Resetting the Epigenetic Histone Code in the MRL ... - ACS Publications

The baseline level of gene expression varies between healthy controls and systemic lupus erythematosus (SLE) patients, and among SLE patients themselv...
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Resetting the Epigenetic Histone Code in the MRL-lpr/lpr Mouse Model of Lupus by Histone Deacetylase Inhibition Benjamin A. Garcia,† Scott A. Busby,† Jeffrey Shabanowitz,† Donald F. Hunt,‡ and Nilamadhab Mishra*,§ Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, Departments of Chemistry and Pathology, University of Virginia, Charlottesville, Virginia 22904, and Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forrest University School of Medicine, Winston-Salem, North Carolina 27157 Received June 22, 2005

The baseline level of gene expression varies between healthy controls and systemic lupus erythematosus (SLE) patients, and among SLE patients themselves. These variations may explain the different clinical manifestations and severity of disease observed in SLE. Epigenetic mechanisms, which involve DNA and histone modifications, are predictably associated with distinct transcriptional states. To understand the interplay between various histone modifications, including acetylation and methylation, and lupus disease, we performed differential expression histone modification analysis in splenocytes from the MRL-lpr/lpr mouse model of lupus. Using stable isotope labeling in combination with mass spectrometry, we found global site-specific hypermethylation (except H3 K4 methylation) and hypoacetylation in histone H3 and H4 MRL-lpr/lpr mice compared to control MRL/MPJ mice. Moreover, we have identified novel histone modifications such as H3 K18 methylation, H4 K31 methylation, and H4 K31 acetylation that are differentially expressed in MRL-lpr/lpr mice compared to controls. Finally, in vivo administration of the histone deacetylase inhibitor trichostatin A (TSA) corrected the site-specific hypoacetylation states on H3 and H4 in MRL-lpr/lpr mice with improvement of disease phenotype. Thus, this study is the first to establish the association between aberrant histone codes and pathogenesis of autoimmune disease SLE. These aberrant post-translational histone modifications can therefore be reset with histone deacetylase inhibition in vivo. Keywords: mass spectrometry • histone • Lupus • post-translational modification • acetylation • methylation • differential expression • MRL/lpr • stable isotope labeling

Introduction Systemic lupus erythematosus (SLE) is a chronic, inflammatory autoimmune disease characterized by the production of autoantibodies and affects the skin, kidneys, joints, lungs, various blood elements, and the central nervous system (CNS). The severity of disease, the spectrum of clinical involvement, and the response to therapy vary widely among the patients.1 These variations may be due to baseline line level of gene expression in SLE patients.2-4 Within the context of human lupus and mouse models of lupus, phenotypic variation has been typically attributed to differences in the genetic background, and the influences of environment on that genetic background. In isogenic populations of mouse models of lupus, however, phenotypic variations persist (the same but different).5 Studies of isogenic organisms showed that epigenetic mechanisms, which involve changes in chromatin structure by * To whom correspondence should be addressed. Tel: (336) 716-6573. Fax: (336) 716-9821. E-mail: [email protected]. † Department of Chemistry, University of Virginia. ‡ Departments of Chemistry and Pathology, University of Virginia. § Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forrest University School of Medicine.

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Published on Web 11/05/2005

covalent modification of histones, DNA methylation, and nucleosome reorganization arising stochastically or in response to physiological stress account for phenotype variation without changing genotype (DNA coding sequences) and can also be transmitted through mitosis and meiosis.6-8 Moreover, mutation of certain genes results in incorrect composition of chromatin (DNA methylation and histone modifications). The genomic rearrangement may cause epigenetic silencing of certain genes that may result in genetic instability and human diseases.9 This epigenetic mechanism might help to explain the female predominance in lupus (X-chromosome inactivation due to histone modification), early and late disease onset and progressive nature of the disease, the role of environment in disease development, and why a genetically identical twin may develop lupus, while the other twin does not.10 The natural variation in human gene expression (gene expression phenotype) is dependent upon a complex network of regulation that includes determinants that influence expression of nearby genes (cis-acting), determinants located on other chromosomes (trans-acting), and hot spots of genetic determinants (master regulators) affecting the expression of many genes.11 The positioning and post-translational modification of 10.1021/pr050188r CCC: $30.25

 2005 American Chemical Society

Resetting the Epigenetic Histone Code

histones, “histone code”, is one of the master regulators in gene expression. These post-translational modifications including acetylation, methylation, and phosphorylation change the structure of chromatin and have been shown to be involved in gene activation or repression. Disruption of post-translational modification of histone proteins perturbs the pattern of gene expression that may result in disease manifestation.12 Although considerable progress has been made in determining the enzymes that alter post-translational modification of histones, only limited information exist regarding the global pattern of histone modifications in disease states. To determine the genome wide aberrant histone modifications in SLE, we performed differential expression analysis of post-translational modification of histones in the splenocytes isolated from the MRL-lpr/lpr mouse model of lupus disease compared to normal MRL/MPJ lymphoproliferation wild-type mice. MRLlpr/lpr mice exhibit (a) onset of an accelerated autoimmune syndrome with polyclonal B cell activation and hypergammaglobulinemia beginning at about 8 weeks of age; (b) serologic evidence of a panoply of autoantibodies, including anti-doublestranded DNA (anti-dsDNA) autoantibodies and hypocomplementemia by 12-16 weeks of age; and (c) clinical signs of arthritis, massive lymphadenopathy, splenomegaly, vasculitis, and glomerulonephritis (GN) by the age of 16-24 weeks. In addition, fifty percent of MRL-lpr/lpr mice die by 24 weeks of age, primarily from renal failure. In contrast, MRL/MPJ lymphoproliferation wild-type mice develop mild kidney disease at very late age. The female mice die at 73 weeks of age and males at 93 weeks.13 Many human diseases including cancer have an epigenetic etiology that has encouraged the development of new therapeutic options termed “epigenetic therapy”. Indeed inhibitors of the histone deacetylase (HDAC) and DNA methylation enzymes are currently being tested for modulation of certain cancers in clinical trials.9 Recently, we have demonstrated that histone deacetylase inhibitors (HDACi) may have a beneficial effect in autoimmune diseases such as SLE.14-16 To determine the distribution of histone modification patterns after treatment of histone deacetylase inhibitors in vivo in MRL-lpr/lpr mice, we performed differential expression analysis of histones after trichostatin A (TSA) treatment using a stable isotope labeling mass spectrometry based approach. Our results suggest that there exist an aberrant histone code and that site-specific hypoacetylation of histone H3 and H4 can be corrected by histone deacetylase inhibition in vivo in the MRL-lpr/lpr mouse model of lupus.

