SETDB1 activity is globally directed by H3K14 acetylation via its Triple Tudor Domain

SETDB1 is a major H3K9 methyltransferase involved in heterochromatin formation and silencing of repeat elements. It contains a unique Triple Tudor Domain (3TD) which specifically binds the dual modification of H3K14ac in the presence of H3K9me1/2/3. Here, we explored the role of the 3TD H3-tail interaction for the H3K9 methylation activity of SETDB1. We generated a binding reduced 3TD mutant and demonstrate in biochemical methylation assays on peptides and recombinant nucleosomes containing H3K14ac analogs, that H3K14 acetylation is crucial for the 3TD mediated recruitment of SETDB1. We also observe this effect in cells where SETDB1 binding and activity is globally correlated with H3K14ac, and KO of the H3K14 acetyltransferase HBO1 causes a drastic reduction in H3K9me3 levels at SETDB1 dependent sites. Further analyses revealed that 3TD particularly important at specific target regions like L1M repeat elements, where SETDB1 KO cannot be efficiently reconstituted by the 3TD mutant of SETDB1. In summary, our data demonstrate that the H3K9me3 and H3K14ac are not antagonistic marks but rather the presence of H3K14ac is required for SETDB1 recruitment via 3TD binding to H3K9me1/2/3-K14ac and establishment of H3K9me3.