SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization

To detect the direct target genes of SOX4 in differentiated human endometrial stromal cells(hEnSC), differentiated hEnSC cells at the presence of MPA, E2 and cAMP are collected and subjected to ChIP-Seq. After aligned to human Hg38 by STAR, peaks are called by MACS2. Our results show that SOX4 is the key regulator for decidualization by regulating FOXO1, STAT3, PRL and FOSL2. This specific enrichment is confirmed by ChIP-qPCR. Additionally, there are also some other direct genes of SOX4, indicating the essential role of SOX4 in decidualization. This ChIP-Seq data provides fundamental information for our further physiological study of SOX4. ChIP was performed according to the reported previousl with slight modification. Briefly, about 2x106 hEnSC cells were cross-linked with 1/15 volume of 16% formaldehyde (CST) at room temperature for 10 minutes and quenched with 1/10 volume of 1.25 M glycine for 15 minutes on ice. Cell lysate in lysis buffer III were sonicated using Bioruptor pico (Diagenode) and then incubated with 4 µg antibody overnight at 4℃ with rotation. Immunoprecipitated complexes were collected with 15 µl Protein A Dynabeads (Invitrogen, 10002D) for 1 hour at 4℃ with rotation. Subsequently, beads were washed sequentially once with low-salt buffer, twice with high-salt buffer, once with LiCl buffer, twice with TE, and then eluted in 400 µl of elution buffer for 30 min at 65℃. The eluates were incubation at 65℃ for 8 h to reverse the cross-linking. Next, eluates were treated with proteinase K for 1 h at 55℃ and then RNase A for 30 min at 37℃ before DNA was extracted and purified. The ChIP libraries were prepared using KAPA HyperPrep Kits (Roche, 07962347001) and then run on the Illumina sequencer Hiseq-Xten PE150.

为鉴定分化态人子宫内膜基质细胞（human endometrial stromal cells, hEnSC）中SOX4的直接靶基因，本研究收集了经MPA、E2及cAMP处理的分化态hEnSC细胞，开展染色质免疫共沉淀测序（ChIP-Seq）实验。测序读段（reads）经STAR软件比对至人类Hg38参考基因组后，通过MACS2进行峰调用。研究结果表明，SOX4通过调控FOXO1、STAT3、PRL与FOSL2，成为蜕膜化过程的关键调控因子。该特异性富集现象经染色质免疫共沉淀定量PCR（ChIP-qPCR）验证。此外，本研究还发现了其他若干SOX4的直接靶基因，提示SOX4在蜕膜化进程中发挥不可或缺的核心作用。本套ChIP-Seq数据为后续开展SOX4的生理学相关研究提供了基础支撑。本实验的ChIP操作参照已发表的经典方案并略作修改。具体而言，取约2×10⁶个hEnSC细胞，于室温下用1/15体积的16%甲醛（CST）交联10分钟，随后加入1/10体积的1.25 M甘氨酸置于冰上淬灭反应15分钟。将细胞重悬于裂解缓冲液III中，使用Bioruptor Pico（Diagenode）进行超声破碎；之后于4℃旋转条件下，加入4 μg特异性抗体孵育过夜。使用15 μl Protein A Dynabeads（Invitrogen, 10002D）于4℃旋转孵育1小时，以收集免疫沉淀复合物。随后依次用低盐缓冲液洗涤1次、高盐缓冲液洗涤2次、LiCl缓冲液洗涤1次、TE缓冲液洗涤2次；之后加入400 μl洗脱缓冲液，于65℃孵育30分钟以洗脱结合的DNA片段。将洗脱液置于65℃孵育8小时以逆转交联反应。接下来，于55℃用蛋白酶K处理洗脱液1小时，再于37℃用核糖核酸酶A（RNase A）处理30分钟，随后提取并纯化DNA。使用KAPA HyperPrep Kits（Roche, 07962347001）构建ChIP文库，随后在Illumina测序仪Hiseq-Xten PE150上进行双端150 bp测序。