Materials and Methods Animals. Eight-week-old female lupus diseased MRL-lpr/lpr and normal MRL/MPJ mice were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA), housed under specific pathogen-free conditions at the Wake Forest University School of Medicine animal facility, and provided with autoclaved food and sterile water ad libitum. Mice were randomly tested, and documented to be serologically negative for common murine pathogens. All animal experiments were approved by the animal ethics committee of Wake Forest University School of Medicine. In Vivo Treatment. Ten-week-old female MRL-lpr/lpr mice (n ) 10) were treated with subcutaneous injections of TSA 0.5 mg/kg body weight in 40 µL of dimethyl sulfoxide (DMSO) daily for 8 weeks. The dose of TSA was based on published results demonstrating a significant inhibition of kidney disease in

research articles MRL-lpr/lpr mice.15 Control MRL-lpr/lpr mice (n ) 10) were injected with 40 µL of DMSO alone. There were no differences between TSA-treated and vehicle-treated mice in age, weight, or food and water consumption. Splenocyte Isolation. Spleens were isolated from MRL-lpr/ lpr and MRL/MPJ mice at 18 weeks of age. Mice were anesthetized with ketamine/HCl/xylazine mixture (Sigma Chemical Co., Milwaukee, WI), and sacrificed by cervical dislocation. Spleens were surgically removed, and kept on ice in sterile Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% FCS and single cell suspensions were prepared as described previously.15 Histone Isolation. For histone preparation, freshly isolated pooled splenocyte cells from the spleens of MRL-lpr/lpr, MRL-MPJ, vehicle, and TSA treated MRL-lpr/lpr mice were thoroughly washed with cold PBS. Histones were isolated as described previously with minor modification.15 Nuclei were resuspended in ice-cold water and sonicated (Branson Sonifier 250). Samples were acidified with 0.4 N sulfuric acid (final concentration), vortexed thoroughly, and incubated on ice for 1 h. Thereafter, samples were centrifuged and the supernatant was precipitated with 1:5 volume of acetone overnight at 20 °C. Precipitated protein was collected by centrifugation at 15 000 × g for 10 min at 4 °C, air-dried, and resuspended in water. Protein concentrations were quantified by the Bio-Rad Protein Assay kit (Bio-Rad Laboratories Inc., Hercules, California, USA) using BSA as the standard. RP-HPLC Fractionation of Bulk Histones. Acid extracted bulk histones were fractionated on a C8 column (250 mm × 4.6 mm i.d, Keystone Scientific, Inc.) using an Agilent 1100 series HPLC (Palo Alto, CA) with a gradient of 0-100% B in 100 min (A ) 5% MeCN in 0.1% TFA, B ) 90% acetonitrile in 0.085% TFA). Fractions were collected in 1 min time intervals. Collected fractions were subjected to SDS-PAGE using 18% TrisHCl gels (Bio-Rad, Hercules, CA) at 150 V for approximately 1 h with Coomassie blue staining to check purity and elution of histone H3 and H4. Sample Preparation of Histone H3 and H4. Purified histone H3 protein from pooled HPLC fractions was digested by Glu-C protease (Promega, Madison, WI) after dilution of the H3 sample with 100 µL of 100 mM ammonium acetate buffer solution (pH ) 4) and incubation (substrate:enzyme ratio of 10:1) at room temperature for 5 h with quenching by freezing. The 1-50 amino terminus of histone H3 produced by Glu-C digestion was then isolated using a HAISIL-300 C18 column (40 × 2.1 mm i.d., Higgins Analytical Inc., Mountain View, CA) on a 130A Series HPLC (Applied Biosystems, Framingham, MA) with a gradient of 0-100% B in 50 min (solvent A ) 0.1% TFA, solvent B ) 60% MeCN in 0.085% TFA). The amino terminal 1-50 polypeptide was then treated with propionylation reagent to convert monomethylated and endogenously unmodified  amino groups on lysine residues and the N-terminus to propionyl amides.17 The propionylation reagent was created using 75 µL of MeOH and 25 µL of propionic anhydride (Aldrich, Milwaukee, WI). Equal volumes of reagent and H3 (1-50 AA) were mixed and allowed to react at 51 °C for 15 min. Propionylated histone H3 (1-50 AA) was then sub-digested with trypsin (Promega, Madison, WI) at a substrate:enzyme ratio of 20:1 for 5 h at 37 °C after dilution of the sample with 100 mM ammonium bicarbonate buffer solution (pH ) 8). The reaction was quenched by the addition of concentrated acetic acid and freezing. A second round of propionylation was then performed to propionylate the newly created peptide N-termini. Journal of Proteome Research • Vol. 4, No. 6, 2005 2033

research articles Purified histone H4 was treated with propionylation reagent, digested with trypsin and then re-propionylated using the same conditions as stated above. Propionic anhydride is a corrosive substance and caution should be taken when using this material. Mass Spectrometry. Propionylated histone digest mixtures were loaded onto capillary precolumns (360 µm o.d. × 75 µm i.d., Polymicro Technologies, Phoenix, AZ) packed with irregular C18 resin (5-20 µm, YMC Inc., Wilmington, NC) and washed with 0.1% acetic acid for 10 min. Precolumns were connected with Teflon tubing to analytical columns (360 µm o.d. × 50 µm i.d., Polymicro Technologies, Phoenix, AZ) packed with regular C18 resin (5 µm, YMC Inc., Wilmington, NC) structured with an integrated electrospray tip as previously described.18 All samples were analyzed by nanoflow HPLC-µ-electrospray ionization on a linear quadrupole ion trap-Fourier Transform Ion Cyclotron Resonance (LTQ-FT-ICR) mass spectrometer (Thermo Electron, San Jose, CA).19 The gradient used on an Agilent 1100 series HPLC solvent delivery system (Palo Alto, CA) consisted of 0-45% B in 90 min, 45-100% B in 15 min (A ) 0.1% acetic acid, B ) 70% acetonitrile in 0.1% acetic acid) or other similar gradients. The LTQ-FT mass spectrometer was operated in the data-dependent mode with the 10 most abundant ions being isolated and fragmented in the linear ion trap. In addition, the LTQ-FT was operated by acquiring MS spectra using the FT-ICR with R ) 100 000 at m/z 400. All MS/MS spectra were manually interpreted. Stable Isotope Labeling for Relative Quantitative Analysis. For a differential expression comparison of histone posttranslational modifications from disease and normal samples, a stable isotope labeling approach based on conversion of peptide carboxylic groups to their corresponding ethyl esters was used.19 First, all samples were dried to dryness by lyophilization. Propionylated histone peptides from MRL/MPJ (or MRL-lpr/lpr-treated with TSA) mice were converted to d0-ethyl esters by reconstituting the lyophilized sample in 100 µL of 2 M d0-ethanol/HCl, while histone peptides from MRL-lpr/lpr (or MRL-lpr/lpr -vehicle treated with DMSO) mice were converted to d5-ethyl esters by reconstituting the lyophilized sample in 100 µL of 2 M d6-ethanol/HCl. Both reaction mixtures were allowed to stand for 1 h at room temperature. Solvent was removed from each sample by lyophilization and the procedures were repeated using a second 100 µL aliquot of 2 M d0-ethanol/HCl or d6-ethanol/HCl, respectively. Solvent was then removed again by lyophilization and samples were dissolved in 20 µL 0.1% acetic acid. Additionally, the use of methanolic/HCl and deuterated methanolic/HCl was also employed. Aliquots of each solution were then equally mixed for comparative analysis by mass spectrometry. Two individual experiments from histones H3 and H4 from pooled splenocyte cells from normal and diseased mice spleens were completed for all differential expression comparisons. Ethanolic or methanolic/HCl is an irritant and corrosive substance and caution should be used during these reactions.

