SETD3 regulates endoderm differentiation of mouse embryonic stem cells through its effect on the canonical Wnt signaling pathway activity
With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable  tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on  precise regulation of gene expression that requires coordination between transcription (TF) and  chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is  a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through  an shRNA screen, we previously identified SETD3 as an important factor in meso/endodermal  lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent  transcriptomic changes during endoderm differentiation of (mESCs) using time-course RNA-seq  analysis. We found that SETD3 is involved in timely activation of the endoderm-related gene network.  Canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence  of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in setd3?  mESCs coincident with a decrease in nuclear pool of the key TF ß-catenin level, though no change  was observed in its mRNA or total protein level. Furthermore, proximity ligation assay found an  interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by  stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC  culture conditions. Our results suggest that alterations in canonical Wnt pathway activity and  subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of  setd3? mESCs. Overall design: To investigate the function of SETD3 in the regulation of endoderm differentiation, we established an  SETD3 knock-out mouse embryonic stem cell (mESC) line using CRISPR-Cas9.  Then, we differentiated the wild-type and SETD3 knock-out mESCs to definitive endoderm for four  days, and performed RNA-sequencing with two mESC lines at four time points.