Molecular Determinants of Totipotency

全能性的分子决定因素

基本信息

批准号：
10926341
负责人：
Sergio Ruiz
金额：
$ 50.97万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

To understand totipotency we used the recently described totipotent-like conversion induced by expression of the homeoprotein DUX. Expression of DUX induces most of features that characterize 2-cell embryos (transcriptional profile, loss of heterochromatin or activation of retroviral dormant sequences). Thus, we generated mESCs carrying a doxycycline (DOX)-inducible DUX cDNA using the KH2 mESC line (hereafter, ESCDux). We verified that DUX is expressed upon addition of DOX along with well-known DUX-dependent downstream ZGA-associated genes. We observed that sustained expression of DUX leads to DNA-damage, revealed by the increased levels of the gH2AX marker, and efficient cell death in a dose dependent manner correlating with the conversion into the 2C-like state. We determined that DNA-damage was taking place at a subset of CTCF-binding sites and hypothesized that CTCF was a barrier to totipotent-like conversion. Interestingly, we observed that CTCF depletion in ESC induced a 2C-like conversion in an spontaneous manner. Finally, we also showed that 2C-like conversion upon CTCF-depletion was dependent on the endogenous activation of the cluster of genes ZSCAN4. Taking all together, we made great progress in this project, and we have this work accepted for publication in Nature Communications. We are also interested in exploring the DUX-dependent induction of DUXBL, a gene that belongs to the DUX family of proteins only found intact in rodents. This gene is part of a triplicated region of the genome spanning more than 300kb with three identical copies of DUXBL. Using published ChIP-seq data for DUX we observed that all three DUXBL copies contain three binding sites for DUX. In fact, induction of DUX in ESCDux cells leads to elevated levels of DUXBL in a dose dependent manner. We performed Cut&Run to identify the putative binding sites in ESCs and found it enriched in different enhancer and promoters. Interestingly, by performing morpholino microinjections in zygotes to downregulate Duxbl, we observed that this protein is essential for development. Indeed, Duxbl-knockdown zygotes arrested at the 2C-stage of development. RNAseq analysis of wild-type and Duxbl-knockdown 2C embryos revealed strong defects in splicing. This project has moved forward during this past FY and these analyses will set the basis to further explore the role of DUXBL during embryonic development.

为了理解全能性，我们使用了最近描述的由同源蛋白 DUX 表达诱导的全能样转换。 DUX 的表达诱导了 2 细胞胚胎的大部分特征（转录谱、异染色质丢失或逆转录病毒休眠序列激活）。因此，我们使用 KH2 mESC 系（以下简称 ESCDux）生成了携带多西环素 (DOX) 诱导型 DUX cDNA 的 mESC。我们验证了 DUX 在添加 DOX 以及众所周知的 DUX 依赖性下游 ZGA 相关基因后表达。我们观察到 DUX 的持续表达会导致 DNA 损伤，这通过 gH2AX 标记物水平的增加来揭示，并且以剂量依赖性方式有效细胞死亡，与转化为 2C 样状态相关。我们确定 DNA 损伤发生在 CTCF 结合位点的子集上，并假设 CTCF 是全能样转化的障碍。有趣的是，我们观察到 ESC 中 CTCF 的消耗会自发地诱导类似 2C 的转化。最后，我们还表明 CTCF 耗尽后的 2C 样转化依赖于 ZSCAN4 基因簇的内源激活。总而言之，我们在这个项目上取得了很大的进展，并且我们的工作已被《自然通讯》接受发表。我们还有兴趣探索 DUXBL 的 DUX 依赖性诱导，DUXBL 是一种属于 DUX 蛋白家族的基因，仅在啮齿类动物中完整存在。该基因是基因组三倍区域的一部分，跨度超过 300kb，具有三个相同的 DUXBL 拷贝。使用已发布的 DUX ChIP-seq 数据，我们观察到所有三个 DUXBL 副本都包含三个 DUX 结合位点。事实上，ESCDux 细胞中 DUX 的诱导导致 DUXBL 水平以剂量依赖性方式升高。我们进行了 Cut&Run 来鉴定 ESC 中的假定结合位点，发现它富含不同的增强子和启动子。有趣的是，通过在受精卵中进行吗啉显微注射来下调 Duxbl，我们观察到这种蛋白质对于发育至关重要。事实上，Duxbl 敲低受精卵在发育的 2C 阶段就被阻止了。对野生型和 Duxbl 敲低 2C 胚胎的 RNAseq 分析揭示了剪接方面的严重缺陷。该项目在上个财年取得了进展，这些分析将为进一步探索 DUXBL 在胚胎发育过程中的作用奠定基础。