Results Mass Spectrometric Survey of Post-translational Modifications on Histone H3 and H4 from Mouse Splenocyte Cells. Presently, mass spectrometry (MS) has become an immensely useful technique for the analysis of histone post-translational modifications both on a global and differential scale.17,20-23 However, before a quantitative differential expression analysis of the covalent modifications on histone proteins between 2034

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normal and lupus diseased mice can be attempted, a detailed account of site specific post-translational modifications must be cataloged. Total histones were isolated from pooled splenocyte cells from normal MRL/MPJ and lupus diseased MRL-lpr/lpr mice, respectively. Histone H3 and H4 were separated from total histones by RP-HPLC using a C8 column (data not shown). H3 and H4 were chosen for mass spectrometry analysis because these two proteins are arguably the most widely modified and studied of the core histones.24 Although mammalian histone H3 possesses three variants, we pooled all H3 peak fractions from the RP-HPLC separation. Since mouse histone H3 variants contain only one amino acid residue change within the first 50 residues (A f S change from H3.1/H3.2 to H3.3, whereas H3.1 and H3.2 have the same first 50 amino acid residues) and all lysine residues are conserved on all H3 variants, we decided to isolate and pool the Nterminal 1-50 residues after Glu-C digest of bulk histone H3. Histone H4 elutes from the C8 column during RP-HPLC analyses with a minor contamination of histone H2A, but this contamination is not a hindrance for MS analyses, as histone H2A and H4 possess decidedly different sequences easily differentiated with a mass spectrometry based approach. To expedite the identification of histone modifications and also simultaneously prepare the sample for differential analysis, an approach based on chemical derivatization and detection by high mass accuracy tandem mass spectrometry was employed.17,19 Briefly, histones were treated with propionic anhydride reagent to convert amino groups on the N-terminus and internal lysine residues (endogenously unmodified and monomethylated residues only) to propionyl amides (Pr, +56 Da) and then digested with trypsin. The result of a trypsin digestion of propionylated histones is proteolysis only C-terminal to arginine as shown for histone H3 (Figure 1A) and histone H4 (Figure 1B). Generating this set of uniform peptides from highly modified proteins allows for straightforward analysis of posttranslational modifications in the mass spectrometer.17,19 Using this methodology, several sites of modifications on MRL/MPJ and MRL-lpr/lpr mice histone H3 were confirmed (Supporting Information Table 1). Monomethylation was the only modification observed on K4 of the 3-8 residue fragment, which was not detected on mouse histone H3 using MALDITOF mass spectrometry in a prior report.25 All degrees of methylation (mono, di and tri) and acetylation were found on K9. The high mass accuracy of the LTQ-FT mass spectrometer can be used to distinguish between trimethylation and acetylation (42.0464 versus 42.0100 Da). Acetylation was the only modification observed on the K14 residue, and this modification was also found on the same peptide together in combination with all the various modifications encountered on K9. Additionally, the detection of methylation on K9 in the absence of K14 acetylation is again in contrast to a previous study using mouse embryonic stem cells.25 Inspection of peptides spanning residues 18-26 resulted in confirmation of acetylation sites at K18 and K23 (both found separately and together), as well as a novel monomethylation site at K18. Peptides spanning residues 27-40 contain K27 and K36 which are both known modification sites on histone H3 from many organisms. As previously noted, the combinations of modifications between these two sites is quite extensive.24 For this reason, it was decided to monitor only the most clearly resolvable modifications occurring on K27 and K36. No post-translational modifications were found on any residues from the 41-50 peptide fragment. Sequence coverage for the 1-50 residue fragment

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Figure 1. (A) Strategy for the mass spectrometry analysis of the amino terminal (1-50 AA) tail of histone H3. The histone protein is first digested by Glu-C, the 1-50 AA fragment isolated by RP-HPLC, propionylated and then sub-digested by trypsin to create fragments containing residues 3-8, 9-17, 18-26, 27-40, and 41-50. (B) A similar approach for the mass spectrometry based analysis of histone H4. After propionylation, the protein is digested with trypsin to create the nine listed peptides.

of histone H3 was 96%. Finally, no modifications were found to be exclusive to either the MRL/MPJ or MRL-lpr/lpr mouse sample. A similar comprehensive analysis of the modifications on histone H4 was also completed (Supporting Information Table 2). Acetylation modifications on K5, K8, K12, and K16 were confirmed on the 4-17 residue fragment. These modifications have been implicated in various important regulatory processes.26 Our results are also in strong agreement with previous biological and mass spectrometric evidence demonstrating a clear sequential pattern to hyperacetylation on histone H4.27,28 Peptides spanning residues 4-17 with one acetylation mark primarily displayed this modification on K16. The di-acetylated 4-17 peptide mainly contained acetyl modifications on K16 and K12, whereas tri-acetylated 4-17 peptide consisted of modifications mostly at K16, K12, and K8. The tetraacetylated peptide (acetyl groups on K16, K12, K8, and K5) was only detected on the MRL/MPJ sample. This pattern is referred to as the “zip” acetylation model and the hypothesis is that the HATs responsible for acetylation of H4 work from K16 down to K5 and HDACs remove the modifications in opposite fashion.28 These acetylated forms of histone H4 can be readily separated by on-line HPLC (different degrees of acetyl and propionyl groups allow the differently acetylated peptides to be resolved), and no prior fractionation of the acetylated peptides is needed before MS analysis. Mono- and dimethylation of K20 was also observed and this particular modification site has been linked to silent regions of chromatin.29 Novel

