Mapping Post-translational Modifications of Mammalian Testicular

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Mapping Post-translational Modifications of Mammalian Testicular Specific Histone Variant TH2B in Tetraploid and Haploid Germ Cells and Their Implications on the Dynamics of Nucleosome Structure Satya Krishna Pentakota,∥,† Sankaran Sandhya,∥,‡ Arun P. Sikarwar,†,§ Nagasuma Chandra,‡ and Manchanahalli R. Satyanarayana Rao*,† †

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, Karnataka 560064, India ‡ Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India S Supporting Information *

ABSTRACT: Histones regulate a variety of chromatin templated events by their post-translational modifications (PTMs). Although there are extensive reports on the PTMs of canonical histones, the information on the histone variants remains very scanty. Here, we report the identification of different PTMs, such as acetylation, methylation, and phosphorylation of a major mammalian histone variant TH2B. Our mass spectrometric analysis has led to the identification of both conserved and unique modifications across tetraploid spermatocytes and haploid spermatids. We have also computationally derived the 3-dimensional model of a TH2B containing nucleosome in order to study the spatial orientation of the PTMs identified and their effect on nucleosome stability and DNA binding potential. From our nucleosome model, it is evident that substititution of specific amino acid residues in TH2B results in both differential histone−DNA and histone−histone contacts. Furthermore, we have also observed that acetylation on the N-terminal tail of TH2B weakens the interactions with the DNA. These results provide direct evidence that, similar to somatic H2B, the testis specific histone TH2B also undergoes multiple PTMs, suggesting the possibility of chromatin regulation by such covalent modifications in mammalian male germ cells. KEYWORDS: mass spectrometry, post-translational modifications, nucleosome models, histone−histone and histone DNA interactions

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INTRODUCTION Mammalian spermatogenesis is a complex and tightly regulated process which involves several mitotic and two rounds of meiotic divisions to form final mature haploid spermatozoa. This developmental process involves extensive structural reorganization of chromatin. Nucleosomes are the basic units of chromatin, consisting of a histone octamer around which ∼146 bp of DNA is wrapped in 1.75 superhelical turns. Each histone octamer is comprised of a single tetramer (H3−H4)2 and two (H2A−H2B) dimers. Nucleosomes are connected to one another by linker DNA which are bound by histone H1. Unlike somatic cells, the DNA of germ cells is packaged with varying amounts of testicular specific histone variants, such as TH2A, TH2B, and TH3, and also by linker histone variants H1t and HILS11,2 in addition to the canonical histones. Some of the histone variants, such as TH3, are incorporated into chromatin at early spermatogonial stages while the majority of the variants are incorporated during the meiotic prophase and during spermiogenesis.3,4 These histone variants are believed to play significant roles in modulating the chromatin architecture, thereby influencing important biological processes. In recent years, it has become increasingly evident that most of the © 2014 American Chemical Society

histones are subjected to several post-translational modifications (PTMs), such as lysine acetylation, methylation, ubiquitinylation, sumoylation, crotonylation, succinylation, malonylation, propionylation, butyrylation, arginine methylation, serine, threonine and tyrosine phosphorylation, serine, and threonine β-N-acetylglusamine (O-GlcNAc).5−8 There is enough evidence in the literature at present to suggest that these histone PTMs play significant roles in many of the DNA templated processes, such as transcription, recombination, chromosome condensation, segregation, and DNA damage and repair.9,10 Histone modifications can modulate chromatin structure directly by altering the net charge on the substrate PTM residues or can influence biological function by recruiting PTM specific interacting proteins at the site of histone modification.11 Over the recent years, mass spectrometry has become an efficient and invaluable tool for the identification of post-translational modifications when compared with the traditional protein microsequencing or antibody based assays. Although antibody based assays are very sensitive, they require Received: June 17, 2014 Published: September 25, 2014 5603

dx.doi.org/10.1021/pr500597a | J. Proteome Res. 2014, 13, 5603−5617

Journal of Proteome Research

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a prior knowledge of the type of modification and the site at which it is modified.12 It is very difficult to characterize the histone modifications individually due to lack of site specific antibodies. On the other hand, mass spectrometry based methods, such as peptide mass fingerprinting, detect the modifications based on their specific molecular weight changes while tandem mass spectrometry scans the modified peptide for accurate site and localization of the PTMs. Thus, mass spectrometric techniques have contributed significantly toward understanding the epigenomic landscape underlying normal developmental processes and also the pathogenesis of various diseases. TH2B, a testis specific histone variant that was discovered almost three decades ago, is expressed in a stage specific manner during mammalian spermatogenesis.1 It shares 85% of sequence homology with somatic H2B, while the majority of differences are at its amino terminus. In vitro transcription assays have revealed the presence of a repressor protein factor which inhibits the transcription of Th2b gene in early premeiotic stages.13 About 80% of nucleosomal H2B is replaced by TH2B during the meiotic prophase.1 Early studies from our laboratory have demonstrated that the TH2B containing pachytene chromatin are less compact in comparison with liver chromatin,14 and the DNase-I site mapping technique revealed the presence of DNase-I hypersensitive regions within the nucleosome core at the H2B−DNA interacting regions.15 A more recent study has demonstrated that octamers reconstituted with hTSH2B exhibit less stability when compared with octamers reconstituted with that of canonical somatic histones.16 Extensive studies on H2B and its post-translational modifications in various organisms have shown that these covalent modifications are implicated in many chromatin template mediated events, such as gene activation, DNA damage and repair, etc. For example, phosphorylation on serine 14 is involved in DNA damage and repair, 17 while phosphorylation on serine 32 is linked to cell transformation,18 and acetylation on lysine 22 is involved in promoting transcription.19 Despite extensive studies on H2B, it is rather surprising that information on the biological functions of TH2B and its post-translational modifications is very scanty. Recent studies on TH2B/TSH2B20 have shown that TH2B dimerizes with H2AL1/H2AL221 and is necessary for nucleoprotamine transition during spermiogenesis.22 Considering the fact that somatic H2B is subject to several diverse PTMs and that TH2B is a major histone variant in tetraploid and haploid male germ cells, the identification of PTMs on TH2B becomes very important in elucidating the epigenomic modifications associated with this major testis specific H2B variant. In this study, we report a comprehensive analysis of posttranslational modifications of TH2B in both tetraploid spermatocytes and haploid spermatids. Since the time of elucidation of the nucleosome core particle structure by Luger et al., many structures of nucleosome core particle containing histone variants have also been determined by X-ray crystallography and also by modeling studies.23 To gain further insights into the possible biological functions of PTMs of TH2B, we have also computationally modeled the TH2B containing nucleosome core particle using the crystal structure of nucleosome core particle 1KX5 as the template. Furthermore, we have also visualized the spatial orientation of these modified residues and inferred their possible implications on protein−protein and protein−DNA interactions.

