Tranposable Element Control by TRIM28 Safeguards PGC Sex Determination Program [RNA-Seq]

Transposable elements are increasingly appreciated as regulatory elements in early mammalian development, especially in cells which exhibit pluripotency. Mammalian primordial germ cells (PGCs) must exit latent pluripotency in a process termed determination before they are competent to differentiate and enter sex-specific developmental programs. Here, we find that determination is marked by large changes to the transposable element (TE) repertoire, a phenomenon driven by TRIM28, after which testicular and ovarian PGCs exhibit district transposable element repertoires. We find that loss of TRIM28 perturbs entry into determination as marked by expression of DAZL in a sex-specific manner, but does not result in a failure to downregulate markers of latent pluripotency, including SOX2 and NANOG in either sex. Despite downregulation of some early PGC markers, both ovarian and testicular PGCs fail to properly enter into their meiotic germ cell or prospermatogonial programs, respectively. Thus, we show that TRIM28-mediated reorganization of the TE repertoire is necessary to establish a PGC epigenome competent for proper germline commitment and gametogenesis. Prdm1-cre, Trim28 delta/flox, males were paired with Trim28 flox/flox, oct4-eGFP females for time mated breeding. Day of plug was marked as E0.5. Resulting embryos were genotyped and control (prdm1-cre -, trim28 +/flox) and TRIM28 knockout (prdm1-cre +, Trim28 delta/delta) kept for library preparation. At the dissected end point, embryos were removed and the embryonic gonads dissected and dissociated before isolation of PGCs via FACS.转座因子（Transposable Element, TE）在哺乳动物早期发育过程中的调控作用日益受到重视，尤其在具有多能性的细胞中。哺乳动物原始生殖细胞（primordial germ cell, PGC）需在被称为“定型”的过程中退出潜伏多能性状态，随后才具备分化并进入性别特异性发育程序的能力。本研究发现，PGC定型过程伴随转座因子组的显著重塑，这一现象由TRIM28介导；定型完成后，睾丸与卵巢来源的PGC展现出截然不同的转座因子组特征。我们发现，TRIM28缺失会以性别特异性方式干扰PGC定型过程（以DAZL的表达作为定型标记），但并不会阻碍两类性别PGC下调潜伏多能性标记物，包括SOX2与NANOG。尽管部分早期PGC标记物得以下调，但卵巢与睾丸来源的PGC仍分别无法正常进入减数分裂生殖细胞程序与精原前体细胞发育程序。综上，本研究证实，TRIM28介导的转座因子组重塑，对于建立可支持正常生殖系定型与配子发生的PGC表观基因组至关重要。实验繁育方案如下：将携带Prdm1-cre、Trim28 delta/flox的雄性小鼠，与Trim28 flox/flox、oct4-eGFP的雌性小鼠进行定时交配繁育。以阴栓检出日定为胚胎发育第0.5天（E0.5）。对所得胚胎进行基因型鉴定，选取对照个体（prdm1-cre -，trim28 +/flox）与TRIM28敲除个体（prdm1-cre +，Trim28 delta/delta）用于文库制备。在预定解剖终点处，取出胚胎并分离胚胎性腺，经细胞解离后通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）纯化得到PGC。