Rinf regulates pluripotency network and Tet enzymes in embryonic stem cells (ESCs)

In this study: (1) We have mapped the genome wide binding distribution and enrichment of Rinf/CXXC5 at genes and regulatory regions in mouse ESCs by ChIP-seq using a specific antibody against Rinf. (2) We have examined the role of Rinf in regulation of ESC gene expression programs by performing transcriptomic analysis of Rinf wild type and knockout ESCs by RNA-seq to identify differentially expressed genes. (3) We have investigated the role of Rinf in differentiation and lineage specification programs of ESCs by analyzing the transcriptomic profile of wild type and Rinf–/– ESCs during differentiation to embroyoid bodies (EBs) at three time points (day 0, 3, 6). Overall design: Rinf ChIP-seq: It was performed on two independent wild type V6.5 mESC lines and one Rinf–/– ESC line (negative control) as previously described (Johnson et al., 2007). To ensure the specificity of the antibody, we also performed ChIP-seq in a Rinf–/– ESC line as a negative control. Briefly, ESCs were cultured on gelatin, harvested, crosslinked, lysed, sonicated and subjected to ChIP using an anti-Rinf/CXXC5 antibody (Cat#84546S, CST). 100bp paired end sequencing was performed at the Einstein Epigenomics core following their established protocols using Illumina HiSeq 2500 platform. Reads were mapped to the mouse genome (mm10) using the software Bowtie2 (VN: 2.2.3) with default (Langmead and Salzberg, 2012). The Rinf binding peaks were called with the software MACS2 using the input as controls and default parameters (Yong Zhang et al., 2008), with the final peaks called from the merged reads of the two biological replicates. Details of ChIP-seq and data analysis are described in the methods sections of the manuscript. The ChIP-seq analysis identified a total of 2,342 Rinf peaks that were mapped to promoters and gene bodies as well as distal regulatory elements and intergenic regions. We examined the role of Rinf in regulation of ESC gene expression programs by performing transcriptomic analysis of Rinf wild type and knockout ESCs by RNA-seq to identify differentially expressed genes. RNA-Seq of ESCs: Two independent ESC of each genotype (Wild type and Rinf KO) were cultured on feeders then pre-plated to remove feeders and seeded on gelatin overnight. Total RNA was extracted (Omega E.Z.N.A Total RNA kit), barcoded and used to prepare libraries. ERCC spike in controls were included. The libraries were subjected to 150 bp paired-end sequencing using Illumina Next Seq 500 platform at the Einstein Epigenomics core following their protocols. We generated ~25 million reads per sample. The reads were trimmed using trim galore (v 0.4.1, https://github.com/FelixKrueger/TrimGalore) to remove adapters and then mapped to mouse genome (mm10) by tophat software (v 2.0.13) with default parameters (D. Kim et al., 2013). Details of RNA-seq and data analysis are described in the methods sections of the manuscript. RNA-seq of EBs: Two independent ESC line of each genotype (Wild type and Rinf KO) were differentiated to EBs following standard hanging drop methods (described in manuscript) and total RNA was isolated (Omega E.Z.N.A Total RNA kit) at three time points (day 0, 3, 6) of differentiation (total of 12 RNA samples). Samples were barcoded and used to prepare libraries. ERCC spike in controls were included. Libraries were subjected to 75bp single-end sequencing using Illumina Next-Seq 500 platform at Einstein Epigenomics core following their protocols. We generated ~30 million reads per sample. The reads were trimmed using trim galore (v 0.4.1, https://github.com/FelixKrueger/TrimGalore) to remove adapters and then mapped to mouse genome (mm10) by tophat software (v 2.0.13) with default parameters (D. Kim et al., 2013). Data analysis and identification of DEGs between the two genotypes for each time point are explained in detail in the manuscript methods section. Details of RNA-seq and data analysis are described in the methods sections of the manuscript.