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MATERIALS AND METHODS

Materials

All chemicals and Dulbecco’s Modified Eagle Medium were purchased from Sigma chemicals (USA). All the synthetic peptides were procured from Stork Bio laboratories (Estonia). Recombinant H2B protein was purchased from New England Biolabs (USA). Polyclonal anti-H2B antibody was purchased from Millipore (07−371). Secondary antibody conjugated with Alexa-488 dye was obtained from Invitrogen (USA). Male Wistar rats were obtained from the animal facility of JNCASR. All procedures for handling animals were approved by the animal ethics committee of the center. Isolation of tetraploid spermatocytes and haploid round spermatids from rat testes and extraction of histones

Spermatocytes and round spermatids were isolated by the use of centrifugal elutriation technique as described earlier.24 Briefly, testes were excised and decapsulated from 35 to 45 old day rats which were rinsed once with phosphate buffer saline (PBS) and then washed with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (v/v) (FBS). To 100 mL of medium, 0.27 mg/mL collagenase type IV and 60ul of 10 mg/mL DNase I were added and incubated for 20 min with intermittent mixing. The cell suspension was filtered through 4 layers of cheese cloth and centrifuged at 900g for 7 min. The supernatant was discarded and the pellet was rinsed with PBS and then centrifuged for 900g for 7 min. The pellet was then resuspended in 10 mL of PBS and loaded onto the elutriator (Beckman coulter JE 5.0 rotor). The elutriation buffer composed of 0.2% bovine serum albumin and 0.1% glucose in Dulbecco’s phosphate- buffered saline (1xDPBS). Both spermatocyte and round spermatid fractions were collected and stored on ice. These fractions were spun at 900g for 5 min and then rinsed with PBS. The purity of these fractions were evaluated by FACS. Histone extraction was carried out as previously described.25 Purification of TH2B by RP-HPLC

The acid extracted histones were subjected to reverse phase high performance liquid chromatography (RP-HPLC). The samples were injected onto Waters XBridge C18 column (19 mm × 150 mm, 5 μm diameter) with mobile phase of solution A (0.1% Trifluoroacetic acid in 5% acetonitrile and 95% water) and solution B (0.1% Trifluoroacetic acid in 90% acetonitrile and 10% water). Histones were separated by employing a two step gradient: 0−29% of mobile phase B in 21 min and 29− 50% of mobile phase B in 168 min at a flow rate of 1.5 mL/min. The eluted fractions were dried and run on a 15% SDS-PAGE. TH2B containing fractions were identified by Western blot by using TH2B specific antibody. TH2B containing fractions were pooled and reloaded onto the same C18 column. The gradient conditions employed during the second run were: 0−38% of mobile phase B in 24 min and 39−55% of mobile phase B in 176 min at a flow rate of 1.5 mL/min. The collected fractions were lyophilized and stored at −20 °C. Enzymatic protein digestion

TH2B purified from spermatocytes and spermatids were resolved on 15% SDS-PAGE and stained with commassie brilliant blue. The TH2B gel bands were excised and washed with 25 mM ammonium bicarbonate. Reduction and alkylation were performed sequentially with 10 mM dithiothreiotol and incubated at 60 °C followed by 50 mM iodo acetamide at room temperature. Digestion was performed either with trypsin 5604



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(sequencing grade, Promega) at 37 °C for 4 h or with chymotrypsin (sequencing grade, promega) at 37 °C for 12 h or with elastase (promega) at 37 °C for 12 h. The reaction was terminated by adding formic acid to a final concentration of 0.1%. The supernatant was used directly for LC-MS/MS analysis.

standard procedure of isotopic labeling which could be used as an internal standard. Therefore, any errors in the relative ionization of modified and unmodified peptides will be consistent between the two samples. Homology model of the nucleosome and minimization

BLAST searches (E-value 95% sequence coverage for spermatocyte and spermatid TH2B. The high mass accuracy of Orbitraps coupled with characteristic formation of immonium ion at an m/z value of 126 for acetylation has allowed us to make an unambiguous PTM assignment.46 LC-MS/MS analysis of TH2B from spermatocytes identified six acetylation, three monomethylation, and one phosphorylation site while that of TH2B from round spermatids identified four acetylation and two monomethylation sites. Modified peptides that are commonly observed in the digestion mixture of TH2B from spermatocytes and spermatids are listed in Table 1A and Supporting Information Table S1. A detailed analysis of the common modifications identified in both spermatocytes and spermatids is described below. The CID spectrum of a peptide with an m/z value at 715.402+ (Figures 2A and S2A) displayed a complete y ion series (y1 to y12) that matched a peptide spanning residues Pro2-Lys14. The increase in mass by 42 amu from y1 to y7 confirms a single

