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.

过去一年的进展包括：黄等人，干细胞，2015 Geminin 是一种多细胞动物特有的双功能蛋白，具有调节基因表达和防止 DNA 再复制的作用。在这里，我们表明，geminin 在哺乳动物发育初期至关重要，可防止多能细胞（以胚胎干细胞为例）中的 DNA 再复制，因为它们会进行自我更新和分化。在通过基因消融或 siRNA 耗尽 Geminin 之前和之后，对胚胎干细胞、胚胎成纤维细胞和永生化成纤维细胞进行了表征。在促进自我更新或分化的条件下，Geminin 的耗尽会迅速诱导 DNA 重新复制，然后是 DNA 损伤，然后是 DNA 损伤反应，最后是细胞凋亡。一旦分化发生，双联蛋白就不再是生存所必需的，尽管它继续有助于防止 DNA 再复制引起的 DNA 损伤。没有检测到双联蛋白的表达和与多能性或分化相关的基因之间的关系。因此，geminin 在哺乳动物发育初期的主要作用是防止 DNA 再复制依赖性细胞凋亡，以前认为这一作用仅在癌细胞中至关重要。这些结果表明，只有在多能细胞分化为双联蛋白对于生存力不是必需的细胞后，才会发生双联蛋白对基因表达的调节。 Adler-Wailes 等人，提交出版以胚胎干细胞（ESC）为代表的多能干细胞对于哺乳动物的发育至关重要，也是癌症干细胞的潜在来源。在体内，ESC 产生所有细胞谱系。然而，当放置在异位部位时，ESC 会产生畸胎瘤（良性）或畸胎癌（恶性）形式的生殖细胞肿瘤。在体外培养时，ESC 需要至少一个双联蛋白等位基因来防止自我更新过程中 DNA 再复制依赖性细胞凋亡，这一特性在 ESC 体外分化后会减弱。为了确定动物体内是否也存在这种情况，给小鼠皮下接种了含有条件 Gmnn 等位基因和他莫昔芬依赖性 Cre 重组酶的 ESC。早期腹腔注射他莫昔芬限制了那些逃脱 Gmnn 消融的 ESC 的肿瘤形成。已形成的肿瘤中的完全 Gmnn 消融消除了多能干细胞，但它并没有阻止肿瘤内分化的 Gmnn /- 细胞的增殖，尽管它们在体外无法存活。因此，geminin 对于 ESC 分化成多种细胞类型至关重要，但对于实体组织内分化细胞的生存能力却不是必需的。这些结果还提供了选择性抑制geminin功能可以将恶性生殖细胞癌转化为良性肿瘤的原理证明。