Oct1 cooperates with Smad transcription factors to promote mesodermal lineage specification [ChIP-seq]

The pathways used by cells to transition from undifferentiated, pluripotent gene expression programs into cell type-specific gene expression programs are incompletely understood. Here we show that the transcription factor Oct1/Pou2f1 recruits histone lysine demethylase complexes to allow for correct induction of silent, developmental lineage-specific genes and “canalize” developmental progression. Using mesodermal differentiation of inducible-conditional Oct1 knockout embryonic stem cells and single-cell gene expression profiling, we show that the potential to progress efficiently through mesodermal development is impaired in the Oct1 deficient condition. Oct1 deficient cells fail to form late presomitic mesoderm and early somite stage populations, and show “leaky” developmental trajectories with inappropriate lineage branching and accumulation of poorly differentiated cells that retain gene expression and metabolic hallmarks of pluripotency. Oct1 directly binds and regulates genes critical for developmental regulation, including genes encoding mesoderm-specific master regulators and components of chromatin regulatory complexes. Cells lacking Oct1 fail to positively resolve gene bivalency and activate gene expression by removing inhibitory H3K27me3 chromatin marks at mesoderm-specific genes. The Oct1 protein interacts with and recruits UTX to lineage-specific bivalent/poised targets, explaining the failure of Oct1 deficient cells to remove H3K27me3. Ectopic Oct1 expression improves the ability of cells to differentiate accurately under mesoderm lineage-inducing conditions. Parental and cKO ESCs are differentatiate toward mesodermal and neuro-ectodermal linegaes (RA) and profiled using ChIPseq. For mesodermal differentiation, depleated ESCs were plated on 0.1% gelatin and diffrentiated as described in Chal et al., 2015.Briefly, cells were differentiated in N2B27medium supplemented with Bmp4 for 2 days, and then switched to DMEM based medium supplemented with Rspo3 and LDN-193189 for 4 days.. For RA differentiation, depleated ESCs were plated on 0.01% gelatin and differentited using DMEM supplemented with 1.5ug/ml final retionoic acid, NEAA, L-glutamine, Pen/Strep.