MPP8 is essential for sustaining self-renewal of ground-state pluripotent stem cells. MPP8 is essential for sustaining self-renewal of ground-state pluripotent stem cells

Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigentic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3. Overall design: MPP8, FLAG and H3K9me3 chromatin immunoprecipitation profiles (ChIP-seq) analyzed in mouse embryonic stem cells which have been engineered to homozygously express MPP8 carrying a C-terminal miniAID (mAID) tag from the endogenous Mphosph8 locus as well as the E3 ligase OsTIR1 exogenously (Mpp8mAID; OsTIR1 cells). Cells were left untreated or treated with auxin (IAA) for indicated time points to remove endogenous MPP8. MPP8, FLAG and H3K9me3 chromatin immunoprecipitation profiles (ChIP-seq) analyzed in the Mpp8mAID; OsTIR1 background additionally expressing FLAG-tagged MPP8 fragments, Mpp8wt, Mpp8112-858 and MPP81-729, respectively (wt corresponds to the full length protein sequence, Npart corresponds to amino acid 1-729, Cpart correspond to amino acid 112-858). Cells were left untreated or treated with auxin (IAA) for indicated time points to remove endogenous MPP8. FLAG ChIPSeq analysis in cells engineered to homozygously express MPP8, TASOR and PPHLN1 carrying a C-terminal FLAG2 tag at the endogenous Mphosph8, Tasor and Pphln1 loci, respectively, or untagged parental control. RNA-seq analysis in Mpp8mAID; OsTIR1 cells, and cells additionally expressing FLAG-tagged MPP8 fragments, Mpp8wt, Mpp8112-858 and MPP81-729, respectively. Cells were left untreated or treated with auxin (IAA) for indicated time points to remove endogenous MPP8.

解析调控多能基态的分子机制，是理解胚胎发育的核心要点。然而，目前学界对基态小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）的表观遗传调控机制尚未完全阐明。本研究鉴定发现，表观遗传蛋白MPP8是维持基态多能性的必需因子。MPP8的敲除会引发细胞周期阻滞与自发分化。既往研究提示，MPP8可通过将人类沉默枢纽（human silencing hub, HUSH）复合体招募至富含H3K9me3的染色质区域，从而抑制长散在核元件1（LINE1 elements）的表达。令人意外的是，本研究发现缺失染色质结构域（chromodomain）的MPP8仍可有效抑制LINE1元件，而未被注释的C端结构域却是其发挥功能的关键结构。此外，本研究证实SETDB1可将MPP8招募至其基因组靶位点，且维持LINE1元件的转录抑制并不依赖于H3K9me3水平的保留。综上，本研究结果表明，MPP8可通过与HUSH核心复合体结合，保护DNA低甲基化的多能基态，这一调控过程独立于可检测的染色质结合与H3K9me3的维持。

本研究的总体实验设计如下：
1. 对在内源Mphosph8基因座纯合表达带有C端miniAID（mAID）标签的MPP8、且外源表达E3连接酶OsTIR1的小鼠胚胎干细胞（命名为Mpp8mAID; OsTIR1细胞），分析MPP8、FLAG及H3K9me3的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）图谱。将细胞分为两组：一组不予任何处理，另一组用生长素（IAA）处理指定时长以清除内源MPP8。
2. 在Mpp8mAID; OsTIR1细胞背景中，分别额外表达FLAG标记的MPP8全长片段（Mpp8wt，对应全长蛋白序列）、Mpp8112-858（氨基酸112-858片段）及MPP81-729（氨基酸1-729片段），并对这三种工程化细胞的MPP8、FLAG及H3K9me3的ChIP-seq图谱进行分析。同样将细胞分为不予处理组与生长素（IAA）处理组，处理时长以清除内源MPP8。
3. 对分别在内源Mphosph8、Tasor及Pphln1基因座纯合表达带有C端FLAG2标签的MPP8、TASOR和PPHLN1的细胞，以及未带标签的亲本对照细胞，开展FLAG ChIP-seq分析。
4. 对Mpp8mAID; OsTIR1细胞，以及分别额外表达FLAG标记的Mpp8wt、Mpp8112-858及MPP81-729的工程化细胞，进行RNA-seq分析。将上述细胞同样分为不予处理组与生长素（IAA）处理组，处理时长以清除内源MPP8。