The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis

Abstract

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.

Figure 1. The WIYLD domain is a ubiquitin-binding domain.

(A) Layout of the SUVR4 full-length protein, showing the different domains/motifs and the regions included in the two constructs SUVR4-WIYLD and SACSET. SP, SUVR pre-SET; I, pre-SET I; II, pre-SET II; SET, SET domain. Black box indicates the post-SET domain .The amino acid sequence of SUVR4 from N₂₅ to K₈₃ encompassing the WIYLD domain, with conserved residues shaded in black, and residues mutated in this work indicated with arrowheads. (B) Yeast two-hybrid interaction test between SUVR4-WYILD and the full-length CDS of UBQ1, as well as the N-terminal ubiquitin (Ub) and the C-terminal ribosomal L40 moieties of UBQ1. –L/-T – medium selective for diploid colonies; -L/-T/-H +3 AT – medium selective for protein-protein interactions. AD, control mating with empty prey vector; BD, control matings with empty bait vector. (C) GST-SUVR4-WIYLD immobilized on glutathione sepharose beads were used to pull down MBP, MBP-UBQ1, MBP-Ub or MBP-L40 from bacterial lysate. Pull-down reactions were separated on SDS-PAGE, blotted onto a PVDF membrane and detected with an anti-MBP antibody. IN, input (5%); GST, GST negative control. (D) [¹H,¹⁵N]-HSQC spectrum of SUVR4-WIYLD in its free form (black), and after the addition of excess ubiquitin to a molar ratio of 1∶3 (red). The assigned amino acid residues are indicated. (E) GST pull-down of H2Bub1. GST-SUVR4-WIYLD was mutated at positions D74, R37 and W61 and used for GST pull-down of core histones from calf thymus. The pull down reactions were blotted onto a PVDF membrane and probed with an antibody against ubiquitinylated H2B (H2Bub1).

Figure 2. SUVR4 HKMTase activity is stimulated by free ubiquitin in vitro.

(A) HKMTase assay on core histones using a construct encompassing the SACSET domain of SUVR4 or the full-length SUVR4 protein without and with the addition of free ubiquitin. (B) Quantification of band intensity from fluorogram in A, relative to the reaction with SUVR4 without adding ubiquitin. The graph represents the average of four independent assays. (C) HKMTase assay with SUVR4 full-length using core histones from calf thymus as substrate, without (left) and with (right) the addition of 5 µg free ubiquitin, respectively. (D) The same assay as (C) but using histone H3 1-21 K9me1, H3 1-21 K9me2 or H3 1-21 K9me3 peptides with and without the addition of 5 µg free ubiquitin. (E) Peptide mass fingerprints of the products of an identical HKMTase assay as in C, using unlabelled SAM as methyl donor and H3 1-21 K9me1 (upper panel) or H3 1-21 K9me2 peptides as substrate (lower panel). Products from assays without (left) the addition of SUVR4 enzyme, containing SUVR4 protein (middle) and SUVR4 protein with the addition of 5 µg ubiquitin (right), were analyzed. The mass spectra of each peptide are shown as bars representing the mass-to-charge ratio (m/z), and the most abundant m/z is set to 100%. The length of the bars indicates abundance of the m/z relative to the most abundant. All enzyme assays were repeated at least 4 times with independent protein samples.

Figure 3. SUVR4 directs H3K9me3 on transposon and repeat sequences.

(A) Immunostaining of nuclei from SUVR4-GFP^OE seedlings with low expression (left panel) or high expression (right panel) of SUVR4-GFP with antibodies against H3K9me1 or H3K9me3. ChIP analysis of (B) SUVR4-GFP^OE and (C) SUVR4 RNAi lines using antibodies against H3K9me1 (left) or H3K9me3 (right). DNA levels from the ChIP experiments (B, C) relative to the input reactions were quantified using real time PCR and normalized to TUB8. The bars represent the average of two independent biological replicates.

Figure 4. SUVR4 HKMTase activity is inhibited by H3K4me3.

(A) HKMTase assay showing SUVR4 activity on peptides covering the first 1-21 aa of histone H3, that are unmodified, monomethylated or trimethylated on K4. (B) Quantification of band intensity from fluorogram in A, relative to the reaction with unmodified H3 1-21 peptide. The bars represent the average of three independent HKMTase assays.

Figure 5. SUVR4 represses transcription of transposons.

(A) Real time RT-PCR quantification of transcripts reversely transcribed from mRNA isolated from 14 day old SUVR4-GFP^OE and SUVR4-RNAi seedlings, respectively. The data were normalized to ACTIN2 and shown relative to wild type. (B, C) Quantification of bisulfite treated DNA from wt, SUVR4^OE and SUVR4 RNAi seedlings for MULE At2g15810 (B) and AtSN1 (C) respectively.

Figure 6. UBP26 directs H3K9me2 and H3K9me3 on transposon sequences.

ChIP analysis of ubp26-1 lines using antibodies against (A) H3K9me2, (B) H3K9me3 or (C) H2Bub1. DNA levels from the ChIP experiments relative to the input reactions were quantified using real time PCR and normalized to TUB8. The bars represent the average of two independent biological replicates. (D) Western blot of nuclear proteins isolated from ubp26-1 and wild type, probed with antibodies against ubiquitin (ub), H2Bub1 or PBA1 (loading control).

Figure 7. Model describing the relationship between free ubiquitin and SUVR4 activity on transposons.

(A) SUVR4 is repressed by H3K4me3 in vitro, and has no activity on genes with high H3K4me3, H3K9me3, H2Bub1 and a low level of H3K9me1. (B) SUVR4′s preference for heterochromatic transposons intercalated within euchromatin is maintained by its specificity for H3K9me1 which is highly enriched at transposons, and its repression by activating marks like H3K4me3. The deubiquitinase UBP26 regulates H3K9me2/me3 at the same targets as SUVR4, and might produce free ubiquitin that stimulates the H3K9me2/me3 activity of SUVR4 at target transposons. Although SUVR4 normally is repressed by H3K9me2 and H3S10ph which is high in pericentric heterochromatin, these regions may be targets for SUVR4 activity when ubiquitin levels are high. Since the transposons also contain a medium level of H3K27me3 in addition to H3K9me3, this could possibly create a binding site for CMT3 in order to repress transcription in a DNA methylation-dependent manner at some transposons. At other transposons, transcription may be repressed in a DNA methylation- independent manner by the MOM transcriptional repressor (See text for details).