Conserved roles for murine mDUX and human DUX4 in activating cleavage stage genes and MERVL/HERVL retrotransposons [RNA-Seq Mouse siRNA]

To better understand transcriptional regulation during human oogenesis and pre-implantation embryonic development, we defined stage-specific transcription, which revealed cleavage stage as highly distinctive. We present multiple lines of evidence that two cleavage-specific homologs, mouse mDUX and human DUX4, each activate hundreds of cleavage-specific endogenous genes (e.g. ZSCAN4, ZFP352, KDM4E) and retroviral elements (MERVL/HERVL-family). Remarkably, mDux expression converts mouse ESCs into two-cell embryo-like (2C-like) cells by binding to MERVL promoters/enhancers and restoring the chromatin landscape (via ATACseq) to the pattern of mouse two-cell embryos We used siRNAs to transiently deplete mESCs of Chaf1a alone and in combination with mouse Dux (mDux). After two consecuitive days of depletion, RNA was collected (2 biological replicates/condition) for sequencing. 125bp libraries were prepped and sequenced in paired-end format. All sequencing was done on the Illumina HiSeq 2500 platform. Chaf1a silencer select siRNA (and negative control siRNA) was purchased from Life Technologies (s77588). mDux siRNAs (si308 and si309) were synthesized as siRNA pools using Giardia Dicer as described below. Briefly, primers were designed to amplify two ~400bp fragments of the endogenous mDux locus from genomic mouse DNA and add T7 handles. mDux (si308) 5'-AACTCCTCCTCCTTGATCAACTG -3', 5'-CTTCTCTCTGTGGCCAAAAGC-3'. mDux (si309) 5'-CTTCTGCAGAGAGTCCCAGAC-3', 5'-GGCAGATCAGGTGTTGTGTC-3'. Purified PCR products were then used as template for in vitro transcription using the MEGAscript® T7 Transcription Kit (ThermoFischer, AM1334). Template DNA was then degraded and the ssRNA was allowed to anneal before dicing. Diced siRNAs were purified using the PureLinkTM Micro-to-Midi Total RNA purification Kit (Invitrogen, 12183-018) with modifications. siRNA concentration was measured with the Qubit® RNA HS Assay Kit (ThermoFisher, Q32852).

为深入解析人类卵子发生（oogenesis）与植入前胚胎发育过程中的转录调控机制，本研究对阶段特异性转录谱进行了系统界定，结果显示卵裂期（cleavage stage）具有极高的独特性。本研究提供多维度证据表明，两种卵裂期特异性同源因子——小鼠mDUX与人类DUX4，均可激活数百个卵裂期特异性内源基因（如ZSCAN4、ZFP352、KDM4E）以及逆转录病毒元件（MERVL/HERVL家族）。值得注意的是，通过结合MERVL启动子/增强子并将染色质景观（通过ATAC测序（ATACseq））重塑至小鼠二细胞胚胎的模式，mDux的表达可将小鼠胚胎干细胞（embryonic stem cells, ESCs）转化为类二细胞胚胎细胞。本研究使用小干扰RNA（siRNAs）分别单独、以及联合小鼠Dux（mDux）对小鼠ESCs中的Chaf1a进行瞬时敲低。在连续两天的敲低处理后，收集各条件下的2个生物学重复样本的RNA用于测序。构建125bp长度的测序文库，并采用双端测序（paired-end format）策略进行测序。所有测序实验均在Illumina HiSeq 2500平台上完成。Chaf1a沉默筛选用siRNA及阴性对照siRNA购自Life Technologies（货号s77588）。mDux特异性siRNA（si308与si309）以贾第虫Dicer（Giardia Dicer）为工具合成siRNA混合池，具体流程如下文所述。简言之，我们设计引物从小鼠基因组DNA中扩增内源mDux基因座的两个约400bp的片段，并在末端添加T7接头（T7 handles）。针对si308的引物序列为：5'-AACTCCTCCTCCTTGATCAACTG-3'、5'-CTTCTCTCTGTGGCCAAAAGC-3'；针对si309的引物序列为：5'-CTTCTGCAGAGAGTCCCAGAC-3'、5'-GGCAGATCAGGTGTTGTGTC-3'。纯化后的PCR产物作为模板，使用MEGAscript® T7体外转录试剂盒（ThermoFischer，货号AM1334）进行体外转录。随后降解模板DNA，将单链RNA（ssRNA）退火，再进行酶切处理。酶切得到的siRNA使用PureLink™ Micro-to-Midi总RNA纯化试剂盒（Invitrogen，货号12183-018）并稍作修改进行纯化。使用Qubit® RNA高灵敏度检测试剂盒（ThermoFisher，货号Q32852）测定siRNA的浓度。