Identification of unique post-translational modifications observed only in tetraploid spermatocytes

The modified peptides observed exclusively in the digestion mixture of TH2B from tetraploid spermatocytes are listed in Table 1B and Supporting Information Table S2. The precursor ion at m/z 575.322+ had a mass increment by 42 amu from b2 ion, indicating the presence of acetylation modification on the 5608



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Figure 3. Post-translational modifications identified exclusively in tetraploid spermatocytes: (A) MS/MS spectrum of the precursor ion with an m/z value at 575.322+. (B) MS/MS spectrum of the precursor ion with an m/z value at 635.342+. (C) MS/MS spectrum of the precursor ion with an m/z value at 616.352+. (D) MS/MS spectrum of the precursor ion with an m/z value at 649.322+.

Table 2. Modified Peptides Were Identified Using Nano-LC/MS/MSa cell typec Spc Spc Spc Spc Spc Spc Spc Spc Spc Spc Spt Spt Spt Spt Spt Spt

peptide 2

13

Pro-Lys Pro-Lys13 8 Glu-Lys18 19 Ala-Lys25 47 Leu-Iso56 111 His-Lys122 2 Pro-Lys13 34 Cys-Lys45 102 Leu-Lys110 2 Pro-Lys13 2 Pro-Lys13 2 Pro-Lys13 8 Glu-Lys18 19 Ala-Lys25 34 Cys-Lys45 102 Leu-Lys110 2

modified peptide (m/z) 630.350 411.741 412.960 409.243 575.318 635.342 616.349 541.945 484.312 649.321 630.350 411.741 412.96 409.243 541.945 484.312

unmodified peptide (m/z)

modified residue 7ac

609.339 390.734 398.913 ND 554.313 614.337 609.339 537.272 477.306 609.339 609.339 390.734 398.913 ND 537.272 477.306

Lys Lys14ac Lys17ac Lys22ac Lys48ac Lys118ac Lys7me Lys36me Lys110me Ser5p Lys7ac Lys14ac Lys17ac Lys22ac Lys36me Lys110me

area modified

area unmodified

stochiometry (%)

× × × × × × × × × × × × × × × ×

5.21 × 10 3.03 × 108 3.38 × 107 NDb 3.56 × 109 2.29 × 109 5.21 × 108 9.77 × 106 1.17 × 1011 5.21 × 108 6.74 × 107 6.50 × 107 2.51 × 107 NDb 4.74 × 104 9.32 × 109

11.42 1.20 11.04 2.26 0.07 1.37 86.08 0.06 1.46 19.55 6.02 20.36

6.72 3.69 4.20 1.15 8.23 1.65 7.24 6.04 6.69 7.71 1.64 4.16 6.17 2.93 1.15 1.38

06

10 1006 1006 1006 1007 1006 1006 1007 1007 1006 1007 1006 1006 1005 1006 1007

8

96.12 0.15

a

Using peak areas of the unmodified peptide and modified peptides, the percentage of each modification was calculated. bOnly modified peptides identified, and the corresponding unmodified peptide was not detected; hence, a % cannot be calculated. cSPC, spermatocytes; Spt, spermatids.

Lys48 within the peptide (Lys47-Iso56) (Figures 3A and S3A). The identity of the peptide with an m/z value of 635.342+ was determined in a similar fashion (Figures 3B and S3B). In the CID spectra of a peptide with an m/z value of 616.352+, the increase in molecular weight of the precursor ion by 14 amu was evidenced by both b ions (b9 to b11) and y ions (y8 to y10), thus confirming a monomethylation modification on Lys7 within the peptide Pro2-Lys13 (Figures 3C and S3C). The CID spectra at an m/z value of 649.322+ displayed a mass increment of +80 amu, indicating a phosphorylation modification. The mass values of the first three N-terminal sequence ions b1 to b3 and the first eight C-terminal sequence ions y1 to y8 were not modified, leaving Ser5 as the only potential site of phosphorylation. Manual analysis of this modified peptide revealed a characteristic neutral loss (y9-80 and y10-80), confirming the presence of phosphorylation modification within the peptide (Figures 3D and S3D).

Quantitative analysis of PTMs of TH2B

PTM quantification was done by considering the peak area of unmodified to the peak area of modified and therefore calculating the % of each modification across two different stages, namely spermatocytes and spermatids, as listed in Table 2. All the N-terminal acetylation modifications (Lys7ac, Lys14ac, Lys17ac) have shown a general increase in their abundance when comparing spermatocyte TH2B with Spermatid TH2B, while Lys118 was modified to a lower level (0.06%) when compared with all other acetylation modifications of TH2B. The abundance of the Lys22ac modification could not be calculated due to the absence of unmodified peptide. Although both Lys48ac and Lys118ac are modified to lower levels in spermatocyte TH2B, their abundance could not be calculated due to the absence of the modified peptides in spermatid TH2B. The most abundant modification was found to be Lys36me, which is 86% for spermatocyte TH2B and 96% for spermatid TH2B. Although Lys110me was shown to be present in both 5609



