Role of the histone H3 lysine 4 methyltransferase, SET7/9, in the regulation of NF-kappaB-dependent inflammatory genes. Relevance to diabetes and inflammation

Abstract

Nuclear factor kappa-B (NF-kappaB)-regulated inflammatory genes, such as TNF-alpha (tumor necrosis factor-alpha), play key roles in the pathogenesis of inflammatory diseases, including diabetes and the metabolic syndrome. However, the nuclear chromatin mechanisms are unclear. We report here that the chromatin histone H3-lysine 4 methyltransferase, SET7/9, is a novel coactivator of NF-kappaB. Gene silencing of SET7/9 with small interfering RNAs in monocytes significantly inhibited TNF-alpha-induced inflammatory genes and histone H3-lysine 4 methylation on these promoters, as well as monocyte adhesion to endothelial or smooth muscle cells. Chromatin immunoprecipitation revealed that SET7/9 small interfering RNA could reduce TNF-alpha-induced recruitment of NF-kappaB p65 to inflammatory gene promoters. Inflammatory gene induction by ligands of the receptor for advanced glycation end products was also attenuated in SET7/9 knockdown monocytes. In addition, we also observed increased inflammatory gene expression and SET7/9 recruitment in macrophages from diabetic mice. Microarray profiling revealed that, in TNF-alpha-stimulated monocytes, the induction of 25% NF-kappaB downstream genes, including the histone H3-lysine 27 demethylase JMJD3, was attenuated by SET7/9 depletion. These results demonstrate a novel role for SET7/9 in inflammation and diabetes.

FIGURE 1.

SET7/9 knockdown inhibits expression of key NF-κB-dependent genes. A, HEK293 cells were transiently transfected with plasmid vectors expressing SET7/9 shRNA (SI) or scrambled sequence (CON). Inflammatory gene expression was determined by RT-PCR using 18S primers as internal control after cells were stimulated with or without TNF-α (10 ng/ml) for various time periods. B, cell lysates were immunoblotted (IB) with SET7/9 and β-actin antibodies to confirm the knockdown of SET7/9 protein. C, THP-1 cells were stably transfected with lentiviral vectors expressing SET7/9 shRNA or scrambled sequence, and RT-PCRs were performed to detect MCP-1, TNF-α, and IL-8 expression after TNF-α treatment (10 ng/ml) for various time periods. D, Western blot to demonstrate SET7/9 knockdown efficiency by the lentiviral vector in THP-1 cells. E, quantification of TNF-α-induced gene expression at the 1 h time point in THP-1 cells. ^*, p < 0.001 SI + TNF-α versus CON + TNF-α, n = 3.

FIGURE 2.

Interaction of SET7/9 with NF-κB p65 and requirement of SET7/9 methyltransferase activity for inflammatory gene regulation. A, SET7/9 does not affect IκBα degradation or p65 nuclear translocation upon TNF-α (10 ng/ml) treatment. Cytoplasmic and nuclear fractions prepared from SET7/9 knockdown THP-1 cells (SI) and control (CON) cells after treatment with TNF-α (10 ng/ml) for various time periods were subjected to immunoblotting (IB) with indicated antibodies. B, SET7/9 and p65 colocalize in the same cellular complex. HEK293 cells were co-transfected with SET7/9 and p65 expression vectors, and cell lysates were immunoprecipitated withβ-actin antibody (left) or p65 antibody (right). Immunopreciptates were immunoblotted with the indicated antibodies on the left. Representative of three separate experiments. C, HVSMC grown on coverslips were stimulated with TNF-α for 2 h, and p65 and SET7/9 were detected by immunofluorescent staining with p65 or SET7/9 antibody, followed by confocal microscopy. D, SET7/9 protein levels measured by Western blotting in control or SET7/9 knockdown THP-1 cells before and after TNF-α treatment. E, HEK293 cells were transiently transfected with an empty vector (CON) or vectors expressing wild type SET7/9 (wtSET7/9) or enzymatically inactive SET7/9 (mSET7/9). Basal and TNF-α (10 ng/ml, 2 h)-induced gene expression were determined by RT-PCR. F and G, quantification of data from E (^*, p < 0.001 methylation mutant of SET7/9 + TNF-α or SI + TNF-α versus CON + TNF-α, n = 3).

FIGURE 3.

Inhibition of TNF-α-induced chromatin events in SET7/9 knockdown THP-1 cells. Wild type (CON) or SET7/9 stable knockdown THP-1 cells (SI) were treated with or without TNF-α for the indicated time periods, and ChIP assays were performed with anti-p65, anti-SET7/9, anti-H3K4-monomethyation, anti-H3K4-trimethylation, or anti-p300 antibodies. ChIP-enriched samples were analyzed by regular PCR using MCP-1 (A) and TNF-α (B) promoter primers. Specific locations of MCP-1 promoter primers are shown in supplemental Fig. 1. Data are representative of three separate experiments. B, real time QPCRs with ChIP-enriched DNA using MCP-1 promoter primers showing significant changes and confirmation of data in A and C (^*, p < 0.005 SI + TNF-α for 1 h versus CON + TNF-α for 1 h, n = 3).

