Probing the competitive antagonism between O-GlcNAcylation and phosphorylation modifications of the nucleosome at histone H2B-Ser36

Abstract

Various nucleosome modifications interact with each other (crosstalk) to ensure a tighter control of chromatin regulation. The molecular details of these crosstalks remain largely unexplored due to a lack of suitable methods for the ready synthesis of modified nucleosomes. Here, we show the versatility of the Dehydroalanine moiety to expedite the synthesis of differently modified nucleosome and demonstrate its application in the study of potential antagonism between O-GlcNAcylation and phosphorylation modifications. MS-based affinity enrichment proteomics with these synthetic nucleosomes allows the identification of preferential protein binding partners for each modification, thus providing a plausible molecular mechanism for their functional outcome. We believe that the generality of the method described herein will accelerate the understanding of several similar crosstalks.

Introduction

The genetic information of eukaryotes is stored in a dynamic polymeric complex called chromatin, comprised mostly of DNA and histone proteins.¹ Chromatin not only serves to package the genomic DNA efficiently but also regulates chromosomal processes such as transcription, replication, and repair through the covalent modifications of its functional unit, the nucleosome.^2,3 These modifications can be posttranslational modifications (PTMs) of histone proteins or nucleobase modification of chromosomal DNA. In a biological context, nucleosomes are decorated with different modifications at multiple sites.⁴ These multiple modifications can establish a ‘crosstalk’ to act in a concerted manner, providing an extra layer of complexity in chromatin regulation as compared to the ‘two-state (on/off)’ regulation afforded by a single modification.⁵ The crosstalk between different histone modifications can occur through various mechanisms.^3,6 In one of the simplest mechanisms, two or more different histone modifications can compete for the same modification site leading to different epigenetic states.^7,8

An intriguing interplay between O-GlcNAcylation and phosphorylation can occur on serine or threonine residues of histone proteins.^9,10 Earlier studies have established a competitive behaviour between these two modifications to occupy the same sites on many different proteins, such as c-Myc oncogene protein, estrogen receptor β protein, RNA polymerase II protein, endothelial nitric oxide synthase protein as well as histone protein H2B in a context-dependent manner.^11-17 Both these modifications are dynamic in nature, regulating in response to cellular stimuli, and modulate the structure as well as interacting protein partners of the target proteins.¹⁸ Interestingly, the regulatory roles of these two modifications are often considered to be intertwined and to some extent reciprocal in nature. Herein, we probe such an interplay between these two modifications of histone protein H2B on a commonly occupied Serine residue (H2B-Ser36) in a nucleosomal context.

H2B-Ser36 was previously found to be a site for O-GlcNAcylation or phosphorylation modifications in two independent reports.^16,17 Bungard et al. found histone protein H2B to be phosphorylated at Ser-36 residue through the catalytic action of adenosine monophosphate-activated kinase (AMPK).¹⁶ AMPK,  a central energy regulator in eukaryotic cells, performs its function by modulation of gene expression through phosphorylation of various transcription factors and coactivators.¹⁹ The modification at H2B-Ser36 was found to facilitate transcription with the modified protein being associated with the promoter as well as transcribed regions of p53 target genes. Phosphorylation of H2B-Ser36 was found to be nutrient-sensitive as would be expected for an AMPK-dependent modification, with the level of modification decreasing in response to increased glucose level.

In a separate study, Sakabe et al. reported H2B-Ser36 as one of the modification sites for O-GlcNAcylation, although no functional consequence of the modification has been established to date.¹⁷ Interestingly, Fujiki et al. have earlier demonstrated a positive correlation between O-GlcNAcylation modification of histone protein and elevated glucose levels.²⁰ In light of such results, a possible competition between these two modifications to occupy the same site in a nutrient-dependent manner seems highly plausible.

