Transcriptional regulation of 15-lipoxygenase expression by histone h3 lysine 4 methylation/demethylation - PubMed (original) (raw)

Transcriptional regulation of 15-lipoxygenase expression by histone h3 lysine 4 methylation/demethylation

Cheng Liu et al. PLoS One. 2012.

Abstract

15-Lipoxygenase-1 (15-LOX-1) oxidizes polyunsaturated fatty acids to a rich spectrum of biologically active metabolites and is implicated in physiological membrane remodelling, inflammation and apoptosis. Its deregulation is involved in the pathogenesis of diverse cancer and immune diseases. Recent experimental evidence reveals that dynamic histone methylation/demethylation mediated by histone methyltransferases and demethylases plays a critical role in regulation of chromatin remodelling and gene expression. In the present study, we compared the histone 3 lysine 4 (H3-K4) methylation status of the 15-LOX-1 promoter region of the two Hodgkin lymphoma (HL) cell lines L1236 and L428 with abundant and undetectable 15-LOX-1 expression, respectively. We identified a potential role of H3-K4 methylation in positive regulation of 15-LOX-1 transcription. Furthermore, we found that histone methyltransferase SMYD3 inhibition reduced 15-LOX-1 expression by decreasing promoter activity in L1236 cells. SMYD3 knock down in these cells abolished di-/trimethylation of H3-K4, attenuated the occupancy by the transactivator STAT6, and led to diminished histone H3 acetylation at the 15-LOX-1 promoter. In contrast, inhibition of SMCX, a JmjC-domain-containing H3-K4 tri-demethylase, upregulated 15-LOX-1 expression through induction of H3-K4 trimethylation, histone acetylation and STAT6 recruitment at the 15-LOX-1 promoter in L428 cells. In addition, we observed strong SMYD3 expression in the prostate cancer cell line LNCaP and its inhibition led to decreased 15-LOX-1 expression. Taken together, our data suggest that regulation of histone methylation/demethylation at the 15-LOX-1 promoter is important in 15-LOX-1 expression.

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Conflict of interest statement

Competing Interests: The study was partially financed by an unconditional support for basic research by Biolipox AB. The company was closed in 2007. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. Association of 15-LOX-1 expression and activity with H3-K4 methylation status in HL-derived cell lines.

(A) 15-LOX-1 mRNA levels in L1236 and L428 cells were measured using real-time PCR (n = 6). The expression levels were calculated relative to that of a calibrator sample (A549 cells ectopically expressing 15-LOX-1), levels of transcripts were expressed as the ratio vs. human β2 microglobulin. Bar, SD; ** p<0.01. (B) 15-HETE was measured as an indication of 15-LOX-1 activity in L1236 and L428 cells (n = 4). Cells were harvested after incubation with exogenous arachidonic acid (40 mM) at 37°C for 5 min. Bar, SD; * p<0.05. (C) ChIP assay for H3-K4 trimethylation at the 15-LOX-1 core promoter in L1236 and L428 cells. Primers B (see Materials and Methods and Fig. 3 A) were used for PCR amplification. Shown is one of four independent experiments.

Figure 2

Figure 2. SMYD3 and SMCX regulate 15-LOX-1 expression in cultured HL-derived cells cells and prostate cancer cells.

(A) Real-time PCR assay of 15-LOX-1 mRNA expression in L1236 and LNCaP cells treated with SMYD3 siRNAs or control siRNA (n = 4). The results were normalized to the mRNA level of beta-2 microglobulin. The efficiency of SMYD3 siRNA knocking down was evaluated using Western blots. Bar, SD; * p<0.05. (B) Real-time PCR assay and Western blot analysis of 15-LOX-1 mRNA and protein expression in L428 cells treated with SMCX siRNAs or control siRNA (n = 4). The real-time PCR data were normalized to the mRNA level of beta-2 microglobulin. The efficiency of SMCX siRNA knocking down was evaluated using Western blot and β-actin served as a loading control. Bar, SD; * p<0.05.

Figure 3

Figure 3. Modulation of the H3-K4 methylation/demethylation balance influences on 15-LOX-1 expression by affecting H3 acetylation and STAT6 occupancy at the 15-LOX-1 promoter.

