Gene Expression At The Beginning Of Mammalian Development

哺乳动物发育初期的基因表达

基本信息

批准号：
9339944
负责人：
Melvin DePamphilis
金额：
$ 83.48万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
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

来源：
https://reporter.nih.gov/project-details/9339944
关键词：
Ablation Alleles Animals Apoptosis Benign Cell Differentiation process Cell Lineage Cells Characteristics Cleaved cell DNA DNA Damage Development Embryo Endometrium Event Fibroblasts GMNN gene Gene Expression Gene Expression Regulation Genes Genome Germ Cell Cancers Germ Cells Human Development Implant In Vitro Inner Cell Mass Intraperitoneal Injections Laboratories Life Malignant - descriptor Modeling Mus Neoplasms Nervous system structure Organism Placenta Pluripotent Stem Cells Publications Role Site Small Interfering RNA Solid Source Stem cells Tamoxifen Teratocarcinoma Teratoma Tissues Totipotent blastocyst cancer cell cancer stem cell cell type egg embryo cell embryo stage 2 embryonic stem cell in vivo monolayer pluripotency prevent protein function recombinase response self-renewal sperm cell stem cell differentiation trophoblast tumor zygote

项目摘要

Progress during the past year includes: Huang et al., Stem Cells, 2015 Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. Here, we show that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability. Adler-Wailes et al., submitted for publication Pluripotent stem cells, exemplified by embryonic stem cells (ESCs), are essential for mammalian development, but also a potential source of cancer stem cells. In vivo, ESCs give rise to all of the cell lineages. However, when placed at ectopic sites, ESCs produce germ cell neoplasia in the form of teratomas (benign) or teratocarcinomas (malignant). When cultured in vitro, ESCs require at least one geminin allele to prevent DNA re-replication dependent apoptosis during self renewal, a characteristic that diminishes after ESC differentiation in vitro. To determine whether or not this is also true within the animal, mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles and a tamoxifen dependent Cre recombinase. Early intraperitoneal injection of tamoxifen limited tumor formation to those ESCs that escaped Gmnn ablation. Complete Gmnn ablation in established tumors eliminated the pluripotent stem cells, but it did not prevent proliferation of the differentiated Gmnn /- cells within the tumor, although they did not survive in vitro. Thus, geminin was essential for ESCs to differentiate into multiple cell types, but not for the viability of differentiated cells within a solid tissue. These results also provide proof of principle that selective inhibition of geminin function could convert malignant germ cell cancer into a benign tumor.

过去一年的进度包括： Huang等人，干细胞，2015年双子素是一种双功能蛋白，是多细胞动物独有的，在调节基因表达和防止DNA再生中作用。在这里，我们表明，双子素在哺乳动物发育开始时至关重要，以防止多能细胞中的DNA重新复制，在胚胎干细胞中示例，因为它们会经历自我更新和分化。胚胎干细胞，胚胎成纤维细胞和永生的成纤维细胞在通过基因消融或siRNA耗尽后和之后表征了胚胎的成纤维细胞。在促进自我更新或分化的条件下，双子素的耗竭迅速诱导的DNA再复制，随后是DNA损伤，然后是DNA损伤反应，最后凋亡。一旦发生分化，双子素不再对生存力至关重要，尽管它继续有助于防止DNA复制引起的DNA损伤。双子素的表达与与多能性或分化相关的基因之间未检测到任何关系。因此，双子素在哺乳动物发育开始时的主要作用是预防DNA再复制依赖性凋亡，这是先前仅在癌细胞中至关重要的作用。这些结果表明，只有在多能细胞分化为多物质对于生存能力至关重要的细胞中，geminin对基因表达的调节才发生。 Adler-Wailes等人提交出版用胚胎干细胞（ESC）举例说明的多能干细胞对于哺乳动物发育至关重要，但也是癌症干细胞的潜在来源。在体内，ESC产生了所有细胞谱系。但是，当放置在异位部位时，ESC以畸胎瘤（良性）或teratocarcinomas（恶性）的形式产生生殖细胞肿瘤。当体外培养时，ESC需要至少一个双子素等位基因，以防止自我更新期间DNA重复依赖性凋亡，该特征在体外ESC分化后会减少。为了确定在动物中是否也是如此，将小鼠接种了含有条件GMNN等位基因和他莫昔芬依赖的CRE重组酶的ESC。早期腹膜内的他莫昔芬的注射有限的肿瘤形成到逃避GMNN消融的ESC。在已建立的肿瘤中，完全的GMNN消融消除了多能干细胞，但并不能阻止分化的GMNN / - 细胞在肿瘤内的增殖，尽管它们在体外没有生存。因此，Geminin对于ESC分化为多种细胞类型至关重要，但对于固体组织中分化细胞的生存能力不是必不可少的。这些结果还提供了原理证明，选择性抑制双子素功能可以将恶性生殖细胞癌转化为良性肿瘤。