Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification

Abstract

We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.

Figure 1

Experimental strategy and results for identified histone PTM sites. (A) Schematic diagram of the experimental design for comprehensive mapping of PTM sites in linker and core histones from HeLa cells. Histone extracts were in-solution trypticly digested without chemical propionylation (Method I), chemically propionylated after in-solution tryptic digestion (Method II), chemically propionylated before in-solution tryptic digestion (Method III), and in-gel digested after SDS-PAGE gel separation. Samples from Methods I and II were further subjected to IEF fractionation to generate 12 fractions. (B) Peptide sequence coverage of linker and core histones in each of the four methods is shown. (C) A table summarizing all the PTM sites identified by this study. Abbreviations: me, monomethylation; me2, dimethylation; me3, trimethylation; fo, formylation; ac, acetylation; oh, hydroxylation; cr, crotonylation. (D) A diagram showing sites of histone PTMs other than Kcr identified in this study. Amino acid residue number is indicated below its sequence. Gray and blank boxes indicate N-terminal and globular core domains, respectively. (E) Illustrations of histone Kcr sites in human HeLa cells and mouse MEF cells. All Kcr sites are shown in red and underlined. Previously reported Kac sites are shown in blue.

Figure 2

Short-chain lysine acylations. (A) An illustration of the enzymatic reactions for lysine acetylation by lysine acetyltransferases (KATs) using acetyl-CoA as a cofactor, and a hypothesized mechanism for Kcr using crotonyl-CoA as a cofactor. (B) Ball-and-stick models of a crotonyl group and an acetyl group. The three-dimensional arrangement of four carbons and one oxygen of the crotonyl group are rigid and located in the same plane (left). The two olefinic carbons of the crotonyl group are shown in yellow. In contrast, the tetrahedral CH₃ in the acetyl group (right) can be rotated such that it is structurally very different from the crotonyl group. (C) Crotonyl-CoA metabolism pathways. Crotonyl-CoA was generated from butyryl-CoA or glutaryl-CoA, and oxidized to acetyl-CoA through multiple steps.

Figure 3

Identification and verification of a Kcr peptide, PEPAK_crSAPAPK (Kcr indicates a crotonyllysine residue). (A, B, C) High-resolution MS/MS spectrum of a tryptic peptide, PEPAKSAPAPK, with a mass of +68.0230 Da at its Lys5 residue identified from in vivo histone H2B (A), its synthetic Kcr counterpart (B), and a peptide mixture of the in vivo-derived tryptic peptide and its synthetic counterpart (C), each showing the same MS/MS fragmentation patterns and the same precursor ion mass. Inset shows their precursor ion masses. (D) Extracted ion chromatograms (XICs) of the in vivo-derived PEPAK_+68.0230SAPAPK peptide, the synthetic Kcr counterpart, and their mixture by nano-HPLC/MS/MS analysis using a reversed-phase HPLC column, showing the co-elution of the two peptides.

Figure 4

Detection of Kcr in histones by Western blotting. (A) Specificity of pan anti-Kcr antibody demonstrated by dot-spot assay using five peptide libraries with indicated amount (ng). Each peptide library contains 13 residues CXXXXXKXXXXXX, where X is a mixture of 19 amino acids (excluding cysteine), C is cysteine, and the 7^th residue is a fixed lysine residue: unmodified lysine (K), Kac, propionyllysine (Kpr), butyryllysine (Kbu), and Kcr. (B) Detection of Kcr in histones. Western blotting was carried out using the histones from HeLa cells with competition of a peptide library bearing a fixed unmodified lysine (K) or Kcr. (C) Dynamics of histone Kcr in response to crotonate. The histone proteins extracted from human prostate cancer cell line Du145 incubated with 0, 50 or 100 mM crotonate for 24 hours, were Western blotted with anti-Kcr pan antibody. (D) MS/MS spectrum of PEPA K_D4-crSAPAPK identified from D₄-crotonate-labeled sample. The mixture of D₄-, D₃- and D₂-crotonyl groups was used for the identification of D₄-crotonyl peptide. (E) Kcr signals in core histones of S. cerevisiae, C. elegans, D. melanogaster (S2), M. musculus (MEF), as well as H. sapiens (HeLa) cells by Western blotting analysis with competition.

Figure 5

Enrichment of histone Kcr on active chromatin. (A) ChIP-seq snapshots of input, H3K4me3, H3K4me1 and Kcr in IMR90 cells. (B) Pie chart showing the genomic distribution of all histone Kcr peaks with annotated genomic regions. TSS is defined as regions +/− 2.5kb around known transcription starting sites in RefSeq database. Enhancers are promoter distal regions associated with H3K4me1 as predictive mark. (C) Curves showing the mean reads density of indicated histone modification around all known TSS. Reads densities are calculated within a 100bp sliding window and normalized by subtracting reads density in the control input ChIP-seq data. RPKM is calculated as the number of reads which map per kilobase of genomic region per million mapped reads. (D) Average normalized read densities of H3K4me3, H3K4me1 and histone Kcr around predicted enhancers are plotted. (E) All RefSeq genes are divided into 5 groups based on their expression level calculated from mRNA-seq data, and the average normalized read densities of Kcr around each group of TSS are plotted.

Figure 6

Correlation of histone Kcr with gene expression in meiotic, post-meiotic male germ cells, and in tissues. (A) Hyper-crotonylation wave in elongating spermatids. Kcr was detected on paraffin mouse testis tubule sections representing different stages of spermatogenesis by immunohistochemistry (IH) using an anti-Kcr antibody. Pre-meiotic spermatogonia (Spg) and meiotic spermatocytes (Spc) cells are present at the periphery and middle of the tubule sections, whereas post-meiotic round (RS), elongating (ES) and condensing (CS) spermatids are near the lumen. The nuclei of ES are positive for Kcr. (B, C) Genes associated with higher Kcr in RS than Spc are mostly post-meiotically activated and show a predominant expression in the testis. The genes associated with Kcr peaks were divided into three categories according to their Kcr levels in Spc and RS: i) Spc=RS, similar Kcr levels between Spc and RS; ii) Spc>RS, lower Kcr levels in RS than Spc (fold change >=2); iii) Spc=2). The expression of these genes in male germ cells (B) and tissues (C) was then compared among the three categories. (B) Expression in male germ cells. Left panel: respective proportions of genes (Y axis) with higher expression either in Spc or RS among the three gene categories (X axis). Right panel: heatmap showing the expression of the third category of genes (Kcr, Spc

Figure 7

Sex chromosomes are highly crotonylated in round spermatids. IH (A) on a testis tubule section and immunofluorescence (IF) (B) of male germ cells showing high crotonylation enrichment in the sex chromosome region (besides the HP1γ enriched chromocenter) in post-meiotic round spermatids. IH is shown without counterstaining for better visualization of the region detected by the antibody. IF: pan-Kcr was detected in green, and is shown in co-staining with HP1γ in bright red fluorescence. Scale bar for IF: 5 micrometers.