Short activation domains control chromatin association of transcription factors

Abstract Transcription factors regulate gene expression with DNA-binding domains (DBDs) and activation domains. Despite mounting evidence to the contrary, it is frequently assumed that DBDs are solely responsible for interacting with DNA and chromatin. Here, we used single-molecule tracking of transcription factors in living cells to show that short activation domains can control the fraction of molecules bound to chromatin. Stronger activation domains have higher bound fractions and longer residence times on chromatin. Furthermore, mutations that increase activation domain strength also increase chromatin binding. This trend was consistent in four different activation domains and their mutants. This effect further held for activation domains appended to three different structural classes of DBDs. Stronger activation domains with high chromatin-bound fractions also exhibited increased binding to the p300 coactivator in proximity-assisted photoactivation experiments. Taken together, these results suggest that activation domains play a major role in tethering transcription factors to chromatin, challenging the traditional view that the DBD is the sole driver of genome binding. Competing Interest Statement X.D. is a co-founder of Eikon Therapeutics, Inc. Footnotes The manuscript has been extensively revised. The introduction has been completely rewritten. Many additional experiments have been added to almost all of the figures. There are 3 new figures. Data availability SMT trajectories (CSV files tabulating all detections surviving filtering steps) are available on Dryad (https://doi.org/10.5061/dryad.41ns1rnqt). We were unable to deposit underlying SMT movies due to their large size. FRAP movies are also available as part of the same Dryad dataset (https://doi.org/10.5061/dryad.41ns1rnqt). CUT&RUN data has been deposited to the Sequence Read Archive and is available as PRJNA1305492. ATAC-seq data from (Oomen et al., 2019) is available on the GEO as Series GSE121840.