FIGURE 4.

SET7/9 does not affect the binding activity of p65 to oligonucleotides containing NF-κB consensus binding sites: EMSA using³²P-labeled oligonucleotide probes corresponding to NF-κB binding site sequence in the MCP-1 promoter (top) or generic NF-κB consensus binding site (bottom) with nuclear extract from control (CON) or SET7/9 knockdown (SI) cells with and without TNF-α treatment. Specificity of DNA-protein complexes was confirmed by competition with cold unlabeled DNA oligonucleotides (lanes 4 and 6). The presence of p65 was confirmed by supershifting with p65 antibodies (lanes 10 and 13).

FIGURE 5.

Role of SET7/9 in inflammatory gene expression related to diabetes. A, regulation of S100b-induced gene expression by SET7/9. THP-1 cells (both CON and SI) were treated with or without the RAGE ligand S100b (40 μg/ml), and gene expression was analyzed by relative RT-PCR using β-actin as internal control. B, quantification of data from three independent experiments (^*, p < 0.001, SI + S100b versus CON + S100b). C, ChIP assays showing recruitment of SET7/9 to the MCP-1 promoter in THP-1 cells stimulated with S100b. Wild type THP-1 cells were treated without or with S100b (40 μg/ml) for the indicated time periods, and ChIP assays were performed using the indicated antibodies. ChIP enriched samples were analyzed using MCP-1 promoter primers. Results are representative of three experiments. D, increased MCP-1 expression in macrophages of diabetic mice. Macrophages derived from control (normal saline; NS) and streptozotocin (STZ)-induced diabetic mice were stimulated with TNF-α (10 ng/ml) for 1 h, and MCP-1 gene expression was analyzed by real time RT-QPCR (^*, p < 0.05 streptozotocin versus normal saline, n = 3). E, enhanced TNF-α induced recruitment of SET7/9 in diabetic macrophages. Control (open bars) and diabetic (filled bars) macrophages were treated with or without TNF-α and then subjected to ChIP assays using SET7/9 antibodies followed by QPCR with MCP-1 promoter primers. Results are expressed as-fold over control after normalization with input samples (^*, p < 0.001 diabetic macrophages with TNF-α for 60 min versus Control macrophages with TNF-α for 60 min, n = 3).

FIGURE 6.

SET7/9 regulates monocyte adhesion but not monocyte differentiation. A, wild type (CON) or SET7/9 knockdown THP-1 cells (SI) were treated with PMA, followed by RT-PCRs to detect markers of monocyte differentiation, scavenger receptor A (SRA), macrophage colony-stimulating factor (MCSF), and CD36. B, adhesion of THP-1 cells (TNF-α-treated or -untreated) to HVSMC monolayers is attenuated in SET7/9 knockdown cells (^*, p < 0.001 SI versus CON or SI + TNF-α versus CON + TNF-α, n = 3). C, adhesion of untreated THP-1 cells to TNF-α-treated HUVEC monolayers is also attenuated in SET7/9 knockdown cells (^*, p < 0.001 SI versus CON, n = 3). Binding assays with HUVEC or HVSMC were performed as described under “Experimental Procedures.” Bound monocytes were quantified with Quantity One software (Bio-Rad).

FIGURE 7.

SET7/9 regulates a subset of TNF-α-regulated genes. A, unbiased microarray screening of SET7/9-regulated TNF-α downstream genes. Expression profiling experiments were performed in triplicate using RNA from both SET7/9 knockdown (shRNA) and control THP-1 cells treated with or without TNF-α (10 ng/ml). Shown are the 153 Affymetrix probe sets divided into four clusters with significant interaction effects (p < 0.05) from two-way analysis of variance analysis (see “Experimental Procedures”). Gene cluster with blue label on the left represent genes up-regulated by TNF-α but whose induction is attenuated by SET7/9 shRNA (Upreg-atten). The orange cluster is down-regulated-attenuated (Downreg-atten); red and purple are clusters up-regulated-enhanced (Upreg-enh) and down-regulated-enhanced (Downreg-enh), respectively. B, Venn diagram showing that a significant fraction of TNF-α downstream genes are affected by SET7/9 shRNA. C, pie chart from the data in A comparing gene numbers of the four different clusters of interaction genes. D, validation of microarray results showing expression changes of six “interaction genes” by real time PCR. Black bars, CON THP-1 cells; gray bars, SET7/9 knockdown THP-1 cells (^*, p < 0.01, SI + TNF-α versus CON + TNF-α, n = 3).

FIGURE 8.

Schematic representation of the proposed regulatory function of SET7/9 on NF-κB and inflammatory gene expression. SET7/9 can stabilize and enhance p65 recruitment to a subset of gene promoters, and this is accompanied by increased H3-K4 methylation and augmented gene expression. Thus, SET7/9 may transform the promoter from a relatively “dormant” to more “active” state and thus allow efficient transactivation by NF-κB.