modifications on K31 from histone H4 were detected on both MRL/MPJ and MRL-lpr/lpr samples. Figure 2 shows the MS/MS spectrum of the [M+2H]2+ ion at 712.3994 m/z from a trypsin digest of propionylated histone H4 from the MRL/MPJ mouse. This ion was fragmented and the corresponding sequence was found to be DNIQGITKAcPAIR (Note that a Pr ) propionyl amide modification is present on the N-terminus). The high mass accuracy (+0.42 ppm) of the FT-MS mass analyzer identified this modification as an acetylation modification and not tri-methylation. Unfortunately, the dynamics of K31 acetylation compared to acetylation at K16, K12, K8, and K5 could not be determined using our “bottom-up MS” approach. A previous report from the analysis of bovine calf thymus H4 described relatively low abundance MS peaks from peptide mapping experiments that suggested acetylation of K31.30 Monomethylation on K31 was also discovered in these mouse samples. No other modifications on the rest of the trypsin generated H4 fragments were detected by our methods. The sequence coverage recorded for histone H4 was 87%, and coverage included every lysine residue in the protein. It should also be noted that although abundant methylation and acetylation modifications were observed on the histone proteins, no phosphorylation modifications were detected. Phosphorylation on histone H3 at S10 and S28, and on histone H4 at S1 are well characterized modification sites important for chromatin segregation and condensation during mitosis.31 Nevertheless, phosphorylated peptides were not even found after histones were isolated in the presence of phosphatase Journal of Proteome Research • Vol. 4, No. 6, 2005 2035

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Table 1. Summary of Differential Expression MS Analysis of Histone H3 (1-50 Residues) from MRL/MPJ and MRL-lpr/lpr Micea residues

peptide sequence

ratio MRL (MPJ/-lpr/lpr)

3-8

TKQTAR TKMeQTAR KSTGGKAPR KMeSTGGKAPR KMe2STGGKAPR KMe3STGGKAPR KAcSTGGKAPR KSTGGKAcAPR KMeSTGGKAcAPR KMe2STGGKAcAPR KMe3STGGKAcAPR KAcSTGGKAcAPR KQLATKAAR KMeQLATKAAR KAcQLATKAAR KQLATKAcAAR KAcQLATKAcAAR KSAPATGGVKKPHR KMeSAPATGGVKKPHR KMe2SAPATGGVKKPHR KMe3SAPATGGVKKPHR KMeSAPATGGVKMeKPHR KMe2SAPATGGVKMeKPHR KMe3SAPATGGVKMeKPHR

1.07 ( 0.28 1.50 ( 0.05 0.71 ( 0.04 0.53 ( 0.04 0.72 ( 0.03 0.60 ( 0.05 1.37 ( 0.11 1.29 ( 0.17 0.71 ( 0.12 1.22 ( 0.11 0.81 ( 0.26 4.85 ( 0.19 0.61 ( 0.03 1.95 ( 0.12 1.89 ( 0.21 2.04 ( 0.04 7.05 ( 0.99 1.87 ( 0.45 0.95 ( 0.27 0.51 ( 0.02 0.17 ( 0.02 0.28 ( 0.05 0.52 ( 0.22 0.36 ( 0.002

9-17

18-26

27-40

ratio MRL (-lpr/lpr MPJ)

0.97 ( 0.25 0.66 ( 0.02 1.41 ( 0.07 1.90 ( 0.16 1.41 ( 0.08 1.67 ( 0.14 0.74 ( 0.06 0.78 ( 0.10 1.42 ( 0.24 0.82 ( 0.07 1.30 ( 0.41 0.21 ( 0.01 1.65 ( 0.09 0.52 ( 0.03 0.53 ( 0.06 0.49 ( 0.01 0.14 ( 0.02 0.55 ( 0.13 1.10 ( 0.31 1.96 ( 0.09 5.68 ( 0.67 3.64 ( 0.68 2.12 ( 0.89 2.80 ( 0.02

a Note- Ac ) acetylation; Me ) methylation; Me2 ) dimethylation; Me3 ) trimethylation. Histone H3 protein was obtained from pooled splenocyte cells derived from the spleens of normal MRL/MPJ and lupus diseased MRLlpr/lpr mice. All data relatively quantified by the use of stable isotope labeling and mass spectrometry during two individual experiments completed. The ratio of normal versus diseased (Ratio MRL (MPJ/-lpr/lpr)) and the inverted ratio of diseased versus normal (Ratio MRL (-lpr/lpr/MPJ)) are shown.

inhibitors and enriched using immobilized metal affinity chromatography.32 Failure in detecting these phosphorylation

Table 2. Summary of Differential Expression MS Analysis of Histone H4 from MRL/MPJ and MRL-lpr/lpr Micea residues

peptide sequence

4-17 GKGGKGLGKGGAKR GKGGKGLGKGGAKAcR GKGGKGLGKAcGGAKAcR GKGGKAcGLGKAcGGAKAcR GKAcGGKAcGLGKAcGGAKAcR 20-23 KVLR KMeVLR KMe2VLR 24-35 DNIQGITKPAIR DNIQGITKAcPAIR DNIQGITKMePAIR

ratio MRL (MPJ/-lpr/lpr)

ratio MRL (-lpr/lpr/MPJ)

0.42 ( 0.06 4.78 ( 0.35 6.60 ( 0.29 2.84 ( 0.32 detected/ not detected 1.06 ( 0.49 0.48 ( 0.05 0.58 ( 0.18 1.01 ( 0.44 2.47 ( 0.26 0.50 ( 0.10

2.42 ( 0.37 0.21 ( 0.02 0.15 ( 0.01 0.35 ( 0.04 not detected/ detected 1.06 ( 0.48 2.11 ( 0.21 1.81 ( 0.55 1.09 ( 0.48 0.41 ( 0.04 2.04 ( 0.41

a Note- Ac ) acetylation; Me ) methylation; Me2 ) dimethylation; Me3 ) trimethylation. Histone H4 protein was obtained from pooled splenocyte cells derived from the spleens of normal MRL/MPJ and lupus diseased MRLlpr/lpr mice. All data relatively quantified by the use of stable isotope labeling and mass spectrometry during two individual experiments completed. No post-translational modifications were found on any peptide sequences following the 24-35 residue peptide. The ratio of normal versus diseased (Ratio MRL (MPJ/-lpr/lpr)) and the inverted ratio of diseased versus normal (Ratio MRL (-lpr/lpr/MPJ)) are shown.

