Essential genes are dominantly activated by single transcription factors

Abstract Cell viability depends on the precise expression of essential genes, which are controlled by CpG-island (CGI) promoters densely bound by transcription factors (TFs). This has led to the prevailing model that TFs cooperate to ensure ubiquitous expression. Here, using rapid and reversible single and combinatorial degradation in murine stem cells, we systematically dissect the regulatory interactions between five key TFs. We uncover an unexpectedly specific architecture in which regulatory dominance, rather than cooperation, is the prevailing mode, where individual TFs autonomously drive chromatin opening and gene activation at largely distinct promoters. Cooperative regulation occurs at a minority of sites with antagonistic or synergistic outcomes modulated by the interplay between nucleosome positioning and TF sensitivity to chromatin. This logic is recapitulated at synthetic sequences and reflected in human genetic variation. These findings reveal that single TFs dominantly activate distinct sets of CGI-linked genes, including essential genes, across development, homeostasis, and disease.