Histone modifications typically achieve their functional outcome either through modulation of nucleosome structure or by promoting preferential binding of interactor proteins.^6,21,22 The modification site in this study is located on the lateral surface of histone octamer in the vicinity of nucleosomal DNA. The serine residue serves to delineate the protruding N-terminal tail of H2B from the nucleosome core (Figure 1a).¹ Given the location of modification site, H2B-Ser36 modifications will most likely not destabilize the octamer structure. However, the same cannot be said for physical effects of the modification on the nucleosome structure. H2B-Ser36 is known to interact with nucleosomal DNA, and modifications such as phosphorylation or O-GlcNAcylation on this site can potentially modulate the octamer-DNA interactions. Herein, to understand both the individual role of these nucleosomal modifications in an epigenetical context and crosstalk between them, we precisely synthesized ‘designer nucleosomes’ (Figure 1b).  The effect of the modifications on the nucleosome structure were probed by biophysical studies. These synthetic nucleosomes were also used in conjugation with quantitative MS to analyze respective nucleosome-binding protein partners to reveal potential effector proteins, providing a molecular basis for functional output of these modifications.

Results

Synthesis of modified H2B-Ser36 proteins

The study of various crosstalk mechanisms of the nucleosome have been impeded by lack of suitable methods for easy access to differently modified histone proteins, and their use to readily assemble designer nucleosomes.²³ Installing site-specific histone modifications through enzymatic means is often challenging due to the promiscuous nature of histone-modifying enzymes resulting in heterogeneous mixtures.^20,24 Recent developments have made the incorporation of phosphoserine into proteins possible using amber codon suppression, however low expression yields of the modified protein remain a concern.²⁵ Although chemical ligation-based protein semi-synthesis methods have been regularly used to assemble designer nucleosomes, the low throughput and time constraints have curbed its potential as a method of choice for assembly of a range of differently modified nucleosomes. To overcome these limitations, we envisaged the installation of a chemical handle at the site of modification which can be used to readily generate differently modified proteins (SI Figure XX). Earlier work in our group has established the versatility of the dehyrdoalanine (Dha) ‘tag’ for synthesis of acetylated, methylated, GlcNAcylated, and phosphorylated functional proteins using a ‘tag-and-modify’ approach.^21,22,26,27

To synthesize modified proteins, a mutant of histone protein H2B bearing a cysteine residue at site 36 was designed. The protein was expressed in E. coli cellsand later purified by standard chromatography techniques.²⁸ A Dha ‘tag’ was site-selectively installed by treating the H2B-S36C protein with 2,5-dibromohexanediamide (DBHDA) under denaturing conditions (SI Figure XX). The H2B-S36Dha intermediate protein bearing an electrophilic (Dha) tag was reacted with suitable nucleophiles (GlcNAc-thiol and Sodium thiophosphate respectively) separately to generate H2B protein bearing either GlcNAc (H2B-S36 GlcNAc protein) or phosphate mimics (H2B-S36 thiophosphate protein) at Ser-36 site (SI Figure XX). The proteins synthesized using this chemical protein modification strategy bear an thioether linkage instead of natural ether linkage, however previous studies have established the utility of such mimics for functional studies of histone PTMs.^21,22,26

In addition to behaving like an electrophile in Thia-michael reactions, Dha ‘tag’ has the potential to function as a radical acceptor. Recent work in our group and in other labs has exploited this mode of reactivity to enable carbon-carbon bond formation on protein motifs for introduction of various posttranslational modifications.^29,30 In earlier work, this C-C bond formation strategy was used to install a Phosphonodifluoromethylene alanine (Pfa) residue on serine-10 site of histone H3 to create a functional mimic of a phosphorylated histone protein. This fluorinated residue has been suggested to be a suitable alternative to phosphoserine residue based on its better mimicry of pKa, polarity and shape.^31,32 A similar approach was used for generation of H2B protein bearing a fluorinated mimic of phosphoserine at the Ser-36 position (H2B-S36 Pfa protein). H2B-S36 Dha protein was treated with (Bromodifluoromethyl)phosphonic acid in presence of sodium borohydride under anaerobic conditions yielding the desired protein (SI Figure XX).

