Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability

Abstract

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H3. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me-which would lead to aberrant gene silencing and epigenetic instability-is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue suggest that the mechanism described here is broadly conserved.

Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

“…Contrasting with prior findings [51] we find that Suv39/Clr4 H3K9 methylation catalysis is directly inhibited by Set1 products, most strongly by H3K4me3. This finding represents a rare example of direct regulation of the Suv39/Clr4 SET domain active site, beyond auto-inhibition [70], but is consistent with the effect H3K4me can have on other H3K9 methylases [52][53][54]. The flipside of an orientation sensitive gene barrier is weaker effectiveness in the other orientation.…”

C. elegansnuclear RNAi factor SET-32 deposits the transgenerational heritable histone modification, H3K23me3

“…Plasmids sequences were confirmed by Sanger sequencing. pGEX-6P-1-GST-Clr4 was a gift from the Danesh Moazed 42 .…”

“…The procedure for protein expression and purification was adapted from 42 . Briefly, E. coli BL21-Gold (DE3) that was transformed with the expression plasmid was cultured in 2xYT at 37°C until OD600 reached 0.6-0.8, followed by incubating on ice for 30 min, and then 30 mins of 18°C in shaker before IPTG induction.…”

“…The HMT assay was performed as described in 42 . Briefly, the [3H]-based HMT assay was carried out in a 10 µl reaction mix containing 2 µM histone or nucleosome, 5.6 µM [3H]-S-adenosyl methionine (SAM), 0.3-1 µM enzyme, and 1x HMT buffer (50 mM Tris-HCl, pH 8.0, 20 mM KCl, 10 mM MgCl 2 , 0.02% Triton X-100, 1 mM DTT, 5% glycerol, and 1 mM PMSF).…”

A non-enzymatic function associated with a putative histone demethylase licenses epigenetic inheritance

“…These conflicting lines of biochemical and genetic data have prompted several alternative explanations for how Epe1 might exert its anti-silencing function in cells. This includes the possibility that Epe1 acts as a protein hydroxylase that targets non-histone proteins such as Swi6 HP1 , regulates the activity of the multisubunit H3K9 methyltransferase CLRC complex, or functions as an H3K9 demethylase when in complex with Swi6 HP1 (Aygun et al, 2013;Iglesias et al, 2018;Trewick et al, 2007;Zofall and Grewal, 2006). Although these models represent attractive possibilities for how Epe1 regulates heterochromatin, there is no direct evidence to suggest that any of these proteins represent bonafide enzymatic targets.…”