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Figure 4. Model of the nucleosome with TH2B replacing H2B: (A) The top-down view of the nucleosome with histone TH2B and H2A sequences (two chains of each in red and yellow). All other chains are colored in shades of gray and labeled. DNA is shown in pale green tint. (B) shows the side view of the nucleosome with the location of residue positions that differ between H2B and TH2B, H2A and TH2A (in black). Panels C to G show interaction differences at residue positions that differ between TH2B and H2B. H2B and TH2B histone chains are colored in red. Residues on H2B and TH2B are indicated as green and pink sticks, respectively. The DNA backbone is shown in gray and bases that interact with H2B/TH2B are shown as sticks. Panels C and D are residue substitutions in TH2B that weaken interaction with DNA, Panels E and F are residue substitutions that show additional and stronger interactions with DNA. Panel G captures many residue interactions between H2B/TH2B with H2A and H2A′ (both colored in yellow). Panel H shows H2A and TH2A in yellow with residues indicated in green and yellow sticks, respectively.

structure. Although the highest sequence similarity was indicated with a human homologue (2CV5),48 these structures did not contain significant portions of the N-terminal tail, in which we were interested for our modeling studies. The rat histone sequences showed over 85% sequence identity with the corresponding template sequences from Xenopus laevis (1KX5). As shown in Figures 4A and S5, the overall structures of the nucleosome models 1, 2, and 3 that incorporate somatic and testicular variants of H2B and H2A were essentially similar to the structure of the nucleosome core particle of the template. It may be pertinent to point out here that TH2B and TH2A comprise 85% and 25% of the total H2B and H2A in germ cell chromatin in mammals. Each of the individual chains adopted the histone-fold-like structure with the well conserved globular core and retained the characteristic unstructured and positively charged N-terminal histone tails that extended out of the nucleosome through the DNA gyres. PSIPRED secondary structure predictions of the regions also showed that these regions occur as coils. It is quite possible that association with DNA induces formation of local secondary structures.49 The N-

spermatocytes and spermatids, it is modified to a much lower extent, 0.06% and 0.15%, for spermatocyte and spermatid TH2B, respectively. The ratio of phosphorylated peptide to unmodified peptide was determined to be 1.46% for Ser5 phosphorylation for spermatocyte TH2B while the modified peptide was not detected in spermatid TH2B. Structural and functional implications of nucleosome models incorporating testicular and somatic histones

Quaternary structure. As detailed above, we have identified several PTMs that we have detected in the testis specific TH2B by mass spectrometry. In order to determine the influence of each of these modifications on the nucleosome core particle structure and dynamics, it is important to position these modifications within the structure of the nucleosome core particle. For this purpose, we modeled the nucleosome core particle structure containing TH2B. In the present study, we have employed computationally generated models of the nucleosome to describe the organization and assembly of the nucleosome using rat somatic histone sequences. The template sequence of Xenopus laevis (1KX5) was used as the reference 5610



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a The column titled C lists whether the residue is conserved among high-scoring homologues. Y indicates conservation across eukaryotic chordates; N implies that the residue is not conserved across the sample set (the multiple alignment of homologues is provided in Figure S4b).

Y TH2B K109, K117 K105, K113 8

K110, K118

Y Y TH2B K28 K35 K24 K31 6 7

R29 K36

Y TH2B,H2B K21 K17 5

K22

TH2B, H2B S15 K16 S11 K12 3 4

F16 K17

K13 K14 K9 2

1

Y Y

Hydrogen bond with DNA backbone in the template and in both NM1 and NM3. Same residue also involved in a main chain−side chain interaction with P7 in the template. On PTM, the interaction with DNA is weakened in both TH2B and H2B. F16 loses bond with the DNA backbone that is seen in S11 in1KX5 and S15 in H2B K12 in the template shows polar contacts with DNA and main chain−side chain interactions with S11. K16 and K17 acetylation in NM3 and NM1 do not interact favorably with DNA. The intrachain interaction with S15 is retained in NM3 alone Do not interact with DNA backbone. Template shows intrachain hydrogen bonds with A13. These interactions are weakened on acetylation at K22 in TH2B (NM1). New interactions with DNA are introduced in the NM1 model containing TH2B. Possibly this tethers this region strongly with DNA. Lies in a region that strongly interacts with DNA. Methylation does not alter interaction with DNA; however, the residue lies on a loop, and increased net positive charge on lysine through the addition of the methyl group could contribute to tighter binding with DNA. Solvent exposed and could play a role in packing with neighboring nucleosomal unit Y

N

TH2B: S5 (phosphorylation) TH2B, H2B: K7, K6 acetylation and methylation TH2B, H2B P4, K6

C (90%) site of PTM H2B TH2B 1KX5 S. No

Table 3. Residue Differences between H2B and TH2B and Impact of PTMs on Nucleosome Structurea