With access to modified histone proteins, one can assemble and purify differently modified histone octamers separately. However, to expedite the synthesis of differently modified nucleosomes even further, we envisioned to perform these mild, bioorthogonal protein chemical reactions at a later stage (at histone sub-complex or even at nucleosome stage) to introduce the desired modification (Figure 1c). If possible, this would save precious starting material as well as time during assembly/purification of histone sub-complexes or/and nucleosome reconstitution. As such, in a proof-of-principle experiment, we assembled and purified a histone octamer complex with Dha as an auxiliary ‘tag’ for late-stage functionalization.

LC-MS/MS analysis of modified proteins

The site-specific nature of protein chemical modification was later verified by LC-MS/MS analysis. The commonly used fragmentation technique, collision-induced dissociation (CID), is not suitable for the analysis of labile PTMs such as glycosylation and phosphorylation.³³ Electron-transfer dissociation (ETD) fragmentation can be the method of choice for glycopeptide analysis as it fragments only the peptide backbone leaving the glycan on side-chain intact. However, data acquisition based solely on ETD provides lower sequence coverage and is not ideal for complex samples.³⁴ Hence, a hybrid method combining HCD and ETD (HCD-PD-ETD ) was optimized and used for glycopeptide analysis. ^35,36 For the analysis of phosphoproteins, another hybrid method based on CID and ETD fragmentation techniques was optimized. Since both these fragmentation techniques are complementary in nature (for e.g. their speed, preference for peptide charge state), a data-dependent decision tree algorithm was designed and optimized to increase the overall sequencing rate.³⁴ It should be noted that these analysis methods are generic, and can be used for analysis of complex biological samples comprising  labile modifications.

The synthesized proteins were digested enzymatically and analyzed on an UHPLC (Ultra-high performance liquid chromatography) coupled to a hybrid Ion Trap-Orbitrap mass spectrometer using afore-mentioned data-acquisition methods. LC-MS/MS analysis confirmed the homogeneous and site-specific nature of the protein chemical modification (SI Figure XX).

Refolding and purification of histone octamers

We then used the wt and modified H2B protein to assemble wt and differently modified histone octamers. H2B proteins were mixed with other canonical wt histone proteins (H2A, H3 and H4) under denaturing condition and dialyzed against high-salt buffer (2 M NaCl) to allow for histone octamer refolding. The resulting histone sub-complexes were purified and analyzed by Size-exclusion chromatography (SEC). All refolded octamers had similar SEC traces suggesting no major disruption of histone-folds upon modification, as would be expected given the location of the modification site (SI Figure XX). The refolded octamers were later analyzed by SDS-PAGE to confirm that each refolded octamer had the correct stoichiometry of constituent proteins (SI Figure XX).

Reconstitution of synthetic nucleosomes

Nucleosomes were reconstituted by the salt-gradient dialysis method using a 186 bp biotinylated DNA fragment containing the strong ‘601’ nucleosome positioning sequence.²⁸ The salt-gradient dialysis based nucleosome reconstitution relies on the sequential binding of H3/H4 tetramer to DNA at higher salt concentration followed by deposition of H2A/B dimers onto the DNA-tetramer structure at lower salt concentration. The reconstituted nucleosomes were analyzed using native-PAGE (SI Figure XX) which revealed similar mobility and reconstitution yield for the modified nucleosomes. Based on native-PAGE results, it appeared that neither GlcNAcylation nor phosphorylation modification of H2B-Ser36 has a substantial effect on nucleosome structure. However, it is of interest to further probe the reconstituted nucleosomes by CD spectroscopy to reveal finer details regarding the effect of the modifications on structure and stability of the assembled biomolecules.