modifications may be a result of phosphorylation being concentrated on histone proteins during specific phases of the cell cycle such as mitosis, and may be undetectable by mass spectrometry in bulk asynchronously harvested histones such as in these samples. Differences in Histone Site-Specific Modifications between MRL-lpr/lpr and MRL-MPJ Mice Determined by Stable Isotope Labeling and Mass Spectrometry. Using mass spectrometry for the quantitative analysis of posttranslational modifications overcomes the problems associated with the use of sitespecific antibodies such as specificity, cross-reactivity and

Figure 2. Identification of an unexpected modification on histone H4. MS/MS spectrum of the doubly charged peptide ion at 712.3994 m/z from a trypsin digest of propionylated histone H4 from the MRL/MPJ mouse. This sequence was characterized as the peptide DNIQGITKAcPAIR with an acetylation modification on K31 and a propionyl amide (Pr, +56 Da) at the N-terminus. The high mass accuracy of the LTQ-FT-MS unambiguously identifies this modification as acetylation and not trimethylation. 2036

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Figure 3. (A) Strategy for the stable isotope based differential expression MS analysis of histone post-translational modifications between MRL/MPJ and MRL-lpr/lpr mice. (B) MRL/MPJ versus MRL-lpr/lpr differential expression ion chromatogram for propionylated histone H3 (1-50 AA) digested by trypsin. Inset MS shows doublet peaks for the 41-50 residue peptide (YRPGTVALRE) which is used to normalize all data since no modifications have ever been detected on these residues. This particular peptide was found in roughly a 1:1 ratio between the samples, showing equal loading. Another inset MS shows the 27-40 residue peptide doublet peaks with K27 di-methylation (KMe2SAPATGGVKKPHR) present approximately 2x higher in the MRL-lpr/lpr sample.

epitope occlusion through interference by neighboring modifications. The last of these problems has been previously well demonstrated to be a potential hindrance to the analysis of highly modified histone proteins.33,34 Additionally, mass spectrometry also allows for the simultaneous detection of multiple modifications on the same peptide and identification of unexpected modifications (as shown in Figure 2 for K31 acetylation) as well. The treatment of histone proteins with propionic anhydride followed by trypsin digest generates fragments that allow for differential expression analysis by mass spectrometry.17 Histone H3 and H4 isolated from pooled splenocyte cells from the spleens of normal MRL/MPJ and lupus diseased MRL-lpr/lpr mice were analyzed by stable

isotope labeling and mass spectrometry as outlined below. To compare post-translational modifications on histone H3 and H4 from MRL/MPJ and MRL-lpr/lpr mice, the propionylated trypsin fragments from each sample were labeled with different isotope labels as shown in Figure 3A, similar to an approach taken by Smith et al. for quantification of H4 acetylation.35 Histone peptides from MRL/MPJ mice were converted to d0-ethyl esters and those from MRL-lpr/lpr mice were converted to d5-ethyl esters. After an MS screening of the samples, aliquots containing equal amounts of the two samples were mixed and analyzed by nano-flow LC coupled to an LTQ-FT mass spectrometer. Stable isotope labeling allows for H3 peptides from the MRL (d0-ethyl ester) and MRL-lpr/lpr (d5-ethylJournal of Proteome Research • Vol. 4, No. 6, 2005 2037

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Figure 4. (A) Full MS of a differential expression analysis of histone H3 peptides from MRL/MPJ (isotopically labeled with D0-ethyl esters) and MRL-lpr/lpr (isotopically labeled with D5-ethyl esters). Zoom inset shows a set of doublet peaks (535.3207 m/z and 537.8181 m/z) with the first peak present in higher abundance (∼5x) in the MRL/MPJ sample. (B) MS/MS spectrum of the [M+2H]2+ peptide ion at 537.8181 m/z identifies this peptide as the 9-17 residue fragment with acetylation at both K9 and K14, and a propionylation (Pr) modification at the N-terminus.

esthers) samples containing identical post-translational modifications to appear as signal peak doublets (chemically identical peptides whose isotopic distribution is separated by 5 Da shifts) in the mass spectrometer (Figure 3B). Although d0- and d5-ethyl ester do have slightly different LC retention times and roughly coelute, the quantification of peptide species is straightforward after identification of the peptide by accurate mass and MS/MS fragmentation on the LTQ-FT mass spectrometer. Quantification of peptides involves measuring the area under the curve for the ion chromatograms (no signal processing used) for each charge state of the peptide ions and normalizing this ion abundance-to-a ratio of the ion abundance observed for the peptide containing the 41-50 residues on histone H3 (residues 46-55 for histone H4). These two peptides were chosen for normalization since no post-translational modifications have yet to be detected on these sequences. Displayed in Figure 3B is a chromatogram example of a differential expression analysis with selected zoom MS insets. The full mass spectrum the of doubly charged peptide of the 41-50 residue fragment (YRPGTVALRE) for the MRL/MPJ sample at 637.3674 2038

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m/z (d0-ethyl-ester) and MRL-lpr/lpr (d5-ethyl-ester) at 642.3984 m/z show a ion abundance ratio of 0.94 (MRL/MPJ/MRL-lpr/ lpr), demonstrating equal loading of the two samples. The MS signal doublets for the [M+2H]2+ ions are separated by 5 Da, as the YRPGTVALRE sequence can incorporate two ethyl ester labels (Glu residue and C-terminus). Another inset MS shows the doublet peaks (829.4924 and 832.0076 m/z) for the set of doubly charged peptide ions spanning residues 27-40 with a dimethylation at K27 (KMe2SAPATGGVKKPHR). The doubly charged doublet peaks are separated by 2.5 Da indicating that the peaks contain only one isotopic label. This particular modification is found twice as abundant in the MRL-lpr/lpr sample. Shown in Table 1 are the results from the differential expression MS analysis of MRL/MPJ and MRL-lpr/lpr peptides from histone H3 (two individual experiments). The ratio of normal versus diseased (Ratio MRL (MPJ/-lpr/lpr)) and the inverted ratio of diseased versus normal (Ratio MRL (-lpr/lpr/ MPJ)) are shown. For the most part, most modified peptides showed little or no change in the abundance levels between samples. However, on the 9-17 residue peptide, mono- and

