ARTICLE https://doi.org/10.1038/s41467-020-20707-x OPEN Short H2A histone variants are expressed in cancer Guo-Liang Chew 1, Marie Bleakley2, Robert K. Bradley 3,4,5, Harmit S. Malik4,6, Steven Henikoff 4,6, ✉ ✉ Antoine Molaro 4,7 & Jay Sarthy 4 Short H2A (sH2A) histone variants are primarily expressed in the testes of placental mammals. Their incorporation into chromatin is associated with nucleosome destabilization and modulation of alternate splicing. Here, we show that sH2As innately possess features similar to recurrent oncohistone mutations associated with nucleosome instability. Through 1234567890():,; analyses of existing cancer genomics datasets, we find aberrant sH2A upregulation in a broad array of cancers, which manifest splicing patterns consistent with global nucleosome destabilization. We posit that short H2As are a class of “ready-made” oncohistones, whose inappropriate expression contributes to chromatin dysfunction in cancer. 1 The Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore. 2 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 3 Computational Biology Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 4 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 5 Department of Genome Sciences, University of Washington, Seattle, WA, USA. 6 Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. 7Present address: ✉ Genetics, Reproduction and Development (GReD) Institute, Université Clermont Auvergne, Clermont-Ferrand, France. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:490 | https://doi.org/10.1038/s41467-020-20707-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20707-x ucleosomes, the fundamental subunit of chromatin, con- contribute to cancer. Previous work showed that expression of sist of octamers of histones (H2A, H2B, H3, and H4) that H2A.B causes increased sensitivity to DNA damaging agents, N 1 22 17,19,22 wrap 147 bp of DNA . Single-allele mutations in histones, shortens S-phase , and alters splicing , each of which are termed “oncohistones”, are found in many different associated oncogenesis. Additional evidence for a role for H2A.B malignancies2,3. Oncohistones comprise small percentages of the in cancer comes from Hodgkin’s lymphoma (HL), where H2A.B total histone pool4,5 and rarely cause cancer by themselves2,6. transcripts have been detected23 and HL cells expressing H2A.B Instead, they synergize with other oncogenes to facilitate the grow faster than H2A.B-negative cells22. Here, through com- development of neoplastic chromatin landscapes2,6. Recent large- parative analyses of germline short H2A sequences and onco- scale cancer genome analyses identified recurrent mutations in histone mutations in canonical H2A, we show that short H2As histones within the highly conserved histone fold domain (HFD) inherently possess oncohistone features. We explore several in many common cancers2,3. These HFDs mutations, including cancer data sets and find H2A.B expression in a diverse array of the well-characterized H2B-E76K substitution, reduce nucleo- malignancies. We also show that many of these cancers possess some stability in vitro and perturb chromatin in vivo2,7. Similarly, unique splicing signatures. We propose that the nucleosome- most H2A HFD oncohistone mutations disrupt either contact destabilizing characteristics sH2As evolved for their role in testis sites with DNA (R29Q) or inter-nucleosomal interactions (acidic result in oncohistone activity in other tissues. patch) (Fig. 1a). Cells co-expressing H2B-E76K and a PI3KCA oncogene showed increased transformation capacity, consistent Results with nucleosome instability, enhancing the cancerogenic potential sH2As have evolved oncohistone features. There are five X- of other oncogenes2. linked sH2A genes in humans: H2A.B.1.1 (H2AFB2), H2A.B.1.2 Short histone H2A variants (sH2A) are a class of histone var- – (H2AFB3), H2A.B.2 (H2AFB1), H2A.P (HYPM), and H2A.Q iants expressed during mammalian spermatogenesis8 11. Regula- (unannotated)8. We compared the amino-acid sequences of tion of sH2A expression in normal testis is unknown12. Unlike sH2As to canonical H2A to assess whether their rapid evolution other histone variants, sH2As are rapidly evolving and possess resulted in oncohistone-like changes. This analysis revealed that highly divergent HFDs, mutated acidic patches, and truncated C- – many of the most common cancer-associated mutations in termini, all of which impact nucleosome stability8,13 16. The best canonical H2A are already present in all wild-type sH2A characterized of these variants, H2A.B, forms unique nucleosomes – sequences (Fig. 1a, Supplementary Fig. 1a, b). This includes R29Q/ that wrap ~120 bp of DNA both in vitro and in vivo15 17. In testis, F substitutions that correspond to the second most frequent H2A.B is incorporated into nucleosomes during meiosis and has mutation in canonical H2A (Fig. 1a, Supplementary Fig. 1a, b)2,3. been shown to interact with