Background/Purpose: To identify new candidate genes regulated by DNA methylation and involved in the pathogenesis of systemic lupus erythematosus (SLE), we integrated genome-wide DNA methylation analysis and mRNA expression profiling in CD4+ splenic T cells derived from MRL/lpr lupus-prone mice (MRL) and C57BL6/J mice (B6) as a control.

Methods: Chromatin immunoprecipitation (ChIP)-PCR was used to investigate the transcription factors binding to CGCG methylation sites. Murine T lymphoma cell line, EL-4, was treated with DNA methyltransferase inhibitor, 5-Azacytidine (5-azaC) or histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA) for in vitro study. To analyze the expression levels of mRNAs, quantitative real-time PCR (qPCR) was performed on a Step One Plus Real-Time PCR System (Applied Biosystems) using the TaqMan Gene Expression assays. To identify the function of Ctse, the Ctse gene was depleted by siRNA in EL4 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and measured IL-10 in cell culture supernatants by ELISA . To examine the expression levels of Il-10 and Ctse in CD4+ T cell subsets in peripheral blood mononuclear cell (PBMC) from 15 SLE patients and 6 healthy controls.

Results: We show that expression levels of Ctse transcripts are elevated in MRL T cells compared to that in B6 T cells and that the 583 bp region in the 1^st intron in the Ctse gene is hypomethylated. Bisulfite sequencing show that the CGCG motif in this region is hypomethylated in MRL cells. Kaiso, known to specifically recognize the methylated DNA motif (mCGCG) through the C2H2 zinc-finger domains, recruits the SMRT (Silencing Mediator of Retinoic acid and Thyroid hormone receptors) / N-CoR (Nuclear hormone receptor Co-Repressor) HDAC3 (Histone deacetylase 3) complex, leading to suppression of its target gene. To demonstrate that Kaiso and HDAC3 are components of the transcriptional complex regulating Ctse expression, we performed ChIP of the endogenous Ctse promoter in CD4+ T cells and observed strong enrichment of a Ctse-derived amplicon including the CGCG motif in both Kaiso and HDAC3 chromatin immunoprecipitates. Moreover, we found that the recruitment of Kaiso to the Ctse promoter was suppressed in MRL cells compared to B6 cells. Additionally, EL4 cells treated with 5-azaC or TSA showed the reduced recruitment of both Kaiso and HDAC3 to the motif. . Moreover, we observed that depletion of the Ctse gene by siRNA in EL4 cells results in reduction of IL-10 expression in cell culture supernatants by ELISA and that the expression level of Il-10 transcripts are up-regulated in MRL T cells compared to B6. Lastly, we observed that In CD4+ T cell subsets in PBMC from SLE, the expression levels of CTSE and IL-10 transcripts were elevated in CD4+ T cells from SLE patients compared to that in healthy controls.

Conclusion: Our present study provides evidence that DNA methylation-mediated recruitment of both Kaiso and HDAC3 to the Ctse promoter regulates expression of Ctse and that demethylation of this promoter and subsequent elevation of Ctse and Il-10 expression may be the pathogenesis of SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-methylation-dependent-regulation-of-cathepsin-e-gene-expression-by-the-transcription-factor-kaiso-in-mrllpr-mice/