CORE PLURIPOTENCY FACTORS AND SMAD SIGNALING ORCHESTRATE HUMAN EMBRYONIC STEM CELL DIFFERENTIATION

OCT4, NANOG, and SOX2 (abbreviated OSN) are the core transcription factors (TFs) regulating pluripotency human embryonic stem cells (hESCs). Although their basic importance in maintaining self-renewal and reprogramming has been demonstrated, their mechanistic functions in differentiation are not well studied. To address this question, we report the integrative genome-wide chromatin accessibility and TF binding data with extensive transcriptomic and epigenomic data across the differentiation of hESC to the three germ layers. Using ATAC-seq (the assay for transposase-accessible chromatin using sequencing) technique, we identify 12,3483 transposase-accessible DNA elements. Integration of ATAC-seq with multi-omics data identifies a substantial number of putative distal enhancers that distinguish cell fate commitments. These data reveal regulatory TF binding motifs, validated by the DNA occupation by respective TF from in-house and published ChIP-seq dataset. For example, SMAD1, HAND1 for mesoderm (Meso), SMAD2/3, GATA6 for definitive endoderm (DE), SOX2 for neural ectoderm (Ecto). To our surprise, Active enhancers are densely occupied by OSN not only in hESCs, but also in early differentiated cells, in a motif independent manner.The co-binding of OSN with extracellular signaling effectors, SMAD1 and SMAD2/3, are in a cell fate specific manner. In hESCs, OSN individually interact with each other and with SMAD1, SMAD2/3. Upon differentiation, interactions within OSN decrease, whereas Activin-A activated SMAD2/3 specifically interacts with NANOG in DE, but not in Meso or Ecto Similarly, BMP4 activated SMAD1 specifically interacts with OCT4 in Meso, but not in DE or Ecto. During Ecto differentiation, dual SMAD signaling inhibition enables SOX2 to release from the OSN-SMAD complex and to re-distribute onto Ecto-specific enhancers. In addition, OSN mediate higher-order chromatin remodeling in respective cell fate. Our studies provide a new model of how OSN function as cell fate specifiers in collaboration with SMAD signaling. INDEX