Histone Isoform H2A1H Promotes Attainment of Distinct Physiological

et al. Epigenetics & Chromatin (2017) 10:48 DOI 10.1186/s13072-017-0155-z Bhattacharya Epigenetics & Chromatin Open Access RESEARCH Histone isoform H2A1H promotes attainment of distinct physiological states by altering chromatin dynamics Saikat Bhattacharya1,4,6, Divya Reddy1,4, Vinod Jani5†, Nikhil Gadewal3†, Sanket Shah1,4, Raja Reddy2,4, Kakoli Bose2,4, Uddhavesh Sonavane5, Rajendra Joshi5 and Sanjay Gupta1,4* Abstract Background: The distinct functional effects of the replication-dependent histone H2A isoforms have been demonstrated; however, the mechanistic basis of the non-redundancy remains unclear. Here, we have investigated the specific functional contribution of the histone H2A isoform H2A1H, which differs from another isoform H2A2A3 in the identity of only three amino acids. Results: H2A1H exhibits varied expression levels in different normal tissues and human cancer cell lines (H2A1C in humans). It also promotes cell proliferation in a context-dependent manner when exogenously overexpressed. To uncover the molecular basis of the non-redundancy, equilibrium unfolding of recombinant H2A1H-H2B dimer was performed. We found that the M51L alteration at the H2A–H2B dimer interface decreases the temperature of melting of H2A1H-H2B by ~ 3 °C as compared to the H2A2A3-H2B dimer. This difference in the dimer stability is also reflected in the chromatin dynamics as H2A1H-containing nucleosomes are more stable owing to M51L and K99R substitutions. Molecular dynamic simulations suggest that these substitutions increase the number of hydrogen bonds and hydrophobic interactions of H2A1H, enabling it to form more stable nucleosomes. Conclusion: We show that the M51L and K99R substitutions, besides altering the stability of histone–histone and histone–DNA complexes, have the most prominent effect on cell proliferation, suggesting that the nucleosome stability is intimately linked with the physiological effects observed. Our work provides insights into the molecular basis of the non-redundancy of the histone H2A isoforms that are being increasingly reported to be functionally important in varied physiological contexts. Keywords: Cancer, Chromatin, Differentiation, Histone, Nucleosome of DNA is wrapped to form the nucleosome core particle (NCP) [1]. Further compaction of the chromatin is achieved with the aid of the linker histone H1 [2]. Background Histones are a class of highly conserved basic proteins that package the genome. e core histones are comprised of H2A, H2B, H3 and H4 which form the octameric protein core of the fundamental repeating unit of chromatin, the nucleosome. Around this core, ~147 bp e canonical histone proteins are synthesized during the S-phase, and to meet up with their high demand during DNA replication, genes that encode them are present in clusters. ere are three clusters of canonical histone genes present in humans at chromosome numbers 1 and 6. Notably, differences in the primary sequence are observed amongst the histone proteins encoded by these genes. For the sake of clarity, these are termed as the histone isoforms in this manuscript. In humans, there are 17 genes for H2A that code for 12 isoforms [3, 4]. Likewise, *Correspondence: [email protected] †Vinod Jani and Nikhil Gadewal contributed equally to this work 1 Epigenetics and Chromatin Biology Group, Gupta Lab, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, MH 410210, India Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<a href="/goto?url=http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a>), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<a href="/goto?url=http://creativecommons.org/publicdomain/zero/1.0/" target="_blank">http://creativecommons.org/ </a><a href="/goto?url=http://creativecommons.org/publicdomain/zero/1.0/" target="_blank">publicdomain/zero/1.0/</a>) applies to the data made available in this article, unless otherwise stated. et al. Epigenetics & Chromatin (2017) 10:48 <ul style="display: flex;"><li style="flex:1">Bhattacharya </li><li style="flex:1">Page 2 of 19 </li></ul>there are 13 genes for H2A in rats that code for 9 iso- cells [10, 11]. Interestingly, later on in a larger cohort of forms (most are “predicted”). samples, H2A1C expression was conversely reported to e histone isoform genes are named based on their be upregulated in CLL [12]. Also, H2A1C was found to identity and location in the genome. In the name of the be upregulated in non-small cell lung carcinoma [13]. gene, the first part refers to the cluster (HIST1—cluster 1, e levels of H2A1C, in particular, have been reported HIST2—cluster 2, HIST3—cluster 3), the second part of to change in other diseases including human papillothe