本研究包含三项核心工作：(1) 利用针对Rinf的特异性抗体开展染色质免疫共沉淀测序（ChIP-seq），绘制了小鼠胚胎干细胞（mouse ESCs）中Rinf/CXXC5在基因及调控区域的全基因组结合分布与富集特征。(2) 分别对Rinf野生型与基因敲除型小鼠胚胎干细胞进行转录组测序（RNA-seq），分析Rinf对胚胎干细胞基因表达程序的调控作用，以鉴定差异表达基因。(3) 选取胚胎干细胞向拟胚体（embryoid bodies, EBs）分化过程中的三个时间节点（第0、3、6天），对野生型与Rinf基因敲除型胚胎干细胞的转录组谱进行分析，探究Rinf在胚胎干细胞分化及谱系特化程序中的功能。

实验整体设计：
1. Rinf ChIP-seq：参照此前报道（Johnson等，2007），本实验设置两株独立的野生型V6.5小鼠胚胎干细胞系，以及一株Rinf基因敲除型胚胎干细胞系作为阴性对照。为验证抗体特异性，我们额外以Rinf基因敲除型胚胎干细胞系作为阴性对照开展ChIP-seq实验。简要实验流程如下：将胚胎干细胞培养于明胶包被的培养底物上，收集细胞后进行交联、裂解、超声破碎，随后使用抗Rinf/CXXC5抗体（货号：84546S，CST公司）进行ChIP实验。后续在爱因斯坦表观基因组学核心实验室按照标准操作流程，使用Illumina HiSeq 2500平台进行100 bp双端测序。测序reads使用Bowtie2软件（版本：2.2.3）默认参数比对至小鼠基因组mm10版本（Langmead和Salzberg，2012）。使用MACS2软件以输入样本为对照、默认参数进行Rinf结合峰的识别，最终结合峰由两个生物学重复的合并reads鉴定得到。ChIP-seq实验及数据分析的详细步骤参见论文的材料与方法部分。本研究通过ChIP-seq共鉴定到2342个Rinf结合峰，这些峰分布于启动子区域、基因本体、远端调控元件及基因间区。

2. 胚胎干细胞RNA-seq：分别培养两株独立的野生型与Rinf基因敲除型胚胎干细胞，使用饲养层细胞培养，随后通过预铺板去除饲养层细胞，接种至明胶包被的培养板中过夜培养。使用Omega E.Z.N.A总RNA提取试剂盒提取总RNA，对RNA进行条形码标记后构建测序文库，并加入ERCC外部RNA标准对照（ERCC spike-in controls）。文库在爱因斯坦表观基因组学核心实验室按照标准流程，使用Illumina NextSeq 500平台进行150 bp双端测序，每个样本平均产出约2500万条reads。使用Trim Galore软件（版本：0.4.1，https://github.com/FelixKrueger/TrimGalore）对reads进行质控修剪以去除接头序列，随后使用Tophat软件（版本：2.0.13）默认参数比对至小鼠基因组mm10版本（D. Kim等，2013）。RNA-seq实验及数据分析的详细步骤参见论文的材料与方法部分。

3. 拟胚体RNA-seq：分别培养两株独立的野生型与Rinf基因敲除型胚胎干细胞，参照标准悬滴法将其诱导分化为拟胚体（详细步骤参见论文），并在分化的第0、3、6天三个时间点收集样本，使用Omega E.Z.N.A总RNA提取试剂盒提取总RNA（共12个RNA样本）。对样本进行条形码标记后构建测序文库，并加入ERCC外部RNA标准对照。文库在爱因斯坦表观基因组学核心实验室按照标准流程，使用Illumina NextSeq 500平台进行75 bp单端测序，每个样本平均产出约3000万条reads。使用Trim Galore软件（版本：0.4.1，https://github.com/FelixKrueger/TrimGalore）对reads进行质控修剪以去除接头序列，随后使用Tophat软件（版本：2.0.13）默认参数比对至小鼠基因组mm10版本（D. Kim等，2013）。针对每个时间点的两种基因型样本间差异表达基因的鉴定及数据分析细节，均参见论文的材料与方法部分。