MS-based interaction proteomics with modified nucleosomes

Since no direct structural perturbation of nucleosomes were observed upon modification, (to be confirmed by variable-temperature CD analysis) we envisioned to probe the nucleosome-binding proteome of modified nucleosome to understand their role in DNA-templated processes. MS-based affinity enrichment proteomics approach was used to identify and quantify nucleosome-binding proteins (Figure 1b).^21,22,29,37 Wt, H2B-S36 GlcNAc modified, and H2B-S36 thiophosphate modified nucleosomes were immobilized separately on streptavidin-coated magnetic beads. These immobilized nucleosomes were incubated with HeLa cell nuclear extract to affinity-enrich their putative interacting protein partners. After incubation, the beads were washed to remove non-specific protein binders. The interacting protein partners for each modified nucleosome were eluted from the beads and were digested enzymatically using the filter-aided sample preparation (FASP) protocol.³⁸ The resulting peptide mixtures from each sample were separated on an UHPLC and sprayed directly into a hybrid quadrupole-orbitrap (Q-Exactive) mass spectrometer operating under a data-dependent method.

The acquired mass spectrometry data were processed in pairs using MaxQuant software (e.g. GlcNAc modified nucleosome vs wt nucleosome). The relative quantification was done using the MaxLFQ algorithm (in-built in MaxQuant).^39-41 Since, the proteomics data is from a single pulldown experiment to avoid complications related to data-analysis, only protein groups identified in both the samples were used for analysis. Note that by doing so, information regarding the proteins which selectively bind to only one of two samples will be lost. Statistically significant protein partners for each batch were determined by significance B analysis based on Benjamini-Hochberg⁴² FDR. The list of all identified proteins for each batch and their ratios are given in (Supplementary Table 1). (Biological replicates need to be performed)

False discovery rate (FDR) based Significance B statistics revealed Serine/Threonine Protein Kinase WNK3 protein (WNK3) as the statistically most significant and most enriched interacting protein partner for GlcNAcylated nucleosome compared to wt nucleosome (Figure 2a). In case of phosphorylated nucleosome versus wt nucleosome, DNA topoisomerase 2 protein (TOP2B), Transformer-2 protein homolog alpha protein (TRA2A), 60S ribosomal protein L22-like 1 protein (RPL22L1), Serine/Threonine protein phosphatase PPP1CC protein (PPP1CC) and Transcription factor AP-2-alpha/epsilon protein (TFAP2A) were among the significantly enriched protein partners for modified nucleosome (Figure 2b).

Discussion

The sub-family of WNK protein kinases is composed of four human genes coding for WNK1, WNK2, WNK3, and WNK4 and is characterized by a lack of highly conserved catalytic lysine in subdomain II, leading to their name, With No K [Lysine].⁴³ These kinases have been implicated in regulation of different ion transporters in both the kidneys and extrarenal tissues.  Recently, there has been growing evidence for additional roles of WNK kinases in signaling cascades related to cancer. WNK3 has been suggested to be involved in the evasion of apoptosis and thus promotion of cell survival in a procaspase-3-dependent pathway.⁴⁴ Interestingly, phosphorylation of H2B at Serine-14 promotes apoptosis in a caspase dependent manner.⁴⁵ An interplay between these two modifications of H2B at Ser14 and Ser36 as a ‘control’ mechanism leading to eventual apoptosis state cannot be ruled out and requires further examination.

Interestingly, for the phosphorylated nucleosome, PPP1CA, a phosphatase, was observed amongst the significant protein partners (Figure 2b). It needs to be investigated if it is indeed the ‘eraser’ protein for this histone mark. Two proteins – DNA topoisomerase 2 protein (most enriched protein partner) and TFAP2A protein (a transcription factor) – previously linked to active transcription were found to be among the most significant interacting protein partners for phosphorylated nucleosome.^46,47 TOP2B protein is believed to remove excessive DNA supercoils during elongation and in turn promote transcription. The observation of these nucleosome-binding proteins that promote active transcription might provide a molecular basis for previously observed H2B-Ser36 phosphorylation promoted transcription.¹⁶