gene name introduces the type of histone (H2A, H2B, maviruses hyperplasia, AIDS and multiple sclerosis [14, H3, H4, H1), and the third part indicates the alphabetical 15]. Collectively, these reports demonstrate the altered order within each cluster (centromere distal to proximal). expression of the H2A isoforms in different pathophysierefore, HIST1H2AB refers to the second histone H2A ological states. e question now is whether the observed gene in the histone cluster 1 and HIST2H2AB refers to changes are merely a consequence of the change in the the second histone H2A gene in the histone cluster 2. state or these isoforms also contribute to the attainment e proteins coded by these genes, however, were not of such states. One report that aims to address this quesreferred to as systematically. Traditionally, the histone tion showed that specific knockdown of H2A1C leads H2A isoforms were broadly classified into two categories, to a marked increase in cell proliferation. is effect is H2A.1 and H2A.2, based on the difference in their mobil- not observed on depleting the other abundant isoforms ity on AUT (acetic acid, urea, Triton X-100)–PAGE gels. like H2A1B/E [10]. However, how the histone isoforms e H2A isoforms that migrated slowly were collectively impart their non-redundant effects remains unclear. <ul style="display: flex;"><li style="flex:1">termed H2A.1 and the isoforms that migrated faster were </li><li style="flex:1">Here, we show that the expression level of the H2A1H/ </li></ul>collectively referred to as H2A.2 [5]. e difference in H2A1C isoform markedly varies in different tissues migration arises due to the L51M alteration in H2A. Leu- in addition to being generally upregulated in many cine binds more Triton X, and hence, the H2A isoforms cancer cell lines. We provide further evidence that with L51 migrate slower than isoforms with M51 residue. H2A1H (encoded by HIST1H2AH, accession number: However, as each of these two bands may be constituted NM_001315492.1) provides a growth advantage to cells; of multiple proteins, this system of referring to isoforms however, this effect is context dependent. Importantly, can be misleading. Especially considering the growing with the help of in vitro and in silico studies, we demonevidence of the changes in the expression level of the iso- strate that H2A1H forms more stable nucleosomes than forms, a better way to name them would be to maintain the H2A.2 isoform H2A2A3 (encoded by HIST2H2AA3, consistency with their gene nomenclature. For example, accession number: NM_001315493.1), and this is specuthe protein coded by HIST1H2AB will be referred to lated to confer the non-redundant functionality. Our as H2A1B. If two genes code for the same protein as in studies reveal that the highly similar histone isoforms the case of HIST1H2AB and HIST1H2AE, the protein can bring about changes in cell physiology by modulating will be referred to as H2A1B/E. Hence, in rats, the pro- chromatin dynamics. teins H2A3, H2A4, H2A1F, H2A1K, H2A1H and H2A1C (H2AE-like, H2A1I, H2A1N) constitute the H2A.1 iso- Results forms, and the H2A2B, H2A2C and H2A2A3 proteins H2A1H/H2A1C expression level varies in cancer cell lines belong to H2A.2 isoforms (see Additional file 1: Figure S1 and amongst different normal tissues <ul style="display: flex;"><li style="flex:1">for the alignment). </li><li style="flex:1">Previously, we have reported the upregulation of H2A.1 </li></ul>e histone isoforms were considered function- isoforms during the progress of hepatocellular carcinoma ally redundant for a long time considering the similar- (HCC) [9]. During the course of development of HCC, ity in their amino acid sequences. Interestingly, though, the animals were under the administration of NDEA. We the H2A isoforms have been reported to be differen- wanted to see whether the increased expression of H2A.1 tially expressed in a variety of physiological states. For persists even without the influence of NDEA. To address instance, the proportion of the H2A.1 and H2A.2 iso- this, a tumor was developed in the liver of Sprague– forms in rats has been shown to decrease during the Dawley rat by feeding NDEA with drinking water. After course of development, differentiation and aging [6–8]. the development of the tumor (105 days since the start An earlier report from our laboratory revealed the over- of NDEA administration), a 3-mm2 tumor tissue was expression of the H2A.1 isoforms during the sequential excised and subcutaneously implanted in a NOD-SCID stages of rat hepatocellular carcinoma [9]. e expression mice. e NOD-SCID mice were not fed with NDEA. level of the isoform H2A1C in humans has been reported Two weeks post-implantation, the animals were sacrito alter in pathological