Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

Abstract

Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

Figure 1. Identification and localisation of methylated H2A glutamine 105

a) Tandem mass spectrum (MS/MS) of the Q104me modified peptide VTIAQGGVLPNIQAVLLPK from H2A. The y and b series indicate fragments at amide bonds of the peptide, unambiguously identifying the methylated glutamine 104 (mammalian cells – yeast: glutamine 105). b) Alignment of the region encompassing Q105 of H2A and its variant H2A.Z. Highlighted in red is Q105 in H2A and the corresponding change to glycine or serine in H2A.Z. c) Analysis of yeast and mammalian cell extracts for the presence of Q105 methylation using a modification specific antibody.

Figure 2. Identification of Nop1/Fibrillarin as the methyltransferase of Q105

a) General strategy. b) Fractions from (a) were assayed on peptides containing glutamine or alanine at position 105 and compared to an unrelated peptide (H3K36me). For this, peptides were bound to Dynabeads, incubated with extract and [³H]-SAM and after extensive washes, analysed by liquid scintillation. Representative data of three independent experiments is shown. c) TAP-tag purification of the Nop1 complex coupled to the same activity assay as in (b) recapitulates the activity as found in the DEAE fraction. d) Recombinant Nop1 was incubated with SAM and recombinant histone H2A. Coomassie stain (CBB) and western blot (WB) of the reaction are shown. e) Tandem mass spectrum (MS/MS) of the Q104me modified peptide from H2A, which unambiguously identifies the methylated glutamine 104. f) Strains carrying thermosensitive alleles (15 and 16) of Nop1 were analysed for loss of Q105 methylation levels upon shift to restrictive temperature. g) The mammalian homolog of Nop1, Fibrillarin was knocked-down by independent siRNAs and probed for loss of Q105 methylation 48 h after transfection. A scrambled siRNA served as control (CTRL). h) Immunofluorescence of cells treated as in g) were stained using the Q105me-specific and anti-Fibrillarin antibodies and counterstained with DAPI as nuclear marker.

Figure 3. Genomic landscape of Q105me

a) ChIP-Seq profile of Q105me and H2A over Chromosome 12. The upper plot represents the normalised tracks for the mapped reads of Q105me/H2A. The magnification of the enriched region shows the rDNA region located on this chromosome. The two blue bars represent the 35S transcripts that are present in the genome annotation. b) Schematic representation of one rDNA repeat with the position of primers used to scan the rDNA region by ChIP qPCR. c) ChIP-qPCR validation of the ChIP-Seq shown in (a). d) Nop1 profile over the rDNA locus. The ChIP-qPCR profile was internally normalised to signal of primer pair “A”. ChiP-qPCR data shows the mean ± SEM of three independent biological experiments.

Figure 4. Unmodified Q105 is part of a recognition motif for FACT

a) Indicated peptides were bound to Streptavidine-Dynabeads and incubated with recombinantly purified Spt16/Pob3 for 4h at 4°C. Input represents 50% of Spt16/Pob3 used for the IP. Bound Spt16/Pob3 was analysed by Coommassie stain (CBB) or western blotting. Peptides were spotted on membrane as loading control. b) Effect of the H2AQ105A mutant on the transcription of a URA3 reporter integrated at the indicated positions in the rDNA repeat (S3 and S6) and R31 outside the rDNA. Strains were assayed for their ability to grow on 5-FOA for three days at 30°C (n≥3, n.d. – not detected) c) The spt16-11 allele was assayed as described in c), but colonies were counted after one week. d) Yeast expressing either wild-type (WT) or Q105A (QA) mutant H2A as sole source of histones were assayed for rDNA transcription by transcriptional run-on and compared to cells that carry an spt16 thermosensitive allele (spt16). RNA levels are expressed as relative levels to actin (* represents p<0.05 and ** p<0.01). e) Differences in histone incorporation rates between WT and Q105A (QA) histones. WT H2A and H2AQ105 were myc-tagged and placed under the control of a galactose inducible promoter in either wild-type or spt16-11 cells. Myc-tagged histones were induced by addition of 2% galactose (t=0). Samples were taken at the indicated time points and chromatin immunoprecipitated with anti-myc and H2A antibodies. f) Nop1 over-expression (Nop1 overexpressed) leads to an increase of Q105 methylation and a concurrent decrease in FACT occupancy at the rDNA locus. Primer positions are indicated and data was presented as the log2 changes compared to an empty vector control (CTRL). ChiP-qPCR data shows the mean ± SEM of at least two independent biological experiments.