Ression by altering histone modification and thereby transcription factor occupation. SMYD3 expression in L1236 cells was knocked down using siRNA and thereafter alterations in H3-K4 mono2/di2/trimethylation at the 15-LOX-1 promoter was examined by ChIP assay. As shown in Fig. 3 B, SMYD3 inhibition leads to decrease H3-K4 diand trimethylation but not monomethylation at the promoterregion of 15-LOX-1, indicating that SMYD3 is required for di- or trimethylation of H3-K4 at the 15-LOX-1 promoter. Promoter H3-K4 di- or tri-methylation provide docking sites for certain protein complexes containing histone acetyltransferase (HAT) activity that in turn leads to increased accessibility for transcriptional activators [32]. We therefore investigated whether abolished H3-K4 di2/trimethylation impedes the 15-LOX-1 promoter occupancy of the transcription factor STAT6, a predominant trans-activator of the gene. We found that after three days of SMYD3 siRNA treatment, histone acetylation was diminished and the STAT6 binding was noticeably reduced at the 15-LOX-1 promoter (Fig. 3 B). Thus, data suggest thatHistone Methylation Regulates 15-LOX-1 ExpressionSMYD3 is required for H3-K4 di2/trimethylation of the 15LOX-1 promoter in L1236 cells, promoting STAT6 access.SMCX Inhibition Affects Histone Modifications and Enhances STAT6 Binding at the 15-LOX-1 Promoter in L428 CellsBecause inhibition of H3-K4 demethylase upregulates 15-LOX1 expression in L428 cells (Fig. 2 B), we sought to delineate the underlying mechanism. To this end, L428 cells were cotransfected with the pGL3-15-LOX-1-WT AZP-531 web reporter plasmid and SMCX siRNA or control siRNA. As shown in Fig. 3 F, after three days of cotransfection, SMCX depletion led to a significant increase of 15-LOX-1 transcriptional activity. To further investigate the regulatory function of SMCX in 15-LOX-1 transcription, ChIP assay was applied. After three days of SMCX siRNA treatment, significant enhanced H3-K4 trimethylation but not di- or monomethylation of the 15-LOX-1 promoter region was detected in the L428 cells (Fig. 3 C). Consistent with the results presented in Fig. 2 B, it was also noted that inhibition of the H3-K4 demethylase with SMCX siRNA leads to a clear upregulation of histone acetylation and STAT6 occupation without IL-4 treatment (Fig. 3C). These observations suggest that H3-K4 demethylase is required to keep the 15-LOX-1 promoter silenced in L428 cells by controlling chromatin folding and the accessibility of STAT6.DiscussionChromatin remodelling including DNA and histone modification has an enormous potential for organizing and controlling information encoded by the genome. The genomic histone methylation/demethylation regulation mediated by the dynamic balance of HMTs/HDMs is a common event which is involved in as diverse cellular biological processes as gene transcription, Xchromosome inactivation, DNA damage repair, telomere function and DNA recombination [36]. H3-K4 methylation has been considered as a positive histone modification for transcription; it increases as gene expression becomes active [22]. H3-K4 hypermethylation is predominantly localized to the promoter region of genes and different lines of evidence suggest that disrupting HMTs or HDMs can modulate gene expression by changing the pattern of histone methylation at the promoter region [24,34]. Because the 15-LOX-1 gene is highly regulated and specifically expressed only in certain types of human cells, we attempted to investigate a pot.Ression by altering histone modification and thereby transcription factor occupation. SMYD3 expression in L1236 cells was knocked down using siRNA and thereafter alterations in H3-K4 mono2/di2/trimethylation at the 15-LOX-1 promoter was examined by ChIP assay. As shown in Fig. 3 B, SMYD3 inhibition leads to decrease H3-K4 diand trimethylation but not monomethylation at the promoterregion of 15-LOX-1, indicating that SMYD3 is required for di- or trimethylation of H3-K4 at the 15-LOX-1 promoter. Promoter H3-K4 di- or tri-methylation provide docking sites for certain protein complexes containing histone acetyltransferase (HAT) activity that in turn leads to increased accessibility for transcriptional activators [32]. We therefore investigated whether abolished H3-K4 di2/trimethylation impedes the 15-LOX-1 promoter occupancy of the transcription factor STAT6, a predominant trans-activator of the gene. We found that after three days of SMYD3 siRNA treatment, histone acetylation was diminished and the STAT6 binding was noticeably reduced at the 15-LOX-1 promoter (Fig. 3 B). Thus, data suggest thatHistone Methylation Regulates 15-LOX-1 ExpressionSMYD3 is required for H3-K4 di2/trimethylation of the 15LOX-1 promoter in L1236 cells, promoting STAT6 access.SMCX Inhibition Affects Histone Modifications and Enhances STAT6 Binding at the 15-LOX-1 Promoter in L428 CellsBecause inhibition of H3-K4 demethylase upregulates 15-LOX1 expression in L428 cells (Fig. 2 B), we sought to delineate the underlying mechanism. To this end, L428 cells were cotransfected with the pGL3-15-LOX-1-WT reporter plasmid and SMCX siRNA or control siRNA. As shown in Fig. 3 F, after three days of cotransfection, SMCX depletion led to a significant increase of 15-LOX-1 transcriptional activity. To further investigate the regulatory function of SMCX in 15-LOX-1 transcription, ChIP assay was applied. After three days of SMCX siRNA treatment, significant enhanced H3-K4 trimethylation but not di- or monomethylation of the 15-LOX-1 promoter region was detected in the L428 cells (Fig. 3 C). Consistent with the results presented in Fig. 2 B, it was also noted that inhibition of the H3-K4 demethylase with SMCX siRNA leads to a clear upregulation of histone acetylation and STAT6 occupation without IL-4 treatment (Fig. 3C). These observations suggest that H3-K4 demethylase is required to keep the 15-LOX-1 promoter silenced in L428 cells by controlling chromatin folding and the accessibility of STAT6.DiscussionChromatin remodelling including DNA and histone modification has an enormous potential for organizing and controlling information encoded by the genome. The genomic histone methylation/demethylation regulation mediated by the dynamic balance of HMTs/HDMs is a common event which is involved in as diverse cellular biological processes as gene transcription, Xchromosome inactivation, DNA damage repair, telomere function and DNA recombination [36]. H3-K4 methylation has been considered as a positive histone modification for transcription; it increases as gene expression becomes active [22]. H3-K4 hypermethylation is predominantly localized to the promoter region of genes and different lines of evidence suggest that disrupting HMTs or HDMs can modulate gene expression by changing the pattern of histone methylation at the promoter region [24,34]. Because the 15-LOX-1 gene is highly regulated and specifically expressed only in certain types of human cells, we attempted to investigate a pot.
