Apoptosis-associated acetylation on histone H2B is an epitope for lupus autoantibodies

doi:10.1016/j.molimm.2009.08.009

Molecular Immunology

Volume 47, Issues 2–3, December 2009, Pages 511-516

https://doi.org/10.1016/j.molimm.2009.08.009 Get rights and content

Abstract

Objective

Nucleosomes have been identified as a key autoantigen in systemic lupus erythematosus (SLE). Nucleosomes are present in the circulation due to a disturbed apoptosis and/or an insufficient clearance in SLE. During apoptosis, histones can be modified, thereby making them more immunogenic. Recently, we showed the importance of apoptosis-induced acetylation of histone H4 in the pathogenesis of SLE. The lupus-derived antibody LG11-2 was previously shown to react with the N-terminal tail of histone H2B, which contains amino acid residues that can be modified including phosphorylation of serine 14, known to occur during apoptosis. Here, we evaluate whether apoptosis-induced histone modifications on H2B exist that are targeted by LG11-2 or lupus-derived plasmas.

Methods

Immunofluorescence staining and western immunoblot analysis of control, apoptotic and trichostatin A-treated cells/chromatin were performed with monoclonal antibody LG11-2. Reactivity of LG11-2 and plasmas from lupus mice and SLE patients with acetylated and/or phosphorylated H2B peptides was determined in competition ELISA.

Results

LG11-2 showed enhanced reactivity with apoptotic and hyperacetylated H2B compared to normal H2B. This enhanced reactivity was due to the acetylation of lysine 12 in H2B. This modification was also recognized by autoantibodies from pre-diseased lupus mice, but to a lesser extent by plasmas of diseased lupus mice and lupus patients.

Conclusions

The apoptosis-induced acetylation on H2BK12 is a target for autoantibodies in SLE. Since the anti-H2BK12ac reactivity was mainly found in pre-diseased lupus mice, this epitope seems important in the early phase of the anti-chromatin autoimmune response with subsequent epitope spreading to unmodified H2B.

Introduction

One of the characteristic features of systemic lupus erythematosus (SLE) is the formation of anti-nuclear antibodies, including anti-histone antibodies. Nucleosomes, composed of an octamer of core histones with DNA wrapped around, are a major autoantigen in SLE. Nucleosomes are released into the circulation due to a disturbed apoptosis and/or insufficient clearance of apoptotic material in SLE (Dieker et al., 2002). Histone modifications take place mainly at the N-terminal tails of the histones, thereby adjusting the structure and function of chromatin. According to the histone code hypothesis distinct (combinations of) histone modifications are related to specific chromatin-related functions and processes (Jenuwein and Allis, 2001). Histone modifications play, for example, a role in gene transcription, DNA repair, mitosis, meiosis, development and most likely in apoptosis. The first identified histone modification linked to apoptosis was the phosphorylation of serine 14 on histone H2B that is catalyzed by the mammalian sterile 20 kinase (Mst1) (Ajiro, 2000, Cheung et al., 2003). Other identified modifications linked to apoptosis include the SLE-associated tri-acetylation of histone H4, as we described previously (Dieker et al., 2007) and the recently identified phosphorylation of threonine 45 on histone H3 (Hurd et al., 2009). We and others hypothesized that histone modifications set during apoptosis makes released apoptotic nucleosomes more immunogenic, leading to an immunogenic presentation by antigen presenting cells, which ultimately could result in autoantibody production. Hence, characterization of epitope specificity of autoantibodies in SLE could reveal histone modifications that occur specifically during apoptosis.

Initially, the epitope of the lupus-derived antibody LG11-2 was roughly mapped in the N-terminal tail of histone H2B (amino acids (aa) 6–18) using peptide ELISA (Monestier et al., 2000). Furthermore, the epitope of LG11-2 seems accessible at the surface of the nucleosome (Muller et al., 1984, Stemmer et al., 1997). The amino-terminal tail of histone H2B appears to be essential for chromatin condensation (de la Barre et al., 2001), and, as mentioned, the phosphorylation of serine 14 on H2B is linked to apoptosis (Ajiro, 2000, Cheung et al., 2003) and also to double strand DNA breaks (Fernandez-Capetillo et al., 2004).