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Resetting the Epigenetic Histone Code Table 3. Summary of Differential Expression MS Analysis of Histone H3 (1-50 Residues) from MRL-lpr/lpr Mice Treated with Trichostatin A and MRL-lpr/lpr Mice Vehicle Treated with DMSOa

residues

3-8 9-17

18-26

27-40

peptide sequence

ratio MRL-lpr/lpr (TSA/Vec)

ratio MRL-lpr/lpr (Vec/TSA)

TKQTAR TKMeQTAR KSTGGKAPR KMeSTGGKAPR KMe2STGGKAPR KMe3STGGKAPR KAcSTGGKAPR KSTGGKAcAPR KMeSTGGKAcAPR KMe2STGGKAcAPR KMe3STGGKAcAPR KAcSTGGKAcAPR KQLATKAAR KMeQLATKAAR KAcQLATKAAR KQLATKAcAAR KAcQLATKAcAAR KSAPATGGVKKPHR KMeSAPATGGVKKPHR KMe2SAPATGGVKKPHR KMe3SAPATGGVKKPHR KMeSAPATGGVKMeKPHR KMe2SAPATGGVKMeKPHR KMe3SAPATGGVKMeKPHR

0.98 ( 0.06 0.94 ( 0.13 0.35 ( 0.01 1.75 ( 0.43 0.54 ( 0.02 0.45 ( 0.01 1.18 ( 0.70 1.33 ( 0.48 0.87 ( 0.31 0.75 ( 0.27 0.62 ( 0.20 2.72 ( 0.10 0.45 ( 0.02 1.19 ( 0.20 0.81 ( 0.14 0.75 ( 0.13 3.46 ( 0.16 0.73 ( 0.06 0.39 ( 0.14 0.66 ( 0.37 0.98 ( 0.44 1.32 ( 0.68 0.60 ( 0.04 0.70 ( 0.12

1.02 ( 0.06 1.07 ( 0.15 2.79 ( 0.04 0.59 ( 0.15 1.85 ( 0.06 2.22 ( 0.05 1.02 ( 0.61 0.80 ( 0.29 1.22 ( 0.44 1.43 ( 0.52 1.72 ( 0.58 0.36 ( 0.01 2.20 ( 0.08 0.85 ( 0.14 1.25 ( 0.22 1.35 ( 0.24 0.29 ( 0.01 1.37 ( 0.10 2.74 ( 0.96 1.81 ( 1.02 1.14 ( 0.52 0.87 ( 0.45 1.65 ( 0.11 1.44 ( 0.24

Note- Ac ) acetylation; Me ) methylation; Me2 ) dimethylation; Me3 ) trimethylation. Histone H3 protein was obtained from pooled splenocyte cells derived from the spleens of lupus diseased MRL-lpr/lpr mice treated with trichostatin A and MRL-lpr/lpr mice vehicle treated with DMSO. All data relatively quantified by the use of stable isotope labeling and mass spectrometry during two individual experiments completed. The ratio of diseased TSA treated versus diseased vehicle treated (Ratio MRL-lpr/lpr (TSA/Vec)) and the inverted ratio of diseased vehicle treated versus diseased TSA treated (Ratio MRL-lpr/lpr (Vec/TSA)) are shown. a

trimethylation on K9 were modestly enriched on the MRL-lpr/ lpr mouse sample. Figure 4A shows a full mass spectrum from a differential expression analysis between the two mouse samples from a propionylated histone H3 trypsin digest. A set of doublet peaks at 535.3027 and 537.8181 m/z were observed and determined to be from the MRL/MPJ and MRL-lpr/lpr samples, respectively. An MS/MS spectrum of the [M+2H]2+ ion at 537.8181 m/z is shown in Figure 4B and identified this peptide as the 9-17 fragment with two acetylation modifications at K9 and K14. More interestingly, this particular peptide was found to be expressed almost 5 times higher in the MRL/ MPJ mouse sample. Inspection of 18-26 peptide fragments also indicated that di-acetylation of this peptide at K18 and K23 were also increased in the MRL/MPJ mouse. Methylation at K27 was also found to be enriched on various 27-40 peptides from the MRL-lpr/lpr sample. A differential expression MS analysis was also performed between peptides from MRL/MPJ and MRL-lpr/lpr histone H4. The results of this comparison are summarized in Table 2 (two individual experiments completed), and the ratio of normal versus diseased (Ratio MRL (MPJ/-lpr/lpr)) and the inverted ratio of diseased versus normal (Ratio MRL (-lpr/lpr/MPJ)) are displayed. Since acetylation was detected to be enriched on histone H3 from MRL/MPJ mice, acetylation on H4 was also closely monitored. Indeed, increased amounts of site specific acetylation were observed from MRL/MPJ histone H4 on mono, di and tri-acetylated fragments from residues 4-17 at K16; K16 and K12; and K16, K12, and K8, respectively. A tetra-acetylated

Table 4. Summary of Differential Expression MS Analysis of Histone H4 from MRL-lpr/lpr Mice Treated with Trichostatin A and MRL-lpr/lpr Mice Vehicle Treated with DMSOa

residues

peptide sequence

4-17 GKGGKGLGKGGAKR GKGGKGLGKGGAKAcR GKGGKGLGKAcGGAKAcR GKGGKAcGLGKAcGGAKAcR GKAcGGKAcGLGKAcGGAKAcR 20-23 KVLR KMeVLR KMe2VLR 24-35 DNIQGITKPAIR DNIQGITKAcPAIR DNIQGITKMePAIR

ratio MRL-lpr/lpr (TSA/Vec)

ratio MRL-lpr/lpr (Vec/TSA)

0.45 ( 0.04 4.96 ( 0.75 4.03 ( 0.69 5.73 ( 0.08 detected/ not detected 1.05 ( 0.05 0.84 ( 0.11 0.95 ( 0.30 0.52 ( 0.08 4.91 ( 0.12 0.99 ( 0.17

2.21 ( 0.19 0.20 ( 0.03 0.25 ( 0.04 0.17 ( 0.002 not detected/ detected 1.10 ( 0.56 1.19 ( 0.15 1.11 ( 0.35 1.93 ( 0.28 0.20 ( 0.01 1.03 ( 0.17

a Note- Ac ) acetylation; Me ) methylation; Me2 ) dimethylation; Me3 ) trimethylation. Histone H4 protein was obtained from pooled splenocyte cells derived from the spleens of lupus diseased MRL-lpr/lpr mice treated with trichostatin A and MRL-lpr/lpr mice vehicle treated with DMSO. All data relatively quantified by the use of stable isotope labeling and mass spectrometry during two individual experiments completed. No posttranslational modifications were found on any peptide sequences following the 24-35 residue peptide. The ratio of diseased TSA treated versus diseased vehicle treated (Ratio MRL-lpr/lpr (TSA/Vec)) and the inverted ratio of diseased vehicle treated versus diseased TSA treated (Ratio MRL-lpr/lpr (Vec/TSA)) are shown.

