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We investigated how interactions between pluripotency transcription factors (TFs) affect cis-regulation. We created hundreds of synthetic cis-regulatory elements (CREs) comprised of combinations of binding sites for pluripotency TFs and measured their expression in mouse embryonic stem (ES) cells. A thermodynamic model that incorporates interactions between TFs explains a large portion (72%) of the variance in expression of these CREs. These interactions include three favorable heterotypic interactions between TFs. The model also predicts an unfavorable homotypic interaction between TFs, helping to explain the observation that homotypic chains of binding sites express at low levels. We further investigated the expression driven by CREs comprised of homotypic chains of KLF4 binding sites. Our results suggest that KLF homologs make unique contributions to regulation by these CREs. We conclude that a specific set of interactions between pluripotency TFs plays a large role in setting the levels of expression driven by CREs in ES cells.

Interactions between pluripotency factors specifycis-regulation in embryonic stem cells