Molecular Determinants of Totipotency

全能性的分子决定因素

• 批准号: 10486994
• 负责人: Sergio Ruiz
• 金额: $ 34.23万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486994
• 关键词: Binding Sites; Cell Death; Cells; ChIP-seq; Communication; Complementary DNA; DNA Damage; Data; Defect; Development; Dose; Doxycycline; Embryo; Embryonic Development; Enhancers; Gene Cluster; Gene Expression Profile; Genes; Genome; Goals; Growth; Heterochromatin; Homeodomain Proteins; Maintenance; Malignant Neoplasms; Microinjections; Molecular; Nature; Protein Family; Proteins; Publications; Publishing; RNA Splicing; Rodent; Role; Running; Totipotency; Totipotent; Whole Organism; Work; developmental plasticity; embryo cell; interest; knock-down; promoter; transcriptome sequencing; zygote

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，我们使用了正配蛋白DUX的表达引起的最近描述的类似动力的转化率。 DUX的表达诱导了表征2细胞胚胎的大多数特征（转录谱，异染色质的丧失或逆转录病毒休眠序列的激活）。因此，我们使用KH2 MESC系（以下为ESCDUX）生成了携带强力霉素（DOX）诱导dux cDNA的MESC。我们验证了添加DOX以及众所周知的DUX依赖性下游ZGA相关基因时表达了DUX。我们观察到，持续的DUX表达会导致DNA破坏，这表明，GH2AX标记的水平增加，并且以剂量依赖性方式有效的细胞死亡与转化为2C样状态相关。我们确定DNA破坏是在CTCF结合位点的子集发生的，并假设CTCF是类似动力的转化率的障碍。有趣的是，我们观察到ESC中的CTCF耗竭以自发的方式诱导了2C样转化。最后，我们还表明，CTCF止血时的2C样转化取决于基因ZSCAN4簇的内源性激活。全力以赴，我们在这个项目中取得了长足的进步，我们接受了这项工作，以供自然传播中出版。我们还有兴趣探索Duxbl的Dux依赖性诱导，该基因属于蛋白质的Dux家族，仅在啮齿动物中发现完整。该基因是跨越300kb的基因组的三式三份区域的一部分，并具有三个相同的duxbl副本。使用已发布的DUX芯片序列数据，我们观察到所有三个DUXBL副本都包含三个用于DUX的结合位点。实际上，ESCDUX细胞中Dux的诱导会导致Duxbl的水平升高，以剂量依赖性方式。我们进行了切割和运行，以识别ESC中的假定结合位点，并发现它富含不同的增强子和启动子。有趣的是，通过在Zygotes中进行吗啡微注射以下调Duxbl，我们观察到该蛋白对于发育至关重要。确实，在2C发展的2C阶段，Duxbl-Knockdown Zygotes被捕。 RNASEQ对野生型和Duxbl-Knockdown 2C胚胎的分析显示，剪接中有很大的缺陷。在过去的FY中，该项目已向前发展，这些分析将为进一步探索Duxbl在胚胎开发中的作用奠定基础。