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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611127
• 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.

表观遗传学是先天和后天的分子交集。它是一个涵盖所有真核生物的研究领域，涉及分析染色质结构和发育过程中的调控以及对环境刺激的反应。我的实验室发表的工作表明，印迹基因Sfmbt2是维持小鼠胚胎外祖细胞群体，从而使胎盘和卵黄囊正常发育所必需的，它编码基因的多梳群(PcG)中的一种染色质蛋白。缺乏功能性Sfmbt2基因的胚胎会因胎盘形成失败而在妊娠中期死亡。我实验室的研究计划将调查这种染色质蛋白锚定的胚外组织发育所涉及的表观遗传学机制。 第一系列实验旨在确定胚胎外细胞中的SFMBT2表观基因组。这将通过首先对胚胎外细胞进行CHIP-SEQ，然后通过实验确认SFMBT2的调节来实现。在操纵胚胎外细胞中的SFMBT2蛋白水平之后，将使用我实验室开发的工具来分析潜在靶点的调节。这些数据将被上传到ENCODE数据库中，以便与其他胚外表观遗传标记进行比较。SFMBT2的交互作用装置将通过CHIP-SEQ测试选定目标的共占性，或者是否存在试剂(即芯片级AB)。 第二系列实验将涉及描绘SFMBT2相互作用组。SFMBT2蛋白在果蝇和哺乳动物中高度保守，具有4个结合特定修饰的组蛋白尾巴的MBT结构域、一个功能未知的结构域(DUF)和一个能够与蛋白质相互作用的SAM/POINT(SAM/PNT)结构域。染色质蛋白通常作为多聚体复合体的一部分。我们计划通过天然复合体的免疫沉淀和质谱学鉴定来研究胚胎外组织中的SFMBT2相互作用组，最初的重点是TS细胞。优秀的候选者将接受与SFMBT2重叠的基因组占有率测试。 计划中的最后一系列实验将包括分析我们的Sfmbt2基因敲除小鼠的卵黄囊表型。从父亲那里继承突变等位基因的胎儿在妊娠中期死亡，胎盘严重减少，通常是无血管卵黄囊。后一种观察结果令人费解，因为卵黄囊的血管系统来自胚外中胚层，而中胚层不表达Sfmbt2。我们推测卵黄囊中表达Sfmbt2的原始内胚层部分在某种信号功能上是有缺陷的。对已被很好描述的血管内皮生长因子信号级联的分析表明，它是完整的，我们突变体中的缺陷存在于其他地方。我们将通过比较突变型和野生型卵黄囊的转录本来初步探索这一点。后续实验将包括测试改变SFMBT2水平对潜在靶基因表达的影响。我们所做的一个观察将告诉我们对结果的解释是SFMBT2蛋白保留在有丝分裂染色体上，这是有丝分裂书签的标志。