The models of the nucleosome incorporating TH2A and TH2B were compared with the models that contained H2B and H2A to study (a) the effect on stability of the nucleosome, (b) the interaction with DNA, and (c) the effect on assembly and packing between the histone chains. We describe a few of the important substitutions here and list other residue positions in Table 3. a. Effect on stability. Alignments of individual histone H2B and TH2B chains and H2A and TH2A chains show that the sequences of H2B and TH2B differ mostly at the N and Ctermini, as shown in Figures 1B and 4C. Likewise, H2A and TH2A also show differences at a few positions in the tail regions (Figures 4C and S4). When mapped onto the nucleosome models, it appears that these residues likely involve regions of the histone chains that interact with DNA. In all, of the 19 residue positions that differed between H2B and TH2B, only three lie on the conserved helix of the globular domain. None of the differences in other residue positions were seen to affect the overall quaternary assembly of the nucleosome (Figures 4A and S4), which is retained even after substitution of H2B with TH2B or H2A with TH2A. Indeed, the tight salt bridge interactions involving H4 Lys91 and H2B′ Glu72 that facilitate the tetramer−dimer interaction and play a critical role in octamer assembly are also observed in the nucleosome models that substitute H2B with TH2B.50 Similar interactions involving H4 Arg92, which also undergoes methylation, and H2B Glu78 are conserved in all three models. This suggests that in these assemblies also these residues are positioned similarly in the nucleosome and are amenable to post-translational modifications, as reported in other studies.51 Supporting Information Table S3 shows the structural and chemical properties at the interfaces in the octamer complex, computed using the PISA server.52 These results show that, overall, in each of the three assemblies, the number of residues at the interface, interface area, and solvation energy gain on formation of the interface are comparable. However, the total number of strong hydrogen bonds formed in the canonical histone model is higher than the other two models incorporating testicular variants. Inclusion of salt-bridge interactions further shows that the total number of strong contacts (hydrogen bond and saltbridge contacts) across all the interfaces of the nucleosome assembly in canonical histones is more stable (158 contacts out of 789 interface residues in model M3 vs 145 contacts out of 778 interface residues in model M1 and 143 contacts out of a total of 777 interface residues in model M2). b. Effect on interactions with DNA. In terms of the overall number of hydrogen bonds formed at the protein− DNA interface, the three models are comparable (Supporting Information Table S4). Two of the substitutions between H2B and TH2B weaken the interactions with the DNA, notably Pro11 in H2B with Ser12 in TH2B and Ser15 in H2B with Phe16 in TH2B. As shown in Table 3 and Figures 4C and 4D, in the former substitution, potential steric clashes with the DNA backbone were observed due to the N-terminal loop drawing closer to the DNA. In the latter case a substitution of the serine

potential structural implications

Effect of residues that differ between somatic and testicular histone sequences (H2B, TH2B and H2A, TH2A)

S5, K7

and C-terminal tail regions were found to be geometrically and chemically compatible with the corresponding atomic positions in the template and, hence, did not deviate significantly from their original positions. Each nucleosome model superposed on the template with RMSD < 0.5 Å.

Located on a loop at the N-terminal end of the histone chains. No interactions with DNA. Highly solvent-exposed and available for potential protein− chromatin interactions.

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Figure 5. Mapping locations of PTMs on histones H2B and TH2B. (A) Side view of nucleosome with the location of PTMs indicated in black. PTMs of H2B (Lys-6,13,16,21) are structurally equivalent to those on TH2B (Lys-7,14,17,22). Panels B, C, and D show modifications in interactions for residues that undergo PTMs. In each case, the interactions of lysine in the unmodified histone chain (in blue stick) with the DNA backbone are compared with the modified lysine of TH2B (orange stick representation) or with the modified lysine of H2B (yellow stick representation).

c. Effect on packing and interactions between the histone chains of the octamer. Two of the substitutions in TH2B were found to destabilize the assembly of the nucleosome due to loss of hydrogen bonds involving other histone chains. These included the substitution of Asn68 in H2B with Thr69 in TH2B and the replacement of Arg78 in H2B with Ser80 in TH2B. The nucleosome model with H2B showed a hydrogen bond between Asn68 in H2B with Gly103, the terminal residue of H4′, which was lost on substitution with Thr69 in TH2B. Further, the substitution of Arg78 in H2B with Ser80 in TH2B (Figure 4G) results in the loss of hydrogen bonds with the side chain hydrogens of Asn39 and Gly38 of H2A′. Finally the hydrogen bond interactions between His82 in H2B and Glu42 in H2A with Asp39 in H2A′ were not observed in the nucleosome model that contained TH2B. These substitutions could result in looser packing between the chains in the model with TH2B. Similarly, in comparisons of H2A with TH2A (Figure S4), nine substitutions were observed that lie largely on the N- and C-terminal tails and are unlikely to affect packing or stability of the nucleosome. Two of the residue substitutions involve residues that could interact with the DNA. The substitution of Thr17 in H2A with Ser17 in TH2A shows stronger interaction with the DNA backbone (Figure 4H). Ser17 lies in the loop that is solvent exposed and in close proximity to Ser20 (H2A) that is replaced by Phe20 (TH2A). Possibly, in H2A the presence of Thr17 and absence of a hydrogen bond with the DNA backbone renders this loop more flexible, and therefore, Ser20 which is solvent exposed is accessible to phosphorylation.

residue that is highly conserved in eukaryotes results in the loss of potential hydrogen bonds with the DNA backbone. Further, this substitution also results in the loss of a residue that has the potential to undergo post-translational modifications in conjunction with the neighboring lysine. Two substitutions that strengthened the interaction with DNA were also observed. These include the substitution of lys28 in H2B with Arg29 in TH2B and glu29 in H2B with lys30 in TH2B. In the former case, Arg29 in TH2B (Figure 4E) is seen to penetrate the DNA gyres and contact consecutive bases through additional polar contacts. In the latter case also, Lys30 makes additional moderately electrostatic hydrogen bonds with the DNA phosphate backbone that tethers the N-terminal loop region with the DNA (Figure 4F). In somatic H2B, the signature “KERKRSRK” (HBR domain) is well conserved among homologues and has been implicated in multiple roles such as transcriptional repression of the N-terminal tail of H2B,53 which is a site of binding of chaperone proteins such as FACT.54 Indeed, the DNA binding signature lies positioned between the two turns of the DNA and is seen to be solvent exposed. Substitution of this residue with lysine brings it into a stronger interaction with DNA, and possibly this prevents association of this region with the DNA binding proteins. Further, this region in TH2B shows another substitution of the conserved Ser33 (H2B) with Cys34 (TH2B). This substitution does not alter the hydrogen bond interaction involving the main chain of Ser33 or Cys34 with the DNA backbone. However, as these residues are solvent accessible and if Ser33 is involved in any post-translational modifications, this may be affected on replacement with TH2B. Interestingly, Ser33 of somatic H2B undergoes phosphorylation in H2B and is involved in cell transformation.18 5612