states. Expression of the H2A1C ficed and the developed tumor was excised. Analysis of isoform was reported to be downregulated in chronic the isolated histones from the tumor resolved onto AUT- lymphocytic leukemia (CLL) and gall bladder cancer PAGE showed a higher expression of H2A.1 isoforms et al. Epigenetics & Chromatin (2017) 10:48 <ul style="display: flex;"><li style="flex:1">Bhattacharya </li><li style="flex:1">Page 3 of 19 </li></ul>compared to the normal liver (Fig. 1a). is suggests that in different tissues. To test this hypothesis, the tranH2A.1 upregulation is indeed a stable alteration that script level of H2A1H in different organs was comoccurs during the process of tumorigenesis. e changes pared. Marked variation in H2A1H level was observed. in H2A composition in HCC were further appreciated A very high level of H2A1H expression was observed by performing reverse-phase HPLC of the extracted his- in rectum (Fig. 2e). On the other hand, in the stomach tones (Fig. 1b) (see Additional file 1: Figure S2 for the and tongue tissues, the expression level was found to complete elution profile). e most prominent difference be particularly low (Fig. 2e). Isoform H2A2A3 exhibin the chromatogram of the control vs tumor histones is ited much lesser variation in expression level (Fig. 2e). the distinct peak at around 84 ml elution volume (Fig. 1c, AUT-PAGE analysis of histones isolated from kidd). Mass spectrometry followed by peptide fingerprint- ney, brain and liver demonstrates that the variations ing of the eluted fractions 84 and 85 ml revealed high observed in transcript level of the H2A.1 isoform scores for the H2A.1 isoforms (H2A1H, H2A3, H2A1C, H2A1H is also reflected in protein expression (Fig. 2f). H2A1K) with the maximum score obtained for H2A1H e brain showed an increased proportion of H2A.1, (Fig. 1e) (see Additional file 1: Figure S3 for the peptides whereas kidney and liver have higher levels of H2A.2 detected in MS). We next performed real-time PCR isoforms (Fig. 2g). to check the transcript levels of the histone isoforms in normal vs tumor liver tissues (see Additional file 1: Fig- H2A1H isoform is functionally non‑redundant from the ure S4). We performed normalization to the widely used H2A2A3 isoform normalization control, glyceraldehyde phosphate dehy- e expression level of H2A.1 isoforms varies in difdrogenase (GAPDH) gene (see Additional file 1: Figure ferent tissues, differentiation status, age and diseases. S4a). e histone isoforms are synthesized during the Based on our results, we wanted to test the effect of S-phase of the cell cycle. As the cells in tumor tissues are overexpressing H2A.1 isoform H2A1H on cell physimore proliferative, to normalize for the overall changes in ology. Two cell lines that are derived from the liver of the histone content, we also performed normalization to NDEA-administered Sprague–Dawley rats were chosen histone H4 genes (see Additional file 1: Figure S4b). e for our studies: CL44 (pre-neoplastic), with an equimoprimers for H4 genes were designed to pick up all the lar ratio of H2A.1 and H2A.2, and CL38 (neoplastic), in H4 transcripts. Irrespective of the normalization control which H2A.1 is naturally elevated (see Additional file 1: used, we found that H2A1H was the most prominently Figure S5). By RT-PCR, we validated that the CL38 cells <ul style="display: flex;"><li style="flex:1">upregulated H2A isoform. </li><li style="flex:1">express higher levels of the H2A1H isoform. Localization </li></ul>In terms of the protein sequence, H2A1C in humans is of YFP-tagged H2A1H/H2A2A3 in CL38 cells suggested the most similar to H2A1H of rat, differing in only the that both the isoforms are incorporated across the entire S16T substitution [see Additional file 1: Figure S7(c)]. e chromatin (see Additional file 1: Figure S5). By isolating altered expression level of H2A1C has been reported in histones from the CL38 cells exogenously overexpressing human cancers [10–13, 16–18]. Our observations in the the isoforms [pcDNA3.1(+) vector] and resolving them rat hepatocellular carcinoma prompted us to investigate on AUT-PAGE, we confirmed that the overexpression of the expression level of H2A1C isoform in human trans- H2A1H leads to its increased abundance in the chromaformed cell lines of the liver (HEPG2). We also included tin (Fig. 3a, b). <ul style="display: flex;"><li style="flex:1">cell lines of skin (A431) and stomach (KATOIII, AGS) </li><li style="flex:1">A marked increase in proliferation was observed in </li></ul>origin and their non-transformed immortalized counter- the CL38 cells on exogenous overexpression of H2A1H parts, that is, HHL5 (liver), HACAT (skin) and HFE145 (Fig. 3c). Similar effects were reflected in the colony for(stomach) in