ABSTRACT
OBJECTIVE To analyze trimethylation of genome-wide histone H3 lysine 4(H3K4met3) induced by silicon dioxide(SiO2)through chromatin immunoprecipitation linked to microarrays(ChIP-chip)in lung fibroblast(LF)of rats. METHODS A primary co-culture model of rat alveolar macrophages (AM)and LF in vitro. AM were exposed to 100 mg · L-1 free SiO2 for 24 h,before LF were collected and the phenotype of LF was determined after transdifferentiation by immunohistochemistry. ChIP-chip was used to profile the variations of trimethylation in H3K4 of lung fibroblasts in CpG island regions. ChIP-qPCR was used to validate the microarray results. The mRNA expression of nfib and kpna3 was analyzed by qRT-PCR. RESULTS Totally 1815 (518 increased and 1297 decreased) genes of H3K4met3 displayed significant differences in SiO2 100 mg·L-1 group compared with control group(Cy3/Cy5 value>2.0 or <0.5,NimbleScan V2.5 software). The results of ChIP-qPCR were quite consistent with those of microarray. CONCLUSION There are significant differences in methylation of genome-wide H3K4 between SiO2 100 mg·L-1 group and control group. These novel candidate genes may become potential biomarkers or new interfered targets.
ABSTRACT
PURPOSE: The purpose of the present study was to investigate the aberrance of histone H3 lysine 4 trimethylation (H3K4me3) in patients with IgA Nephropathy (IgAN). MATERIALS AND METHODS: In this study, H3K4me3 variations in peripheral blood mononuclear cells (PBMCs) from 15 IgAN patients and 15 healthy subjects were analyzed using chromatin immunoprecipitation linked to microarrays analysis (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Expression analysis by quantitative real-time PCR (qRT-PCR) revealed correlations between mRNA and H3K4me3 levels. DNA methylation status was analyzed by quantitative methylation-specific PCR. RESULTS: We found that 321 probes displayed significant H3K4me3 differences in IgAN patients compared with healthy controls. Among these probes, 154 probes displayed increased H3K4me3 and 167 probes demonstrated decreased H3K4me3. For further validation, we selected 4 key relevant genes (FCRL4, GALK2, PTPRN2 and IL1RAPL1) to study. The results of ChIP real-time PCR coincided well with the microarray data. Quantitative RT-PCR revealed the correlations between the mRNA expression and the methylation levels of H3K4me3. Different degrees of DNA methylation alterations appeared on the selected positive genes. CONCLUSION: Our studies indicated that there were significant alterations in H3K4me3 in IgAN patients. These findings may help to explain the disturbed immunity and abnormal glycosylation involved in IgAN patients.
Subject(s)
Adult , Female , Humans , Male , Young Adult , Case-Control Studies , Chromatin Immunoprecipitation , Glomerulonephritis, IGA/genetics , Histones/metabolism , Leukocytes, Mononuclear/metabolism , Lysine/metabolism , Methylation , Oligonucleotide Array Sequence Analysis/methods , Real-Time Polymerase Chain ReactionABSTRACT
Objective To study histone H3 lysine 4 trimethylation (H3K4me3) in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus(SLE) patients.Methods PBMCs were isolated by density gradient centrifugation from 10 active SLE patients,7 inactive SLE patients and 8 healthy volunteers.Chromatin immunopreeipitation linked to mieroarrays (ChIP-chip) was used to profile the variations in H3K4me3 in CpG island regions in PBMCs of SLE patients and controls.ChIP-qPCR was used to validate the mieroarray results.To confirm correlations between H3K4me3 and gene expression,expression analysis by qRT-PCR was performed on three randomly selected H3K4me3 candidates.Results 413 (137 increased and 276 decreased H3K4me3) and 393 genes (112 increased and 281 decreased H3K4me3) displayed significant differences in H3K4me3 between active and inactive SLE when compared with healthy SUhjeets.The results of ChiP-qPCR were consistent with microarray.ConclusiOn There are significant differences in H3K4me3 profiling between SLE and healthy subjects.These novel candidate genes may be potential biomarkers for future therapeutic targets.The ChIP-chip technology can help further reveal SLE molecular mechanisms and discover new therapeutic targets.