Transcription factor antagonism regulates heterogeneity in embryonic stem cell states

Gene expression heterogeneity underlies the formation of cell states, and switching between states contributes to developmental robustness. While gene expression variation can arise from stochastic transcriptional processes, whether it can be regulated and coordinated by an underlying program is unclear. We characterize the regulatory program underlying heterogeneity and switching between murine embryonic stem cell (mESC) states. Using nascent transcriptome assays, we identified a subset of differentially active and transcribed enhancers (DATEs) across states. DATEs regulate variably expressed gene targets and contribute to cell state formation. Further, DATEs are distinguished by co-binding of Kruppel-like transcription factors Klf4 and Zfp281. In contrast to other transcription factors that form a positive feedback network stabilizing ES cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of their ability to bind DATEs dampens variation in gene expression and reduces heterogeneity. Single cell analysis reveals competing Klf4 and Zfp281 gene programs that define mESC state, consistent with a model whereby Klf4 and Zfp281 generate transcriptional heterogeneity through functional antagonism at DATEs. These results show how antagonistic circuits of TFs and enhancers contributes to gene expression variation and cell state, with potential implications for the generation of diverse cell types during mammalian development. (1) Examination of nascent transcriptome by mPROseq in three mESC states defined by Nanog- and Sox2-defined subpopulations (States 1, 2, and 3) in three biological replicates; (2) Examination of nascent transcriptome by mPROseq in WT, Klf4KO, Zfp281KO mESC in three biological replicates; (3) Examination of steady state transcriptome by RNAseq in WT, Klf4KO, Zfp281KO mESC in nine biological replicates; (4) Examination of chromatin accessibility by ATACseq in WT, Klf4KO, Zfp281KO mESC in three biological replicates; (5) ChIPseq of Klf4 and Zfp281 in WT, Klf4KO, Zfp281KO mESC; (6) Single cell RNA sequencing of two biological replicates each of WT, Klf4KO, Zfp281KO mESC (7) CUT&RUN of Klf4 and Zfp281 in States 1, 2, and 3