BioProject

Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. More...

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. Less...