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.

表观遗传学是先天与后天的分子交叉。这是一个涵盖所有真核生物的研究领域，涉及在发育过程中以及对环境刺激的反应中染色质结构和调控的分析。我的实验室发表的研究表明，印迹基因Sfmbt2编码多梳组（Polycomb Group, PcG）基因中的一种染色质蛋白，它是维持胚胎外祖细胞群以及小鼠胎盘和卵黄囊正常发育所必需的。缺乏功能性Sfmbt2基因的胚胎在妊娠中期由于胎盘失败而死亡。我实验室的研究项目将研究由这种染色质蛋白锚定的胚胎外组织发育的表观遗传机制。