4-17 residue peptide species containing acetylation at K16, K12, K8, and K5 was also observed only on the MRL/MPJ mouse sample. A modest increase in K31 acetylation on the MRL/MPJ sample was also recorded. It should also be pointed out that a slight increase in methylation at K20 and K31 were found on the diseased MRL-lpr/lpr source. As mentioned earlier, silent chromatin is characterized by methylation at lysine 9 and 27 on histone H3 or K20 on histone H4, while acetylation at any site is correlated with active chromatin.24,36 Results from these analyses seem to indicate that post-translational modifications associated with active chromatin reside on MRL/MPJ mice, and modifications found on silent chromatin regions are somewhat enriched on the MRL-lpr/lpr mouse. Restoration of Site-Specific Histone Acetylation on MRLlpr/lpr Mice by in Vivo Administration of Trichostatin A. HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) or trichostatin A have been previously determined to trigger terminal differentiation of malignant cells and suppress the growth of prostate cancer cells.37,38 HDAC inhibitor effect on histone hyperacetylation has been quantified by MS from acute myeloid and chronic lymphocytic leukemia cell lines.39 The treatment of MRL-lpr/lpr mice with SAHA or trichostatin A has also been demonstrated to modulate renal disease in the MRL-lpr/lpr murine model of lupus.15,16 Compared to vehicletreated control mice, mice treated with these inhibitors have a significant decrease in the clinical signs of this disease including lowered glomerulonephritis and proteinuria symptoms. Although the mechanisms for these processes are unclear, the accumulation of hyperacetylated histone proteins has been linked to these events.14-16 For these reasons, it was decided to also monitor individual acetylation sites on histone H3 and H4 from MRL-lpr/lpr mice after in vivo treatment with trichostatin A or a control vehicle DMSO solution. Table 3 summarizes the results of stable isotope labeling differential expression MS analysis of histone H3 from trichostatin A or vehicle treated MRL-lpr/lpr mice (two individual experiments carried out). The ratio of diseased TSA treated Journal of Proteome Research • Vol. 4, No. 6, 2005 2039

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Garcia et al.

Figure 5. (A) Full MS of a differential expression analysis of histone H4 peptides from MRL-lpr/lpr TSA-treated (isotopically labeled with D0-ethyl esters) and MRL-lpr/lpr vehicle treated with DMSO (isotopically labeled with D5-ethyl esters). Zoom inset shows a set of doublet peaks (775.9577 m/z and 778.4728 m/z) with the first peak present in higher abundance (∼5×) in the MRL-lpr/lpr TSA-treated sample. (B) MS/MS spectrum of the [M+2H]2+ of the peptide ion at 775.9577 m/z identifies this peptide as the 4-17 residue fragment with acetylation at both K16 and K12. Note propionylation (Pr) modifications at endogenously unmodified K8 and K5 residues, and the N-terminus.

versus diseased vehicle treated (Ratio MRL-lpr/lpr (TSA/Vec)) and the inverted ratio of diseased vehicle treated versus diseased TSA treated (Ratio MRL-lpr/lpr (Vec/TSA)) are shown in Table 3. Mono-acetylation sites at K9 and K14 on the 9-17 residue fragment were not affected by the trichostatin A treatment. However, there was a small 2-fold increase in H3 peptides containing di-acetylation at those two sites (K9 and K14) in the MRL-lpr/lpr trichostatin A treated mouse. Enrichment of di-acetylation at K18 and K23 (but not monoacetylation) on the 18-26 residue fragment from the MRL-lpr/ lpr trichostatin A treated sample was also observed (approximately 3× higher). As expected, the methylation status on the vast majority of detected sites did not change in response to trichostatin A treatment (Table 3). The same differential expression MS analysis was performed on peptides from histone H4 from trichostatin A and vehicle treated MRL-lpr/lpr mice for two individual experiments (Table 4) with the ratio of diseased TSA treated versus diseased vehicle 2040

Journal of Proteome Research • Vol. 4, No. 6, 2005

treated (Ratio MRL-lpr/lpr (TSA/Vec)) and the inverted ratio of diseased vehicle treated versus diseased TSA treated (Ratio MRL-lpr/lpr (Vec/TSA)) being displayed. Again, an increase (up to 5-fold) in site-specific acetylation was noted for multiple peptides spanning the 4-17 amino acid regions on K16, K12 and K8 from TSA treated MRL-lpr/lpr mice. Figure 5A shows a full MS spectrum from the differential expression analysis of propionylated H4 peptides from TSA and vehicle treated MRLlpr/lpr mice. Zoom inset focuses on a pair of [M+2H]2+ ion doublet peaks at 775.9577 and 778.4728 m/z. The difference of only 2.5 Da indicates that only one ethyl ester label is incorporated on the peptides. The d0-ethyl ester peak (775.9577 m/z) was found about 5 times more abundant in the TSA treated MRL-lpr/lpr sample than the vehicle treated MRL-lpr/ lpr sample. The MS/MS spectrum of the [M+2H]2+ ion at 775.9577 m/z is shown in Figure 5B and was characterized as the 4-17 fragment with acetylation modifications at K16 and K12 (GKGGKGLGKAcGGAKAcR, note propionyl modifications at

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Resetting the Epigenetic Histone Code

the N-terminus and endogenously unmodified K5 and K8 residues). In addition, the fully tetra-acetylated (K5, K8, K12 and K16) 4-17 peptide could also now be observed in the MRLlpr/lpr sample, but only in the trichostatin A treated sample. An increase in acetylation on the novel modification site K31 was also seen in the TSA treated MRL-lpr/lpr H4 sample. Similar to the histone H3 treated samples, no significant apparent changes in the levels of methylation of K20 and K31 on histone H4 were found between the trichostatin A and vehicle treated MRL-lpr/lpr mice. Our results here are consistent with accumulation of total acetylation on histone H3 and H4 from MRL-lpr/lpr mice treated with the HDAC inhibitor trichostatin A as seen in earlier reports.14,15 Nevertheless, our MS based differential expression methods allow for quantification of these acetylation changes to specific sites on the histone proteins after TSA treatment.

