Pioneer activity distinguishes activating from non-activating SOX2 binding sites

Genome-wide transcriptional activity involves the binding of many transcription factors to thousands of sites in the genome. Determining which sites are directly driving transcription remains a challenge. Here we use acute protein depletion of the pioneer transcription factor SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of pioneer factors. To understand the relationship with transcription we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to all other SOX2 binding sites. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted largely at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.