splicing factors at actively transcribed – In addition, all wild-type sH2As have a C-terminal truncation that genes17 20. Germline disruption of H2A.B-encoding genes in mice removes E121, the most common mutation in canonical H2A revealed that H2A.B loss is associated with chromatin dysfunction (Fig. 1a, Supplementary Fig. 1a, b)2,3. Phylogenetic analyses in and splicing changes in testis18. primates showed that despite their rapid evolution, these Though a role for sH2As in cancer has yet to be determined21, oncohistone-like changes are highly conserved (Fig. 1b, Supple- the emerging literature on nucleosome instability as cancer mentary Fig. 1a–c)8. This conservation implies functional con- driver2,3 along with H2A.B’s potent ability to destabilize – sequences as many of these residues are critical contact points for nucleosomes13 16 prompted us to investigate whether sH2As may histone-DNA or histone-histone interactions1,13–16. These data Fig. 1 H2A.B possesses oncohistone features that are conserved throughout primates. a Schematic of common oncomutations found in human core H2A and their status in H2A.B. Marked sites on core H2A show WT amino acid position followed by its most common cancer-specific substitution in TCGA (pink). Associated sites found in WT short H2As are shown in purple. b Protein alignment of core H2A, testis-specific H2A (TH2A), and H2A.B paralogs from Human and representative primates. Substitutions corresponding to oncohistone mutations in H2A (see Fig. 1.) are shown in pink. 2 NATURE COMMUNICATIONS | (2021) 12:490 | https://doi.org/10.1038/s41467-020-20707-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20707-x ARTICLE show that sH2As contain oncohistone features similar to cano- Tissue Expression database (Supplementary Table 1). The range nical H2A mutations in cancers. of expression varies widely, with H2A.B-encoding transcripts present at >100 TPMs in two specimens (Supplementary Data 2). H2A.Bs are reactivated in a broad array of cancers. The Although many tumors reactivate H2AFB1 alone, most tumors oncohistone properties inherent in sH2As indicate that they may that express H2AFB2 also express H2AFB3 (Fig. 2a). This finding play a role in cancer simply through upregulation. We focused on may result from transcriptional co-regulation due to their geno- the expression of H2A.B paralogs, as they are well annotated and mic proximity (Supplementary Fig. 2b) or inability to distinguish have been shown to impact both nucleosome stability and cell these near-identical paralogs by short-read mapping8. Despite cycle progression22. To investigate whether H2A.Bs are reacti- their similarity, we were able to distinguish these two genes in a vated in different cancers, we first used transcriptomic data from few tumor samples (Fig. 2a). The Cancer Genome Atlas (TCGA). This analysis showed that Across the TCGA data set, diffuse large B-cell lymphomas H2A.B paralogs are activated (at a threshold of >1.5 transcripts (DLBCLs) showed the highest frequency of aberrant H2A.B per million (TPM)) in numerous individual tumors across cancer expression at 50% (Fig. 2a). A recent analysis of DLBCL genomes types (Fig. 2a, Supplementary Data 1, Supplementary Data 2), but identified five distinct molecular subtypes24, including a favorable never in adjacent normal tissue (Supplementary Data 1), and very prognosis-germinal center (FP-GC) subtype associated with rarely (<1.5%) in non-testes tissue samples from the Genotype- histone mutations. We investigated whether H2A.B expression Fig. 2 H2A.B is expressed in a broad array of cancers. a Heat map illustrating the co-expression of H2A.B paralogues in individual tumors that express any one H2A.B paralogue (at >1.5 TPM as measured by RNA-seq), for cancer types with at least 10 tumors expressing any H2A.B paralogue. Percentages of tumors for each cancer type that express any H2A.B paralogue are shown. b Expression levels (TPM) of H2A.B-encoding transcripts in three independent B-acute lymphoblastic leukemia data sets, horizontal line demarcates 1.5 TPM as measured by RNA-seq. Only samples with non-zero expression of any H2A.B paralog are shown: 18, 5, and 1 samples are omitted from Liu et al. 2016, Qian et al. 2017, and Yasuda et al. 2016, respectively. c As in b, but for cancer cell lines from CCLE, grouped by their lineage. Only lineages with any sample >1.5 TPM, and samples with non-zero expression of any H2A.B paralog are shown. NATURE COMMUNICATIONS | (2021) 12:490 | https://doi.org/10.1038/s41467-020-20707-x | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20707-x was restricted to the FP-GC subtype. We queried the 37 DLBCL H2A.Bs are associated with cancer-specific, rather than pan- samples for mutations associated with the FP-GC subtype cancer gene expression programs. H2A.B proteins encoded by including linker H1 and core histones, immune evasion genes, H2AFB1 and H2AFB2/3 are nearly identical in sequence.