transcriptome data provide unique opportunities to understand human iPSC reprogramming.;Reprogramming to pluripotency is also accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in iPSCs are highly similar to those in embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern for using iPSCs in a clinical setting. Thus, it is critical for future applications of iPSCs to find factors that regulate DNA methylation states in reprogramming. We asked whether non-coding RNAs that regulate both de novo methylation and demethylation would facilitate reprogramming. Through target prediction analysis and screening, we found that miR-29 family targets de novo DNA methyltransferases DNMT3A and DNMT3B, demethylases TET1, TET3 and TDG, as well as histone lysine demethylases such as KDM5B. The suppression of miR-29 family improved, while its overexpression decreased the efficiency of human somatic cell reprogramming. Through global DNA methylation and hydroxymethylation analysis, we found that DNA demethylation is a major event mediated by miR-29a in early reprogramming, and that iPSCs derived from miR-29a depleted fibroblasts are epigenetically more similar to established ESCs than control ones. Furthermore, miR29a treated iPSCs give rise to more complex three germ layer --- tissue differentiation as assessed by teratoma formation. Our findings uncover an important miRNA-based approach in generating iPSCs with altered epigenetic states.
机译：体细胞的重编程产生诱导性多能干细胞（iPSC），这是生物医学研究中的宝贵资源。已经研究了转录和表观遗传变化，以增进我们对重编程过程的理解。在这里，我们通过对由多个细胞表面标记物组合定义的细胞进行RNA-seq扩展了先前的转录组研究。我们发现，在重新编程的早期诱导过程中，转录组的变化与Yamanaka因子OCT4，SOX2，KLF4和MYC致密染色质的打开无关。我们的数据还显示，多个细胞信号通路显示出双相变化：重编程的早期抑制和最后阶段的诱导。 RNA-seq使我们能够在重编程期间发现等位基因特异性表达的转换，并鉴定在重编程进行阶段独特表达的基因的多种剪接形式。特别是，我们发现了CCNE1的多能性特异性剪接形式，可大大增强重编程能力。我们的转录组数据为了解人类iPSC重编程提供了独特的机会。重编程为多能性还伴随着全球基因组和表观基因组的变化。 iPSC中的组蛋白修饰和DNA甲基化状态与胚胎干细胞（ESC）中的高度相似。但是，iPSC和ESC之间仍然存在表观遗传差异。特别是，在iPSC中发现的异常DNA甲基化状态是在临床环境中使用iPSC的主要问题。因此，对于iPSC的未来应用而言，找到在重编程中调节DNA甲基化状态的因素至关重要。我们询问了同时调节从头甲基化和去甲基化的非编码RNA是否有助于重编程。通过靶标预测分析和筛选，我们发现miR-29家族靶向从头DNA甲基转移酶DNMT3A和DNMT3B，脱甲基酶TET1，TET3和TDG以及组蛋白赖氨酸脱甲基酶，例如KDM5B。 miR-29家族的抑制作用得到改善，而其过表达则降低了人类体细胞重编程的效率。通过全局DNA甲基化和羟甲基化分析，我们发现DNA脱甲基化是miR-29a在早期重编程中介导的主要事件，并且从miR-29a耗尽的成纤维细胞衍生的iPSC在表观遗传上比已建立的ESC与对照更相似。此外，经miR29a处理的iPSC可产生更复杂的三个胚层-通过畸胎瘤形成评估组织分化。我们的发现揭示了一种重要的基于miRNA的方法，可用于产生表观遗传状态发生变化的iPSC。

著录项

  • 作者

    Hysolli, Eriona.;

  • 作者单位

    Yale University.;

  • 授予单位 Yale University.;
  • 学科 Genetics.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 206 p.
  • 总页数 206
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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