SUV39 SET domains mediate crosstalk of heterochromatic histone marks

Abstract

<jats:title>Abstract</jats:title><jats:p>The SUV39 class of methyltransferase enzymes deposits histone lysine 9 di- and trimethylation (H3K9me2/3), the epigenetic hallmark of constitutive heterochromatin, which serves as the central recruitment platform for the heterochromatic silencing machinery. How these enzymes are regulated to mark specific genomic regions as heterochromatic is not well understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast <jats:italic>S.pombe</jats:italic> and recent evidence suggests that ubiquitination of lysine 14 on H3 tail (H3K14) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remains to be determined. Here we present a structure-function approach to understanding how the H3K14ub mark stimulates Clr4 activity <jats:italic>in cis</jats:italic>. These results show that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby activates the enzyme by 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to a <jats:italic>clr4</jats:italic> deletion. Our work reveals a sensor for H3K14 ubiquitylation in the SET domain of Clr4, which mediates the licensing of heterochromatin formation by an epigenetic cross-talk. This sensor is also active in the human SUV39H2 enzyme.</jats:p>

The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin

“…Furthermore, ChIP-seq analysis shows that H3K9me3 levels are restored at all major heterochromatin domains in hht3-K9MK14R cells (Figure 2H). In contrast, hht1-K14R hht3-K9M cells are still defective in the silencing of otr::ura4 + , consistent with the fact that H3K14ub function in cis to regulate the activity of Clr4 on H3K9 (Oya et al, 2019;Stirpe et al, 2020) (Figures S1C and S1D).…”

“…We generated a recombinant Clr4 SET domain containing the F256A, F310A, and F427A mutations (3FA). That mutation strongly reduced the interaction between Clr4 and H3K14ub and has little effect on the structure of Clr4 ( Stirpe et al, 2020 ). Peptide pull-down assays, thermal-shift assays, and BLI, all demonstrate that the 3FA mutation strongly reduces the interaction between Clr4 and H3K9MK14ub, either in the presence or in the absence of SAM ( Figures 4B , 4C , S3D – S3F , S4B , and S4C ).…”

“…Hydrogen/deuterium exchange coupled to mass spectrometry analysis shows that the interaction between Clr4 and H3K14ub is Report ll OPEN ACCESS mediated by a hydrophobic surface of Clr4 that includes residues F256, F310, and F427 (Stirpe et al, 2020) (Figure 4A). We generated a recombinant Clr4 SET domain containing the F256A, F310A, and F427A mutations (3FA).…”

“…The recent identification of CLRC as an E3 ubiquitin ligase of H3K14ub and the association of H3K14ub with heterochromatin ( Oya et al, 2019 ; Stirpe et al, 2020 ) prompted us to check whether H3K14ub promotes the sequestration of Clr4 by H3K9M ( Figure 2A ). Interestingly, ChIP analyses show that Clr4 sequestration at pericentric repeats is abolished in rik1Δ hht3-K9M , raf1Δ hht3-K9M , and raf2Δ hht3-K9M cells ( Figure 2B ), suggesting that CLRC-mediated H3K14 ubiquitylation might indeed contribute to the sequestration of Clr4 at pericentric repeats.…”

“…Clr4 associates with an E3 ubiquitin ligase complex, composed of Cul4, Rik1, Raf1, and Raf2 ( Hong et al, 2005 ; Horn et al, 2005 ; Jia et al, 2005 ), to form CLRC. CLRC ubiquitylates histone H3K14 (H3K14ub), and H3K14ub is associated with H3K9me3 containing heterochromatin, but not H3K4me3 containing euchromatin ( Oya et al, 2019 ; Stirpe et al, 2020 ). We found that mutating either CLRC or H3K14 abolished the sequestration of Clr4 by H3K9M.…”