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Figure 6. Biological validation of the TH2B Lys17 ac modification. (A) Primary sequence alignment of H2B and TH2B showing the conservation of Lys17 (highlighted in red). (B) Immunoblotting of affinity purified TH2BK17ac antibody against the liver and testicular nuclear extracts showed a specific reactivity only to testicular nuclear lysate. The preimmune serum did not show any reactivity. (C) Specificity of TH2BK17ac antibody was demonstrated by peptide competition assay. Western blot analysis was performed using acid extracted testicular histones. Preincubation of the TH2BK17ac antibody with TH2BK17ac peptide resulted in the loss of the signal at ∼15 kDa (Lane 2). Preincubation of TH2BK17ac antibody with TH2B unmodified peptide or no peptide showed no difference in the reactivity toward testicular histones (Lanse 1 and 3). (D) Immunoblot analysis using TH2BK17ac antibody against acid extracted histones from a pure population of tetraploid spermatocytes and haploid spermatids confirms the presence of this TH2BK17 acetylation modification across both the stages, which is in good agreement with our mass spectrometry data. (E and F) ×100 immunofluorescence images of spermatocytes and spermatid nucleus stained individually with TH2B (green) (top panel, E and F) and TH2BK17ac antibodies (green) (bottom panel, E and F). Nuclei were visualized by DAPI staining. Merged images are shown as indicated. Scale bars: 5 μm.

Structural and functional implications of post-translational modifications in TH2B and H2B

We compared the structures of the nucleosomes in the presence and absence of each PTM to assess the impact of each modification depending on its effect on interaction with the neighboring histones and also DNA. As shown in Figure 5A, the PTMs at S5, K7, K14, and K17 on TH2B and K6, K13, and K16 mapped to the solvent accessible histone tails. Modifications at K48, K110, and K118 in TH2B mapped to the solute accessible face of the nucleosome and could alter higher-order chromatin structure and chromatin−protein interactions. K13, K16, K21 (H2B) and K14, K17, K22, K36 modifications mapped to the lateral surface of the histones and are capable of affecting histone−DNA interaction. None of the modifications were found in the histone−histone interfaces, and hence, they are unlikely to disrupt intranucleosomal interactions. Further, the four residues that undergo modification in H2B, namely, Lys6, Lys13, Lys16, and Lys21, were found to be structurally equivalent to Lys7, Lys14, Lys17, and Lys22 of TH2B; therefore, the interactions effected with DNA are likely to be comparable between the two chains. Of the ten modifications examined in TH2B, three modifications were observed to weaken the interaction with DNA. These included acetylations at Lys14 in TH2B; Lys13 in H2B (Figure 5B), Lys17 in TH2B; Lys16 in H2B (Figure 5C) and Lys22 in TH2B; Lys21 in H2B (Figure 5D). In each case, the weakening of the

The availability of models of the nucleosome core particle incorporating testicular variants of H2B and H2A through the present study presented us an opportunity to map the location of the observed post-translational modifications in the different histone chains. For the histone variants investigated here, their spatial location and interactions with neighboring histone chains or with DNA might offer clues to explain their structural and biological roles in the germ cell chromatin. Each of the observed PTMs reported here was incorporated into the nucleosome assemblies one at a time to assess its potential impact on interaction with other histone chains or DNA. For histone H2B, multiple acetylation sites have been identified at Lys6, Lys13, Lys16, and Lys21.47 For TH2B, a single phosphorylation is observed at Ser5, multiple acetylation at Lys 7 , Lys 14 , Lys 17 , Lys 22 , Lys 48 , and Lys 118 , and few monomethylations at Lys7, Lys36, and Lys110. Both acetylation and phosphorylation can alter the charge of the tails and, therefore, have the potential to influence chromatin through electrostatic mechanisms. Studies have also shown that these modifications primarily alter the interaction between chromatin and nonhistone proteins.55,56 5613



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counterparts to a different extent among, which TH2B replaces H2B on a genome wide scale. Since histones are known to exert their functions by PTMs, it is very likely that PTMs might play a significant role in the biological function of TH2B in germ cells. To begin with, we confirmed that TH2B is expressed in tetraploid spermatocytes and haploid spermatids, and the purified TH2B proteins from these two different cell types were extensively characterized for PTMs by mass spectrometry. We have identified four acetylation sites (Lys7, Lys 14, Lys17, and Lys22) and two monomethylation sites (Lys36 and Lys110) as common modifications observed across both spermatocytes and spermatids. In addition to this, we have also identified two acetylation sites (Lys48 and Lys118), one monomethylation site (Lys7), and one phosphorylation site (Ser5) only in tetraploid spermatocytes. However, we cannot exclude the possibility that these specific modifications may be less abundant, and hence, we were not able to identify them in round spermatids. We would also like to mention here that we are surprised that Lu et al. described the PTMs of TH2B isolated from spermatogonial cells,58 although it is very clear from earlier reports as well that the experiments with TH2B antibodies presented here clearly show that TH2B is not expressed in spermatogonia. A comparison of the PTMs on canonical H2B reveals that 5 out of 10 PTMs were identified exclusively on TH2B (Figure 7). Interestingly, acetylation modifications found on the N-

interaction with DNA involves residues that lie in loop regions and result in moving away from the DNA.57 Other PTMs reported in this study are located on the solvent accessible nucleosomal face as shown in Figure 5A. Notably the location of K48, K118 that undergo acetylation and K110 that undergoes methylation may play a role in the higher order chromatin packing and inter-nucleosomal packing. Further, it is unclear whether these modifications/histone marks occur such that all these residues are modified in a regulated or coordinated manner. Certainly, their spatial location and accessibility seem to suggest that this could be possible. Biological validation of TH2BK17 acetylation