Importantly, the epitope of LG11-2 is located within an identified lupus T cell epitope (Lu et al., 1999). Furthermore, LG11-2 has been used for the detection of apoptotic nucleosomes in ELISA and binds to apoptotic blebs (Radic et al., 2004, Salgame et al., 1997). Moreover, it has been shown that apoptotic material can activate dendritic cells and subsequently T cells, while administration of apoptotic cells or dendritic cells loaded with apoptotic material induces/enhances autoantibody production (Mevorach et al., 1998, Tzeng et al., 2006).

Autoantigen modifications set during apoptosis seem important in these immunogenic processes (Utz et al., 2000). Therefore, LG11-2 with an epitope in H2B encompassing a potential apoptosis-modified residue, is an interesting candidate to further characterize. In this report, we evaluate whether apoptosis-induced modifications on H2B occur that are targeted by the lupus-derived autoantibody LG11-2 and by plasmas from lupus mice and SLE patients.

Section snippets

Cell cultures and induction of apoptosis

Jurkat cells were cultured in complete medium (RPMI 1640 Dutch modification, 10% fetal calf serum, 1% penicillin–streptomycin, 1% glutamax and 1% pyruvate). To inhibit histone deacetylases, cells were treated with 100 ng/ml trichostatin A (Enzo Life Sciences, Raamsdonksveer, the Netherlands). Apoptosis was induced by treating cells with 2 μg/ml camptothecin (Sigma-Aldrich, Zwijndrecht, the Netherlands). Apoptosis was determined by analyzing Annexin V-fluorescein isothiocyanate (FITC) (ITK

Identification of a modification in the epitope of the lupus mouse-derived monoclonal autoantibody LG11-2

Possible modifications within the epitope of the lupus-derived autoantibody LG11-2, originally found to react with histone H2B (Monestier et al., 2000), were evaluated by determining the reactivity of LG11-2 with synthetic histone H2B peptides (aa 1–18) in a peptide inhibition ELISA. Unmodified H2B peptide was coated onto the plates and LG11-2 was pre-incubated with each of the (modified) H2B peptides prior to incubation with the coated unmodified peptide. The reactivity of LG11-2 pre-incubated

Discussion

We demonstrate that the lupus-derived antibody LG11-2 preferably reacts with H2B acetylated at lysine 12, which is formed during apoptosis, located within the epitope of LG11-2 as mapped with unmodified peptides previously (Monestier et al., 2000).

The higher reactivity of LG11-2 with apoptotic histones supports previous findings where LG11-2 was shown to react with apoptotic blebs and nucleosomes (Radic et al., 2004, Salgame et al., 1997).

We previously showed that hyperacetylation of histone H4

Acknowledgements

This work was supported by the Dutch Kidney Foundation (grant C05.2119), the PhD student program of the Radboud University Nijmegen Medical Centre and the French Centre National de la Recherché Scientifique (CNRS). Dr. T. Radstake (Department of Rheumatology, Radboud University Nijmegen Medical Centre) and Dr. W. van Venrooij (Department of Autoimmune Biochemistry, Radboud University Nijmegen) are greatly acknowledged for providing RA and SSc patients’ sera.

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Apoptosis-associated acetylation on histone H2B is an epitope for lupus autoantibodies

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Cell cultures and induction of apoptosis

Identification of a modification in the epitope of the lupus mouse-derived monoclonal autoantibody LG11-2

Discussion

Acknowledgements

J. Biol. Chem.

Cell

J. Immunol. Methods

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Mech. Dev.

J. Biol. Chem.

The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation

EMBO J.

A SmD peptide induces better antibody responses to other proteins within the small nuclear ribonucleoprotein complex than to SmD protein via intermolecular epitope spreading

J. Immunol.

Apoptosis-induced acetylation of histones is pathogenic in systemic lupus erythematosus

Arthritis Rheum.

Triggers for anti-chromatin autoantibody production in SLE

Lupus

Posttranslational modifications of self-antigens

Ann. N. Y. Acad. Sci.

Phosphorylation of histone H2B at DNA double-strand breaks

J. Exp. Med.

Resetting the epigenetic histone code in the MRL-lpr/lpr mouse model of lupus by histone deacetylase inhibition

J. Proteome Res.