(A) Schematic presentation of the 15-LOX-1 promoter and PCR primer locations (relative to ATG) for the ChIP assay in relation to the three potential STAT6 binding motifs and SMYD3 binding site in the 15-LOX-1 promoter region. (B) Quantative ChIP assay for H3-K4 tri−/di−/monomethylation, acetylation, STAT6 and SMYD3 occupancy at the 15-LOX-1 promoter in L1236 cells treated with the SMYD3 siRNA or control siRNA. (C) Quantative ChIP assay for H3-K4 tri−/di−/monomethylation, acetylation, and STAT6 occupancy at the 15-LOX-1 promoter in L428 cells treated with the SMCX siRNA or control. Omission of antibodies (No Ab) was included in the whole experimental procedure, together with the PCR amplification of unrelated GAPDH gene, as appropriate controls. Data shown are from four independent experiments. Mean value of ChIP signals are normalized to 2% input. Input control is from non-immunoprecipitated total genomic DNA. Bar, SD.

Figure 4

Figure 4. SMYD3 and SMCX regulates 15-LOX-1 expression at the transcriptional level.

(A) SMYD3 depletion is associated with decreased 15-LOX-1 promoter activity. SMYD3 siRNA or control siRNA were contransfected with wild type (WT) pGL3-15-LOX-1 reporter plasmid into L1236 cells (n = 4). Variation in transfection efficiency was normalized by thymidine kinase-driven Renilla luciferase activity. Bar, SD; * p<0.05. (B) 15-LOX-1 transcription is induced by SMYD3 ectopic expression. SMYD3 expression vectors pcDNA-SMYD3 or empty vector pcDNA were cotransfected with WT pGL3-15-LOX-1 reporter plasmid into L428 cells (n = 4). Bar, SD; * p<0.05. (C) Sequence of the 15-LOX-1 core promoter region. A putative SMYD3 binding site is underlined. The sequence that was mutated in the transcriptional activity analysis of _cis_-acting elements is indicated by dots and substitutions are given above. −1 indicates the first nucleotide upstream of the transcription start site; the arrow indicates the first nucleotide of the first exon. (D and E) Mutation of the SMYD3 binding motif at the 15-LOX-1 promoter attenuates transcriptional activity in 15-LOX-1 positive cells. WT pGL3-15-LOX-1 (WT) or SMYD3 motif mutant reporter (MUT) were transfected into L1236 or L428 cells (n = 4). Bar, SD; * p<0.05. (F) SMCX knockdown leads to enhanced 15-LOX-1 promoter activity. SMCX siRNA or control siRNA were contransfected with wild type (WT) pGL3-15-LOX-1 reporter plasmid into L428 cells (n = 4). Bar, SD; * p<0.05.

Figure 5

Figure 5. A model for HMT-mediated 15-LOX-1 transcriptional activation and HDM-mediated gene silencing through chromatin remodelling.

In the 15-LOX-1 negative cell line L428, the 15-LOX-1 promoter region is occupied by HDM SMCX. Because H3-K4 is hypomethylated and H3 is hypoacetylated, the 15-LOX-1 promoter is not accessible to the transcriptional activator STAT6, and the gene transcription is repressed. Inhibition of SMCX with siRNA results in H3-K4 hypermethylation and subsequent H3 hyperacetylation through the recruitment of transcription complexes containing HAT activity, leading to an accessible promoter for STAT6. Promoter-bound STAT6 then recruits more HATs that in turn catalyze more H3 acetylation. These sequential events lead to transcriptional activation of the 15-LOX-1 gene (A). In L1236 cells with abundant 15-LOX-1 expression, the binding of SMYD3 to its motif in the 15-LOX-1 promoter region results in H3-K4 hypermethylation and 15-LOX-1 activation via a similar mechanism (B).

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Grants and funding

The study was supported by grants from the Swedish Cancer Society, Stockholm County Council, Karolinska Institutet, an unrestricted grant from Biolipox AB, National Natural Science Foundationg of China (30901497); the Shandong Province Natural Science Foundation (Y2003C08); Promotive research fund for excellent young and middle-aged scientists of Shandong Province (BS2010YY027); the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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