Among the several PTMs that we have observed in the present study, we were interested to validate at least one PTM in vivo by the antibody approach. By primary sequence alignment of TH2B and H2B N-terminal tails from rat, mouse, and humans, we have found that the Lys17 is well conserved (Figure 6A). Interestingly, the same Lys residue is acetylated in H2B as reported.44 The specificity of α-TH2BK17ac antibody that was generated in a female rabbit was demonstrated by an immunoblotting experiment performed with liver and testicular nuclear extracts and also recombinant TH2B. The affinity purified antibody shows a clear signal toward testis nuclear extract but showed no reactivity toward liver or to recombinant TH2B (Figure 6B, upper panel). As demonstrated in Figure S6, although in vivo acetylated H2B is present in both liver and testis (Figure S6, lower panel), α-TH2BK17ac antibody showed reactivity only toward testicular histones, indicating the absence of cross reactivity with the acetylated H2B (Figure S6, lower panel). To further characterize this modification specific antibody, we have performed a peptide competition assay using TH2BK17 ac peptide and the backbone peptide. The finding that the modified antibody is specific for acetylation on Lys17 on TH2B, as preincubation of the α-TH2BK17ac antibody with an unmodified peptide did not alter its reactivity (Figure 6C, lane3), while α-TH2BK17ac antibody when preincubated with TH2BK17ac peptide resulted in complete loss of the signal (Figure 6C, lane2). Western blot analysis with acid extracted histones from tetraploid spermatocytes and haploid round spermatids using α-TH2BK17ac antibody demonstrated the presence of Lys17 modification in both tetraploid spermatocytes and haploid spermatids (Figure 6D). Additionally we also performed immunostaining using backbone α-TH2B antibody and modified antibody separately on total testicular cells. As can be seen from Figure 6E and F, the α-TH2BK17ac antibody is restricted to a few chromatin domains when compared with the backbone antibody staining that was observed along the entire length of chromosomes.

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Figure 7. Summary of the post-translational modifications identified on H2B and TH2B. (A) Illustration of already identified PTMs on somatic H2B from the literature. (B and C) Identification of different PTMs of TH2B from tetraploid spermatocytes and haploid spermatids, respectively. Amino acid residue numbers are indicated below the sequence.

terminal tail of H2B are conserved even in TH2B across both spermatocytes and spermatids. Studies conducted on the Nterminal tail of H2B reveal that it is highly required for chromosome condensation,59 and acetylation on the Nterminal tail of somatic H2B is known to relieve the repressed state of chromatin.19 Therefore, it is very likely that acetylation on the N-terminal tail of TH2B also has a similar function in germ cell chromatin. By now there is an increasing amount of evidence to support the idea that even histone variants are known to influence nucleosomal dynamics. Hence, models incorporating these histone variants should provide a scaffold to identify these subtle changes leading to functional differences. In the present study we have computationally modeled nucleosome containing TH2B, replacing the somatic H2B (model 1). Since germ cell chromatin also contains 25% of total H2A being replaced by the testis specific H2A variant, TH2A, we have also modeled the nucleosome containing TH2A and TH2B (model 2) and compared both these models with model 3, containing somatic

DISCUSSION

The present study describes various post-translational modifications that are identified on a testis specific histone H2B variant, TH2B, at two different stages of male germ cell differentiation in mammals, namely tetraploid spermatocytes and haploid round spermatids. Tetraploid spermatocytes are characterized by unique meiotic chromatin functions, including sex chromosome inactivation, in addition to housekeeping functions, such as transcription of autosomes. On the other hand, haploid round spermatids get ready for their nucleosomal histones to be finally replaced by protamines (more than 90%) in mature spermatozoa. During the process of spermatogenic differentiation, many histone variants replace their somatic 5614



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spermatocytes (86%) and spermatid (96%) structurally this does not appear to directly affect the interaction of this lysine residue with the DNA backbone. It is possible that the addition of the methyl group can influence the interaction of the HBR domain with DNA, given the importance of HBR domain in FACT-dependent removal of H2A/H2B from nuclesomes.54 It will be interesting to study whether this modification influences the binding of the FACT complex to histone TH2B, thereby influencing dimer−DNA interactions by the HBR domain. In addition to this we have also observed two different modifications (Lys7ac and Lys7me1) on the same lysine residue. It is very likely that such bivalent modifications may be relevant for chromatin dynamics and functions in a context dependent manner. A recent study by Montellier et al.22 has shown that somatic H2B is overexpressed in TH2B knockout mice and also identified a unique histone modification map such as crotonylation of H4K77 and H3K122 and also methylation on H4R35, H4R55, and H2BR72. These modifications are wellknown to affect either histone-DNA or histone−histone contacts. Moreover, Shinagawa et al. have demonstrated the presence of both TH2A and TH2B in oocytes and their role in activation of paternal genome post-fertilization.61 Given the importance of PTMs, it would be interesting to see if these PTMs contribute toward a conserved mechanism of the destabilization of nucleosomes in germ cells and oocytes. In summary, our present study has shown that the packing of the histone octamer within the nucleosome is comparable in all three models (TH2B/H2A, TH2B/TH2A, and H2B/H2A), with tighter packing seen with the model of canonical histones. A few residue substitutions weaken interactions with the DNA due to the introduction of incompatible side chains in the region, and a few appear to strengthen the interaction due to chemical compatibility to form new hydrogen bonds. Of the ten PTMs identified on TH2B, three modified residues markedly affect interaction with the DNA by bringing in the side chains to close proximity of the DNA. It would be really challenging to examine the biological functions of these modifications either singly or in combination that influence several chromatin template mediated DNA-transaction processes. In this direction, we have begun to raise modification specific antibodies. One of the (Lys17ac) antibodies as shown here reveals that indeed specific chromatin domains are stained by this antibody, and these specific chromatin domains are being investigated presently in our laboratory.

