Molecular Genetics and Epigenetics of Development

发育的分子遗传学和表观遗传学

• 批准号: RGPIN-2014-04589
• 负责人: Varmuza, Susannah
• 金额: $ 3.86万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587899
• 关键词: Molecular; Genetics; Epigenetics; Development

Epigenetics is the molecular intersection of Nature and Nurture. It is a field of study that encompasses all eukaryotes, and involves analysis of chromatin structure and regulation during development and in response to environmental stimulus. Published work from my lab has revealed that the imprinted gene Sfmbt2, which encodes a chromatin protein in the Polycomb Group (PcG) of genes, is required for maintenance of the extraembryonic progenitor cell population, and consequently proper development of the placenta and yolk sac in mice. Embryos lacking a functional Sfmbt2 gene die by the middle of gestation due to placentation failure. The research program in my lab will investigate the epigenetic mechanisms involved in extraembryonic tissue development that are anchored by this chromatin protein. The first series of experiments is aimed at defining the SFMBT2 EPIGENOME in extraembryonic cells. This will be accomplished by initially performing ChIP-seq on extraembryonic cells, followed by experiments to confirm the regulation by SFMBT2. Regulation of potential targets will be assayed after manipulating SFMBT2 protein levels in extraembryonic cells, using tools developed in my lab. The data will be uploaded into the ENCODE database for comparison with other extraembryonic epigenetic marks. Interactors of SFMBT2 will be tested for co-occupancy at selected targets, or if reagents exist (ie ChIP grade Ab) by ChIP-seq. The second series of experiments will involve delineation of the SFMBT2 INTERACTOME. SFMBT2 protein, highly conserved from flies to mammals, possesses four MBT domains that bind to specific modified histone tails, a DUF (domain of unknown function), and a SAM/Pointed (SAM/PNT) domain capable of protein-protein interaction. Chromatin proteins typically act as parts of multimeric complexes. We plan to investigate the SFMBT2 interactome in extraembryonic tissues by immunoprecipitation of native complexes followed by mass spectrometry identification, focusing initially on TS cells. Good candidates will be tested for genome occupancy that overlaps with SFMBT2. The last series of planned experiments will involve analysis of the YOLK SAC PHENOTYPE in our Sfmbt2 knockout mice. Fetuses inheriting the mutant allele from their fathers die by mid-gestation with severely reduced placenta and often avascular yolk sac. This latter observation is puzzling because the yolk sac vasculature is derived from extraembryonic mesoderm, which does not express Sfmbt2. We hypothesize that the primitive endoderm portion of the yolk sac, which does express Sfmbt2, is defective in some kind of signalling function. Analysis of the well described Vegf signalling cascade indicates that it is intact, and that the defect in our mutants lies elsewhere. We will pursue this initially by comparing the transcriptomes of mutant and wild type yolk sacs. Follow-up experiments will involve testing the effect of altering SFMBT2 levels on expression of potential target genes. One observation we have made that will inform our interpretation of results is the retention of SFMBT2 protein on mitotic chromosomes, a hallmark of mitotic bookmarks.

表观遗传学是自然和养育的分子交叉。它是一个涵盖所有真核生物的研究领域，涉及染色质结构的分析和发育过程中的调节以及对环境刺激的反应。从我的实验室发表的工作表明，印记基因Sfmbt 2，它编码的多梳组（PcG）的基因中的染色质蛋白，是维持胚胎外祖细胞群，从而在小鼠胎盘和卵黄囊的正常发育所必需的。缺乏功能性Sfmbt 2基因的胚胎由于胎盘形成失败而在妊娠中期死亡。我实验室的研究项目将调查由这种染色质蛋白锚定的胚外组织发育所涉及的表观遗传机制。 第一系列实验旨在定义胚外细胞中的SFMBT 2表位基因组。这将通过最初对胚外细胞进行ChIP-seq，然后通过实验确认SFMBT 2的调节来实现。在操纵胚外细胞中的SFMBT 2蛋白水平后，将使用我实验室开发的工具分析潜在靶点的调节。这些数据将被上传到ENCODE数据库中，以与其他胚胎外表观遗传标记进行比较。将通过ChIP-seq检测SFMBT 2的相互作用物在选定靶标上的共占用率，或是否存在试剂（即ChIP级Ab）。 第二系列实验将涉及SFMBT 2相互作用的描述。SFMBT 2蛋白是一种从果蝇到哺乳动物高度保守的蛋白质，具有4个与特定修饰的组蛋白尾部结合的MBT结构域、1个DUF（domain of unknown function）和1个SAM/Pointed（SAM/PNT）结构域，能够与蛋白质相互作用。染色质蛋白通常作为多聚体复合物的一部分。我们计划通过免疫沉淀天然复合物，然后通过质谱鉴定，初步研究胚外组织中的SFMBT 2相互作用组，重点是TS细胞。将测试好的候选物与SFMBT 2重叠的基因组占有率。 最后一系列计划的实验将涉及我们的Sfmbt 2敲除小鼠中卵黄囊表型的分析。从父亲那里遗传了突变等位基因的胎儿在妊娠中期死亡，胎盘严重缩小，卵黄囊常常无血管。后一种观察结果令人困惑，因为卵黄囊脉管系统来自不表达Sfmbt 2的胚外中胚层。我们推测，原始内胚层部分的卵黄囊，这并不表达Sfmbt 2，是有缺陷的某种信号功能。对描述良好的VEGF信号级联的分析表明，它是完整的，并且我们的突变体中的缺陷位于其他地方。我们将通过比较突变型和野生型卵黄囊的转录组来追求这一点。后续实验将涉及测试改变SFMBT 2水平对潜在靶基因表达的影响。我们所做的一个观察将为我们对结果的解释提供信息，即SFMBT 2蛋白在有丝分裂染色体上的保留，这是有丝分裂书签的标志。