Unique Trophoblast Chromatin Environment Mediated by the PcG Protein SFMBT2 [RNA-seq]

Sfmbt2 is a paternally imprinted gene critical for the establishment and maintenance of trophoblast stem cells in mice (Miri K et al., 2013). Inheritance of a paternal Sfmbt2 gene trap null allele (Sfmbt2gt/gt) results in embryonic lethality by e12.5 due to reduced numbers in all trophoblast cell types. Of note, SFMBT2 is characterized as a Polycomb group (PcG) protein and is assumed to act as a master transcriptional repressor. Through RNA-seq analysis of extraembryonic tissues acquired from both wild-type and Sfmbt2gt/gt embryos, we aim to elucidate differentially expressed genes critical for the dramatic phenotype observed in mutant embryos. Sfmbt2gt/gt embryos were generated by intercrossing heterozygous Sfmbt2gt males and females. Embryos were extracted at e7.5 and individually dissected into embryo and extraembryonic (i.e. ectoplacental cone and extraembryonic ectoderm) portions. The embryonic portion was used for genotyping, and total RNA was extracted from extraembryonic portions of both wild-type (+/+) and mutant (gt/gt) embryos using Trizol. Total RNA from pools of 12 embryos was extracted and processed by The Centre for Applied Genomics (TCAG) at The Hospital for Sick Children in Toronto. The RNA-seq library was generated using the Illumina TruSeq protocol and was subject to paired-end next-generation sequencing on the Illumina HiSeq 2500 platform. Two biological replicates of each were analyzed.Sfmbt2是一种父本印记基因（paternally imprinted gene），在小鼠滋养层干细胞的建立与维持过程中发挥关键调控作用（Miri K等，2013）。父本Sfmbt2基因陷阱纯合无效等位基因（Sfmbt2gt/gt）的遗传会导致胚胎在e12.5阶段致死，原因是所有滋养层细胞类型的细胞数量均显著减少。值得注意的是，SFMBT2属于多梳家族（Polycomb group, PcG）蛋白，被认为是一类主转录抑制因子。本研究通过对野生型与Sfmbt2gt/gt胚胎的胚外组织开展RNA测序（RNA-seq）分析，旨在阐明与突变胚胎中观测到的显著表型相关的差异表达基因。Sfmbt2gt/gt胚胎通过杂合子Sfmbt2gt雌雄小鼠交配获得。研究人员于e7.5阶段提取胚胎，并将每个胚胎分别解剖为胚胎本体与胚外组分（即胚外胎盘锥ectoplacental cone与胚外外胚层extraembryonic ectoderm）。胚胎本体用于基因型鉴定，研究人员使用Trizol试剂从野生型（+/+）与突变型（gt/gt）胚胎的胚外组分中提取总RNA。将12枚胚胎混合样品的总RNA提取完成后，交由加拿大多伦多病童医院应用基因组学中心（The Centre for Applied Genomics, TCAG）进行文库构建与测序处理。采用Illumina TruSeq试剂盒构建RNA测序文库，并在Illumina HiSeq 2500平台上开展配对末端下一代测序。每个组别均设置两个生物学重复进行分析。