Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis

Abstract

Antineutrophil cytoplasmic autoantibody (ANCA) causes vascular injury that leads to small-vessel vasculitis. Patients with ANCA aberrantly express neutrophil granule-encoding genes, including 2 that encode autoantigens: proteinase 3 (PR3) and myeloperoxidase (MPO). To uncover a potential transcriptional regulatory mechanism for PR3 and MPO disrupted in patients with ANCA vasculitis, we examined the PR3 and MPO loci in neutrophils from ANCA patients and healthy control individuals for epigenetic modifications associated with gene silencing. We found that levels of the chromatin modification H3K27me3, which is associated with gene silencing, were depleted at PR3 and MPO loci in ANCA patients compared with healthy controls. Interestingly, in both patients and controls, DNA was unmethylated at a CpG island in PR3, whereas in healthy controls, DNA was methylated at a CpG island in MPO. Consistent with decreased levels of H3K27me3, JMJD3, the demethylase specific for H3K27me3, was preferentially expressed in ANCA patients versus healthy controls. In addition, we describe a mechanism for recruiting the H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) to PR3 and MPO loci mediated by RUNX3. RUNX3 message was decreased in patients compared with healthy controls, and may also be under epigenetic control. DNA methylation was increased at the RUNX3 promoter in ANCA patients. These data indicate that epigenetic modifications associated with gene silencing are perturbed at ANCA autoantigen-encoding genes, potentially contributing to inappropriate expression of PR3 and MPO in ANCA patients.

Figure 1. PR3 gene is actively transcribed in ANCA patients.

(A) Schematic of PR3 gene and processed PR3 mRNA. Arrows mark the location of forward and reverse primers (FP and RP, respectively) used for RT-PCR analysis of RNA immunoprecipitated with anti-RNA polymerase II antibody. (B) Ethidium bromide–stained agarose gel showed RT-PCR product specific for PR3 mRNA present in 4 of 6 ANCA patients. Lane 1, 100-bp DNA ladder; lane 2, blank; lanes 3–8, ANCA patients; lane 9, water-only control.

Figure 2. PR3 and MPO genes are depleted for H3K27me3 in neutrophils of ANCA vasculitis patients.

(A–C) Quantitative ChIP analysis for H3K27me3 enrichment on chromatin isolated from neutrophils of healthy controls (white circles; n = 23) and ANCA vasculitis patients (black diamonds; n = 15) is shown for PR3 (A), MPO (B), and MYO-D (C). Levels of H3K27me3 immunoprecipitated chromatin are reported as percent of input DNA. 4 of 5 ANCA patients were clinically in remission (dashed ellipse); and 9 of the remaining 10 had active disease (Supplemental Table 1 and Supplemental Figure 3). (D) Pearson correlation analysis of logarithmically transformed H3K27me3 levels at PR3 promoter versus logarithmically transformed PR3 expression levels among ANCA patients (black diamonds, active; gray diamonds, remission) and healthy controls (white circles) showed a modest inverse correlation (r = –0.494; P = 0.0019). (E) Pearson correlation analysis of logarithmically transformed H3K27me3 levels at MPO promoter versus logarithmically transformed MPO expression levels among ANCA patients and healthy controls (symbols as in D) trended toward an inverse correlation, but was not statistically significant (r = –0.189; P = 0.264). Only 4 ANCA patients in remission are shown because expression data for PR3 and MPO were not obtained for ANCA patient no. 1 (Supplemental Table 1).

Figure 3. Expression of autoantigen and potential regulators.

(A–D) Relative mRNA levels from total leukocytes of healthy controls (white circles; n = 49) and ANCA vasculitis patients (black diamonds; n = 97) were determined by quantitative RT-PCR with SYBR green (A and B) or Taqman (C and D). Mean fold change (horizontal bars) for patients versus controls was 1.77 ± 1.76 versus 1.00 ± 0.38 for JMJD3 (A), 0.67 ± 0.54 versus 1.00 ± 0.63 for RUNX3 (B), 19.77 ± 60.08 versus 1.00 ± 1.58 for PR3 (C), and 9.27 ± 24.85 versus 1.00 ± 0.78 for MPO (D). An outlier and its value (in parentheses) is shown for A. (E and F) Spearman ranked analysis of Affymetrix microarray data of healthy controls (white circles; n = 16) and ANCA patients (black diamonds; n = 25) showed an inverse correlation between RUNX3 mRNA and PR3 mRNA (P < 0.001; E) and between RUNX3 mRNA and MPO mRNA (P < 0.001; F).

Figure 4. Inverse correlation between RUNX3 expression and PR3 or MPO expression results from coordinated changes in gene expression.