H2A and H2B. This comparison has resulted in valuable information regarding nucleosomal structural dynamics, specifically with respect to histone−DNA and histone−histone interactions. High sequence conservation of TH2B and H2B resulted in generating a quaternary assembly of the nucleosome models that did not differ much from the template. Out of the 19 residue differences between rat TH2B and H2B, four residues (Ser12, phe16, Arg29, and Lys30) were shown to result in differential interactions with DNA. The modeling study presented here shows that packing of the histone octamer is similar in all three models and the stability of the octamer is retained in all three models, suggesting that such conservation has evolved to ensure robustness, given the important role of nucleosomes, the fundamental unit of eukaryotic chromatin. A few residue substitutions weaken interactions with DNA, due to the introduction of incompatible side chains in the region, and a few residues appear to strengthen the associations with the DNA due to chemical compatibility to form new hydrogen bonds. Such subtle changes may bring about perturbations in nucleosome dynamics, facilitating various chromatin templated processes through association with many other chromatin binding proteins. It is also worth mentioning here that Li et al.16 have shown that histone octamers prepared with hTSH2B are less stable compared to an histone octamer containing somatic H2B. In our model 3, we did not find any evidence to explain such loosened H2A/TH2B interactions within the nucleosome structure. It is possible that such destabilization of the histone octamer may be apparent in the absence of DNA when nucleosomes are unwound during chromatin templated DNA transaction processes. Earlier studies from our laboratory had also demonstrated that nucleosome core particles from pachytene chromatin are hypersensitive to DNase-I and micrococcal nuclease at the H2B/TH2B interacting region of nucleosomal DNA.14,15 In comparisons of our three models, we observed that the total number of strong hydrogen bonds formed in the canonical histone model is higher than those in the other two models incorporating testicular variants. When salt-bridge interactions were also considered, the total number of strong contacts (hydrogen bond and salt-bridge contacts) across all the interfaces of the nucleosome assembly in canonical histones was found to be more stable. It is quite possible that the residue substitutions in TH2B in combination with the identified PTMs might together contribute to loosened nucleosome core particles. The assembly of a computationally derived model of a nucleosome containing TH2B has also helped us to spatially map the PTMs of TH2B that we have identified in this study. All the N-terminal modifications were mapped onto the solvent accessible face of the nucleosome. The locations of residues that undergo PTMs in TH2B are structurally equivalent to those of residues that undergo PTMs in H2B. A comparison of the effects of the modified residues within the nucleosome core particle has demonstrated an impact on interaction with the DNA. The highly acetylated N-terminal tail of TH2B may serve as a docking site for the recognition by the Bromo domain containing testis specific factor (Brdt), which is highly expressed in spermatocytes and spermatids and has also been shown to be a master regulator of gene expression in spermatogenesis.60 Other modifications (Lys48ac, Lys110me1, and Lys118ac) identified in this study were mapped onto the solvent accessible face of the nucleosome. Although monomethylation on Lys36 remains most abundant across both

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ASSOCIATED CONTENT

S Supporting Information *

Purification of in vivo TH2B; fragmentation tables (related to Figure 2); fragmentation tables (related to Figure 3); multiple sequence alignment of Rat TH2A and TH2B; models of the nucleosome; specificity of TH2BK17ac antibody; modified peptides identified in the digestion mixture of TH2B protein from both spermatocytes and spermatids; modified peptides identified exclusively in the digestion mixture of TH2B protein from both spermatocytes; features of histone−histone chain interfaces determined by using the PISA server; features of DNA−histone interfaces determined by using the PISA server. This material is available free of charge via the Internet at http://pubs.acs.org. 5615



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AUTHOR INFORMATION

Corresponding Author

*Tel: +91-80-2208-2864. Fax: +91-80-2208-2766. E-mail: [email protected]. Present Address §

(A.P.S.) Weill Cornell Medical College (WCMC-Q), Education City, Doha 24144, Qatar. Author Contributions ∥

S.K.P. and S.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.S. is grateful for financial support through the Women Scientist Scheme-A, of the Department of Science and Technology, Government of India. Research in the laboratories of M.R.S.R. and N.C. is supported by grants from the Department of Biotechnology, Government of India. M.R.S.R. acknowledges the Department of Science and Technology for a J. C. Bose and SERB Distinguished Fellowship. We thank B. S. Suma for help with confocal microscopy. We thank Richard Jones from MS Bioworks for help with the MS/MS analysis.

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ABBREVIATIONS MS/MS, tandem mass spectrometry; CID, collision-induced dissociation; RP-HPLC, reversed-phase high performance liquid chromatography; m/z, mass-to-charge ratio; Ac, acetylation; Me1, monomethylation; p, phosphorylation

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