Next, the interacting protein partners for the GlcNAcylated nucleosome and the phosphorylated nucleosome were compared (Figure 2c). Certain proteins such as serine/threonine-protein kinase WNK3 protein, mortality factor 4-like protein, 5’-3’ exoribonuclease 2 protein and interleukin enhancer-binding factor 2 were amongst proteins significantly enriched for GlcNAcylated nucleosome as compared to phosphorylated nucleosome. Intriguingly, previous studies have found association between 5’-3’ exoribonuclease 2 protein and transcription termination.^48-50 Proteins encoded by genes VPS13B, TMPO, PA2G4 and KTN1 were those were selectively enriched for the phosphorylated nucleosome as compared to the GlcNAcylated nucleosome.

(Based on the proteomics results obtained after triplicates, possible molecular mechanism will be drawn which will be represented in Figure 3. The validation of molecular mechanism will be represented in Figure 4.)

(Conclusion)

In summary, we have here utilized the versatility of the Dha-based chemical protein modification method to readily install different histone PTMs starting from a single precursor, and in turn expedited the assembly of differently modified nucleosomes. The biophysical characterization did not reveal any significant structural change upon modifications. The synthetic nucleosomes coupled with MS-based proteomics were used for probing potential histone modifications crosstalk. The proteomics results, involving the discovery of novel interacting protein partners, provided the basis for a molecular mechanism for H2B-Ser36 phosphorylation mediated transcription. The multiplex approach used here is general and provides a simple yet powerful route to study of histone modification crosstalk which has previously lagged due to the low throughput and time constraints of methods for accessing modified histone proteins and designer nucleosomes.

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Figures (These figures are temporary, from the thesis (highly inspired from S112 work!) They will be updated in due course.)

(a)

(b)

(c)

Figure 1: (a) Nucleosome structure (adapted from 1KX5) showing the modification site, H2B-Ser36. The site has been enclosed in black box. H2A and H2B proteins are shown in different shades of red, H3 and H4 proteins in different shades of green, and DNA in grey. (b) Schematicforprobing the potential competitive antagonism of histone modifications at H2B-Ser36 using quantitative MS. H2B-S36Dha protein (intermediate protein) was synthesized which was later reacted to yield differently modified histone proteins. The modified histone proteins were assembled into designer nucleosomes using other canonical histone proteins and biotinylated DNA. Immobilized synthetic nucleosome were used for affinity enrichment of nucleosome-binding proteins. Pooled proteins from each sample were enzymatically digested and identified by LC-MS/MS analysis. The identified protein partners were quantified against each other using MS-based Label-free Quantification (LFQ). H2A/B dimers shown in red, H3/H4 tetramer shown in green, GlcNAc shown as a blue square, Thiophosphate shown as orange circle, Phosphofluoroalanine shown as dark brown circle. (c) Late-stage functionalization strategy for ready synthesis of multiply modified histone sub-complex and nucleosome starting from histone octamer (left) and nucleosome (right) bearing a Dha ‘tag’.

(All these will be replaced by volcano plots obtained after triplicate experiments)

(a)           (b)

(c)

Figure 2: Quantitative MS-based proteomics allowed identification and relative quantification of nucleosome-binding protein partners among each pair of differently modified nucleosomes. The proteins identified in both the samples have been shown as a square in a scatter plot. x axis, logarithmized ratio of LFQ intensity of protein groups in the bait over control pulldown experiment; y axis, log10-transformed summed up intensity of protein groups. Significant protein partners identified by Significance B analysis are shown in red with their gene name. (a) Scatter plot for proteins identified and quantified for GlcNAc modified nucleosome versus wt nucleosome. (b) Scatter plot for proteins identified and quantified for Phospho modified nucleosome versus wt nucleosome. (c) Scatter plot for proteins identified and quantified for GlcNAc modified nucleosome versus phosphor modified nucleosome.

Figure 3:Will depict the molecular mechanism proposed based on identified proteins from proteomics experiments.

Figure 4:Will show the results of validation experiment performed to support the mechanism proposed in Figure 3.