(A–C) Relative mRNA levels for PR3, MPO, and RUNX3 from undifferentiated (white bars) or PMA-differentiated (gray bars) human acute promyelocytic leukemia cell line HL60 (A), acute monocytic leukemia cell line THP-1 (B), and histiocytic lymphoma (monocyte) cell line U937 (C) were determined by quantitative real-time RT-PCR with SYBR green (RUNX3) or Taqman (PR3 and MPO). Data represent median of 4–12 separate experiments, with exact values shown below graphs. *P < 0.05, **P = 0.001 versus untreated, paired t test. (D) PR3 protein levels were determined by flow cytometry on undifferentiated (black histogram) or PMA-differentiated (gray histogram) U937 cells. (E) RUNX3 protein levels (arrow) were detected by Western blot in U937 cells that were undifferentiated or differentiated with PMA for 24 and 48 hours. (F and G) H3K27me3 levels (F) and RUNX3 occupancy (G) at PR3 were determined by quantitative ChIP analysis in undifferentiated (white bars) and PMA-differentiated (black bars) U937 cells. Data (mean ± SD) represent percent DNA input from 2 experiments, with exact values shown below graphs.

Figure 5. Overexpression of RUNX3 in U937 myeloid cells silences PR3 expression.

(A) Relative RUNX3 and PR3 mRNA levels in undifferentiated U937 cells and undifferentiated U937/P44 cells were determined by quantitative real-time RT-PCR with SYBR green (RUNX3) and Taqman (PR3). (B and C) Western blot analysis was performed to detect RUNX3 (B) and PR3 (C) protein in undifferentiated U937 and U937/P44 cells and in PMA-differentiated U937 cells as indicated. Lanes in B were run on the same gel but were noncontiguous (white line). (D) H3K27me3 levels at PR3 were determined by quantitative ChIP analysis in undifferentiated U937 and U937/P44 cells. Data show percent DNA input for 3 separate experiments, with exact values shown above bars.

Figure 6. RUNX3 binds PR3 and MPO genes in myeloid cell lines and in ANCA patients in remission.

(A) Consensus RUNX3 binding sites in PR3 intron 4 and MPO intron 7 and 3′ UTR. (B) ChIP analysis for RUNX3 binding to PR3 intron 4 and MPO intron 7 in undifferentiated or PMA-differentiated U937 cells under the indicated immunoprecipitation conditions. Normal rabbit serum was used in mock immunoprecipitation. Lanes were run on the same gel but were noncontiguous (white lines). (C–E) ChIP analysis for RUNX3 binding to MPO intron 7 and 3′ UTR (C and D) or to MPO intron 7 and PR3 intron 4 (E) in neutrophils from an active ANCA vasculitis patient (C) and from ANCA vasculitis patients in remission (D and E). Relative mRNA levels were determined by Taqman quantitative RT-PCR for MPO and PR3 from total leukocytes of ANCA vasculitis patients and calculated as fold change relative to healthy control, with exact values shown above bars. Lanes in E were run on the same gel but were noncontiguous (white line).

Figure 7. RUNX3 directly interacts with PRC2 subunits EZH2 and EED.

Western blot analysis on immunoprecipitated proteins from undifferentiated and PMA-differentiated U937 cells (A and B) and THP-1 cells (C and D). Cell extracts were immunoprecipitated with anti-RUNX3 (A and C) or anti-EZH2 (B and D) Ab, and Western blots were performed with anti-EZH2 (A and C), anti-RUNX3 (B and D), and anti-EED (A–D) Abs. Normal IgG was used in mock immunoprecipitations. Lanes in B were run on the same gel but were noncontiguous (white line).

Figure 8. Mpo message level in peripheral neutrophils is insensitive to anti-MPO IgG.

Mouse Mpo mRNA levels from peripheral leukocytes were measured by quantitative real-time PCR, and ΔCt values were plotted against a standard curve of Mpo-positive cells serially diluted with Mpo-negative cells (see Methods and Supplemental Figure 5). Shown is Mpo message level for C57BL/6 and 129 mice not injected with anti-MPO IgG as well as for 129 mice 6 days after injection with anti-MPO IgG. Data represent average of 2 biological replicates of Mpo levels from leukocytes that were purified and pooled for each category prior to isolating RNA.

Figure 9. Model for PR3 and MPO gene silencing in normal neutrophils and disruption in ANCA vasculitis patients, highlighting the role of the H3K27me3 modification and enzymes that regulate this histone mark.

(A) Granulocyte genes are silenced in normal mature circulating neutrophils. We hypothesize that maintenance of a transcriptionally silent state is a dynamic process. Inflammatory cytokines can induce JMJD3 (26). Our present demonstration of an interaction between RUNX3 and PRC2 subunits suggests RUNX3 can recruit EZH2 and reestablish H3K27me3. This epigenetic modification would maintain the transcriptionally silent state. (B) In neutrophils of ANCA vasculitis patients, H3K27me3 is depleted at PR3 and MPO because of an unknown process, perhaps during neutrophil development. An alternative, but not mutually exclusive, possibility is that increased JMJD3 in ANCA patients reverses the silent state at PR3 and MPO by erasing the histone mark necessary for PcG-mediated silencing. Low levels of RUNX3 fail to recruit the EZH2 necessary to reestablish H3K27me3 and maintain transcriptional silence. Furthermore, DNA methylation at MPO may also contribute to maintaining the transcriptionally silent state (not shown). An intriguing possibility is that PR3 and MPO are in a transcriptionally poised state marked by both H3K27me3 and H3K4me3 (59) in neutrophils of healthy individuals. In neutrophils of patients with ANCA vasculitis, depletion of H3K27me3 allows transcription of PR3 and MPO.