Refined read‐out: The hUHRF1 Tandem‐Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3

UHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem‐Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1‐K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read‐out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H‐bond capacity of H3K4. Individual TTD mutants showed up to a 10,000‐fold preference for the double‐modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1‐K9me2 regions in human chromatin demonstrated in our TTD chromatin pull‐down and ChIP‐western blot data suggests that it has specific biological roles. Chromatin pull‐down of TTD from HepG2 cells and full‐length murine UHRF1 ChIP‐seq data correlate with H3K4me1 profiles indicating that the H3K4me1‐K9me2/3 interaction of TTD influences chromatin binding of full‐length UHRF1. We demonstrate the H3K4me1‐K9me2/3 specific binding of UHRF1‐TTD to enhancers and promoters of cell‐type‐specific genes at the flanks of cell‐type‐specific transcription factor binding sites, and provided evidence supporting an H3K4me1‐K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1‐TTD binding to H3K4me1‐K9me2/3 double marks in a cellular context.