our study, as the expression level of H2A1C mation assay, with H2A1H overexpressing colonies subin these cell lines has not been previously reported. stantially larger (Fig. 3d, e). Associated upregulation in An increase in the relative expression of H2A1C was proliferation markers Ki67 and PCNA was also noted observed in HEPG2 and A431 (Fig. 2a, b). We did not by qRT-PCR (Fig. 3f). To see the effect of the isoforms find any significant changes in the levels of the isoform overexpression on the cycling of cells, we studied the cell H2A2A3 (identical to rat H2A2A3). e two isoforms did cycle profile of G1-enriched H2A1H/H2A2A3 CL38 cells not show any significant alteration in expression in both post-72-h serum release. Overexpression of H2A1H led the transformed cell lines of the stomach with respect to to a discernible increase in the mitotic cell population their immortalized counterpart, that is, HFE145 (Fig. 2d). (12%) compared to the vector control (4%) (Fig. 3g). We We also found upregulation of H2A1C in MCF7 consist- also observed an increase in the mitotic cell population <ul style="display: flex;"><li style="flex:1">ent with a previously published report (Fig. 2c) [17]. </li><li style="flex:1">with H2A2A3 overexpression (7%) compared to the vec- </li></ul>We speculated that if H2A1H has some specific tor control (4%). is was also reflected in the proliferanon-redundant function, then its expression may vary tion assays (Fig. 3c, d). No significant difference in the et al. Epigenetics & Chromatin (2017) 10:48 <ul style="display: flex;"><li style="flex:1">Bhattacharya </li><li style="flex:1">Page 4 of 19 </li></ul>Fig. 1 H2A.1 isoform, H2A1H’s expression predominantly increases during hepatocellular carcinoma. a AUT-PAGE analysis of histones (silver stained) isolated from xenograft tumor (marked transplantable HCC) along with histones from different stages of NDEA-induced liver cancer in rat. b RP- HPLC chromatogram of histones isolated from control (normal) and tumor tissues of rat liver. c Overlay of RP-HPLC profiles of histones isolated from control and tumor tissues of rat liver to depict major differences observed in the H2A region. d Bar graph depicting the area under the curve in the chromatogram for H2A isoforms. The total area under the curve was measured and considered as 100%. Then, the intensity of the peak at 84–85 ml (H2A.1, as per observed difference in mobility on AUT-PAGE) was measured and plotted as percentage of the total area. Error bar represents SEM of three independent experiments. e Table listing the H2A isoforms identified (with protein scores) in MASCOT search performed after mass spectrometry of the respective fractions of RP-HPLC. Scores greater than 52 are significant (p < 0.05). For the list of unique peptides identified, please see Additional file 1: Figure S3 et al. Epigenetics & Chromatin (2017) 10:48 <ul style="display: flex;"><li style="flex:1">Bhattacharya </li><li style="flex:1">Page 5 of 19 </li></ul>Fig. 2 H2A.1/H2A1C expression varies drastically in different contexts. a–d Quantitative real-time PCR data showing the relative expression levels of H2A1C and H2A2A3 in different human cell lines (see text for more details). Error bar represents SEM of three independent experiments. e Graph showing the relative levels of H2A1H and H2A2A3 in various normal rat tissues, monitored at transcript level normalized to GAPDH by qRT-PCR. Error bar represents SEM of three independent experiments. f H2A and H2B region of AUT-PAGE analysis of histones (silver stained) isolated from the normal kidney, brain and liver tissues. g Quantitative analysis of the isoforms enrichment in the chromatin. Quantification of bands of H2A.1 and H2A.2 was performed by using the software GelAnalyzer. Normalization was done with respect to H2B as it appears as a single discrete band on AUT-PAGE. The data were plotted after taking the densitometric readings of three independent experiments. Error bars represent SEM of three independent experiments closure of the wound in scratch assays performed with upon H2A1H overexpression (see Additional file 1: FigCL38 cells on H2A1H overexpression was perceived ure S6b). Importantly, during liver regeneration post-par(see Additional file 1: Figure S6a) in comparison with tial hepatotectomy, H2A.1 expression was not found to H2A2A3 overexpression. Notably, we did not observe alter [8]. Taken together, these data suggest that although any significant change in the proliferation of CL44 cells H2A1H expression provides a growth advantage to cells, et al. Epigenetics & Chromatin (2017) 10:48 <ul style="display: flex;"><li style="flex:1">Bhattacharya </li><li style="flex:1">Page 6 of 19 </li></ul>et al. Epigenetics & Chromatin (2017) 10:48 <ul style="display: flex;"><li style="flex:1">Bhattacharya </li><li style="flex:1">Page 7 of 19 </li></ul>(see figure on previous page.) Fig. 3 H2A1H overexpression leads to increase in cell proliferation. a AUT-PAGE analysis (silver stained) showing the enrichment of the H2A isoforms in chromatin upon their overexpression in CL38 cells. b Quantitative analysis of the isoforms enrichment in the chromatin. Quantification of bands of H2A.1 and H2A.2 was performed by using the software GelAnalyzer. Normalization was done with respect to H2B as it appears as a single discrete band on AUT-PAGE. The data were plotted after taking the densitometric readings of three independent experiments. Error bars represent SEM of three independent experiments. c Cell proliferation curves by MTT assay of H2A1H and H2A2A3 overexpressing CL38 cells in comparison with control CL38 cells. Error bars represent SEM of six independent experiments. d Colony formation assay of CL38 cells upon H2A1H and H2A2A3 overexpression. e Quantitative analysis of the colony sizes of 20 colonies each performed using ImageJ. Error bar represents SEM. f qRT-PCR for the cell proliferation markers Ki67 and PCNA on H2A1H and H2A2A3 overexpression normalized to 18S rRNA. Error bars represent SEM of three independent experiments. g Cell cycle analysis of the CL38 cells exogenously overexpressing H2A isoform post-serum starvation and release. h The expression level analysis of the CL38 cells expressing H2A1H single or double mutants with anti-FLAG antibody. i Bar graph depicting the proliferation of the CL38 cells expressing H2A1H single mutants by the MTT assay. Error bars represent SEM of 6 independent experiments. j Bar graph depicting the proliferation of the CL38 cells expressing H2A1H double mutants by MTT assay. Error bars represent SEM of six independent experiments. VC—vector control. H2A1H, H2A2A3 and their mutants in the figure are the genes cloned and expressed as FLAG tagged proteins in pcDNA3.1(+) vector its expression is not always correlated with proliferation H2A is involved in interactions with the minor groove <ul style="display: flex;"><li style="flex:1">(discussed later). </li><li style="flex:1">of DNA in the NCP, residue 51st lies in the dimer inter- </li></ul>face with H2B, and residue 99th of H2A interacts with the H4 tail in the octamer core (see Additional file 1: Figure S7b). erefore, potentially the alterations at these Leu51 and Arg99 are important in conferring the non‑redundant functionality to the H2A1H isoform e H2A isoforms, H2A1H and H2A2A3, differ in three residues can alter the stability of the nucleosome and its residues in their primary amino acid sequence (see Addi- subcomplexes. tional file 1: Figure S7a). To understand which residue(s) are important for the non-redundant functionality of The H2A1H‑H2B dimer is less stable than the H2A2A3‑H2B H2A1H, we substituted the residues of H2A1H to the dimer corresponding ones of H2A2A3. MTT assays performed To investigate the possibility discussed above, we comwith CL38 cells suggested that mutating R99K of H2A1H pared the in vitro stability of the H2A1H-H2B with drastically reduced the pro-proliferative effect observed the H2A2A3-H2B dimer reconstituted using purified on its overexpression (Fig. 3i). Mutating L51M also recombinant histones. Equilibrium unfolding of the negatively affected cell proliferation and had a synergis- reconstituted full-length H2A–H2B dimers, which was tic effect when substituted alongside R99K (Fig. 3j). e previously described [19], was used to perform the staassays were conducted with populations showing similar bility analysis. For details pertaining to the structural and levels of overexpressed proteins to rule out possible vari- stability characterization of the dimers, please refer to the ations resulting from any differences in the expression “Methods” section. Once the equilibrium unfolding level (Fig. 3h). Notably, the 16th residue where the rat curves for both H2A1H-H2B and H2A2A3-H2B dimers H2A1H and human H2A1C differ did not have any sig- were obtained, a comparative analysis of their stability nificant effect on the non-redundant effects of H2A1H in was carried out (Fig. 4a). Co-plotting the Fapp (apparent <ul style="display: flex;"><li style="flex:1">the assays performed by us (Fig. 3i, j). </li><li style="flex:1">fraction unfolded) of the H2A1H-H2B and H2A2A3-H2B </li></ul>dimers against the increasing temperature/denaturant concentration shows a hysteresis, suggestive of the difference in the propensity to unfold in response to the denaturant (Fig. 4a–c). e temperature of melting (Tm) for

Histone Isoform H2A1H Promotes Attainment of Distinct Physiological

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