Discussion We have previously demonstrated that in vivo administration of histone deacetylase inhibitors such as trichostatin A and SAHA modulate kidney disease in MRL-lpr/lpr mice without changing autoantibody levels. Moreover, we have reported that HDAC inhibitors modulate key gene expressions that are known to play pathogenic roles in splenocytes and mesangial cells in MRL-lpr/lpr mice and human lupus T cells.15 This study was carried out to first test the hypothesis as to whether any aberrant global post-translational modifications of histones in MRL-lpr/lpr mice exist, and second to determine whether the improvement of disease phenotype in lupus mice with histone deacetylase inhibition is associated with the resetting of aberrant epigenetic histone codes in this model. Our results support this hypothesis by demonstrating that an aberrant global sitespecific hypermethylation and hypoacetylations in H3 and H4 histones isolated from splenocytes of MRL-lpr/lpr mice exist. These modifications may play a role in pathogenesis of lupus because correction of histone hypoacetylation of H3 and H4 by histone deacetylase inhibitors affords improvement of kidney disease in these mice. Using a stable isotope labeling differential expression mass spectrometry analysis, we report here the principal findings as follows: (1) Compared to healthy wild-type MRL/MPJ mice, MRL-lpr/lpr mice have global site specific hypermethylation (approximately 2-fold increase in H3 K9-diMe, H3 K9-triMe, H3 K27-diMe; approximately 6-fold increase H3 K27-triMe, increases in simultaneous methylation on both K27 and K36 and approximately 2-fold increase on H4 K20 methylation). (2) The novel modification H3 K18-Me is slightly higher in MRL/ MPJ splenocytes compared to MRL-lpr/lpr mice. Similarly, modest increases exist in H3 K4-Me on MRL/MPJ compared to MRL-lpr/lpr mice. This finding suggests that the novel H3 K18-Me modification may possibly play a similar role as H3 K4-Me (active transcription associated modification) in other eukaryotic organisms such as Saccharomyces cerevisiae.40 (3) The novel modification H4 K31-Me is approximately 2-fold increased in MRL-lpr/lpr mice. (4) There exist a global H3 and H4 site-specific hypoacetylation in MRL-lpr/lpr compared to MRL/MPJ mice that can be corrected by histone deacetylase inhibition (Tables 1-4). Finally, we have also confirmed a novel acetylation site on histone H4 at K31 and this site was shown to be hyperacetylated on MRL-lpr/lpr mice with in vivo HDAC inhibitor treatment. It is currently unclear how these global changes in histone hypermethylation and hypoacetylation in splenocytes from

MRL-lpr/lpr mice are associated with pathogenesis of SLE. Recently, Baxter et al. reported increased H3 methylation in activated B lymphocyte and decreased histone methylation in quiescent lymphocytes.41 Autoreactive B lymphocyte and T lymphocytes have been known to be present in spleens from MRL-lpr/lpr mice.42 Thus, it may be proposed that increased histone methylation and histone hypoacetylation may serve as novel markers of enhanced epigenetic plasticity of autoreactive lymphocytes in MRL-lpr/lpr mice. We are currently in the process of performing chromatin immunoprecipitation assays to reflect these global modifications of histones to local levels at the individual gene level. Moreover, experiments to determine which enzymes cause and are recruited to these novel modifications at H3 K18-Me, H4 K31-Me and H4 K31-Ac need to be fully explored. It is believed that these studies will help eventually identify the mechanisms into why several anti-lupus genes are suppressed and pro-lupus genes are overexpressed in lupus disease. It is worthy to note that previous work using human lymphoid cell lines has demonstrated a 1-5% difference in gene expression between trichostatin A treated and untreated cells resulting in hyperacetylation of histone proteins.43 Thus, chromatin remodeling through histone acetylation may pose as a possible regulatory mechanism governing the expression of certain lupus related genes. Finally, our results demonstrate that mass spectrometry can be utilized to detect differences in the post-translational modifications of histone proteins from an in vivo animal disease model. These observations may eventually provide the foundation for the use of combination epigenetic therapy using methylase inhibitors plus HDAC inhibitors for the treatment of SLE.

Acknowledgment. This work was supported in part by Alliance for Lupus Research (N.M.), NIH grant GM37537 (D.F.H.), and Ford Foundation Fellowship (B.A.G.) Supporting Information Available: Tables showing selected peptides from tryptic digests of propionylated histone H3 and H4 from MRL/MPJ mice splenocyte cells. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Merrill, J. T.; Erkan, D.; Buyon, J. P. Nat. Rev. Drug Discov. 2004, 3, 1036-1046. (2) Crow, M. K.; Kirou, K. A.; Wohlgemuth, J. Autoimmunity 2003, 36, 481-490. (3) Banchereau, J.; Pascual, V.; Palucka, A. K. Immunity 2004, 20, 539-550. (4) Baechler, E. C.; Batliwalla, F. M.; Karypis, G.; Gaffney, P. M.; Ortmann, W. A.; Espe, K. J.; Shark, K. B.; Grande, W. J.; Hughes, K. M.; Kapur, V.; Gregersen, P. K.; Behrens, T. W. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 2610-2615. (5) Raman, K.; Mohan, C. Curr. Opin. Immunol. 2003, 15, 651-659. (6) Wang, Y.; Fischle, W.; Cheung, W.; Jacobs, S.; Khorasanizadeh, S.; Allis, C. D. Novartis Found. Symp. 2004, 259, 3-17; discussion 17-21, 163-169. (7) Sarmento, O. F.; Digilio, L. C.; Wang, Y.; Perlin, J.; Herr, J. C.; Allis, C. D.; Coonrod, S. A. J. Cell Sci. 2004, 117, 4449-4459. (8) Bjornsson, H. T.; Fallin, M. D.; Feinberg, A. P. Trends Genet. 2004, 20, 350-358. (9) Egger, G.; Liang, G.; Aparicio, A.; Jones, P. A. Nature 2004, 429, 457-463. (10) Cho, K. S.; Elizondo, L. I.; Boerkoel, C. F. Curr. Opin. Genet. Dev. 2004, 14, 308-315. (11) Morley, M.; Molony, C. M.; Weber, T. M., Devlin, J. L., Ewens, K. G., Spielman, R. S.; Cheung, V. G. Nature 2004, 430, 743-747. (12) Sims, R. J., 3rd; Reinberg, D. Nat. Cell Biol. 2004, 6, 685-687. (13) Hahn, B. H. Dubois’ Lupus Erythematosus;Wallace, D. J., Hahn, B. H., Eds; Williams & Wilkins: Baltimore 1997, pp 339-380. (14) Mishra, N.; Brown, D. R.; Olorenshaw, I. M.; Kammer, G. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2628-2633.

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