Nuclear factor -B (NF-B)-regulated inflammatory genes, such as TNF-␣ (tumor necrosis factor-␣), 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-B. Gene silencing of SET7/9 with small interfering RNAs in monocytes significantly inhibited TNF-␣-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-␣-induced recruitment of NF-B 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-␣-stimulated monocytes, the induction of 25% NF-B 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.NF-B is a pleiotropic transcription factor that plays a pivotal role in regulating multiple biological functions, such as inflammation, immunity, cell proliferation, and apoptosis (1, 2). NF-B plays an important role in the regulation of proinflammatory genes, such as TNF-␣ (tumor necrosis factor ␣) and MCP-1 (monocyte-chemoattractant protein-1), that are associated with several inflammatory diseases, including atherosclerosis, insulin resistance, metabolic syndrome, and diabetes and its complications (3-7). These genes also lead to monocyte activation associated with these inflammatory diseases.NF-B consists of homo-or heterodimers of different subunits, such as p50, p52, p65/RelA, RelB, and c-Rel, with p65/ RelA and p50 being the most common and well studied (1,8). In most unstimulated cells, NF-B resides in the cytoplasm in an inactive latent form complexed with its inhibitor subunit, IB␣. Multiple extracellular stimuli, including inflammatory cytokines, such as TNF-␣, and ligands of the receptor for advanced glycation end products (RAGE), 2 can induce NF-B activation by promoting IB␣ phosphorylation and its proteasomal degradation (6, 9). The released p65-p50 dimer then translocates to the nucleus, where it binds to the promoters of NF-B-dependent inflammatory genes, such as TNF-␣, MCP-1, and IL-6 (interleukin-6), to induce their expression (2). p65 protein is a key transcriptionally active component of NF-B whose transactivation potential is enhanced by several coactivators, including CREB-binding protein/p300, p/CAF, and SRC1 (10), which have histone acetyltransferase activity, and CARM1, which has arginine methyltransferase activity (11,12). Recently, we showed that histone H3 lysine acetylation is enriched...

The transcription factor NF-B (NF-B) plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators including histone acetyltransferases (HATs) such as CBP/p300 and p/CAF. Evidence shows that high glucose (HG) conditions mimicking diabetes can activate the transcription of NF-B-regulated inflammatory genes. However, the underlying in vivo transcription and nuclear chromatin remodeling events are unknown. We therefore carried out chromatin immunoprecipitation (ChIP) assays in monocytes to identify 1) chromatin factors bound to the promoters of tumor necrosis factor-␣ (TNF-␣) and related NF-B-regulated genes under HG or diabetic conditions, 2) specific lysine (Lys (K)) residues on histone H3 (HH3) and HH4 acetylated in this process. HG treatment of THP-1 monocytes increased the transcriptional activity of NF-B p65, which was augmented by CBP/ p300 and p/CAF. ChIP assays showed that HG increased the recruitment of NF-B p65, CPB, and p/CAF to the TNF-␣ and COX-2 promoters. Interestingly, ChIP assays also demonstrated concomitant acetylation of HH3 at Lys 9 and Lys 14 , and HH4 at Lys 5 , Lys 8 , and Lys 12 at the TNF-␣ and COX-2 promoters. Overexpression of histone deacetylase (HDAC) isoforms inhibited p65-mediated TNF-␣ transcription. In contrast, a HDAC inhibitor stimulated gene transcription and histone acetylation. Finally, we demonstrated increased HH3 acetylation at TNF-␣ and COX-2 promoters in human blood monocytes from type 1 and type 2 diabetic subjects relative to nondiabetic. These results show for the first time that diabetic conditions can increase in vivo recruitment of NF-B and HATs, as well as histone acetylation at the promoters of inflammatory genes, leading to chromatin remodeling and transcription.

We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term followup Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.T he landmark Diabetes Control and Complications Trial (DCCT; 1983-1993 clearly showed that intensive (INT) glycemic control profoundly reduces the development and progression of microvascular complications in type 1 diabetes (T1D). The DCCT participants were subsequently followed in the Epidemiology of Diabetes Interventions and Complications (EDIC) Study (1994 to present), during which all subjects were advised to practice INT treatment. Surprisingly, those previously assigned to conventional (CONV) therapy continued to develop complications, such as nephropathy, retinopathy, and macrovascular diseases, at significantly higher rates than the previous INT therapy group, despite nearly similar HbA1c levels during the EDIC Study (1-3). This persistence of benefit from early application of INT therapy, called "metabolic memory," is an enigma in the field of T1D: recent studies have suggested the involvement of epigenetic mechanisms (4-9).Epigenetics is the study of mostly heritable changes in gene expression and phenotype that occur without alterations in the underlying DNA sequence. Epigenetic states are affected by environmental factors, such as aberrant nutrition and metabolic states (4, 6-8, 10). DNA methylation (DNA-me; the classic epigenetic mark) and posttranslational modifications (PTMs) of histo...

OBJECTIVE-The complexity of interactions between genes and the environment is a major challenge for type 1 diabetes studies. Nuclear chromatin is the interface between genetics and environment and the principal carrier of epigenetic information. Because histone tail modifications in chromatin are linked to gene transcription, we hypothesized that histone methylation patterns in cells from type 1 diabetic patients can provide novel epigenetic insights into type 1 diabetes and its complications. RESEARCH DESIGN AND METHODS-We used chromatin immunoprecipitation (ChIP) linked to microarray (ChIP-chip) approach to compare genome-wide histone H3 lysine 9 dimethylation (H3K9me2) patterns in blood lymphocytes and monocytes from type 1 diabetic patients versus healthy control subjects. Bioinformatics evaluation of methylated candidates was performed by Ingenuity Pathway Analysis (IPA) tools.RESULTS-A subset of genes in the type 1 diabetic cohort showed significant increase in H3K9me2 in lymphocytes but not in monocytes. CLTA4, a type 1 diabetes susceptibility gene, was one of the candidates displaying increased promoter H3K9me2 in type 1 diabetes. IPA identified two high-scoring networks that encompassed genes showing altered H3K9me2. Many of them were associated with autoimmune and inflammation-related pathways, such as transforming growth factor-␤, nuclear factor-B, p38 mitogen-activated protein kinase, toll-like receptor, and interleukin-6. IPA also revealed biological relationships between these networks and known type 1 diabetes candidate genes. CONCLUSIONS-The concerted and synergistic alteration of histone methylation within the identified network in lymphocytes might have an effect on the etiology of type 1 diabetes and its complications. These studies provide evidence of a novel association between type 1 diabetes and altered histone methylation of key genes that are components of type 1 diabetes-related biological pathways and also a new understanding of the pathology of type 1 diabetes. Diabetes 57:3189-3198, 2008

We assessed whether epigenetic histone posttranslational modifications are associated with the prolonged beneficial effects (metabolic memory) of intensive versus conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term Epidemiology of Diabetes Interventions and Complications (EDIC) study. We performed chromatin immunoprecipitation linked to promoter tiling arrays to profile H3 lysine-9 acetylation (H3K9Ac), H3 lysine-4 trimethylation (H3K4Me3), and H3K9Me2 in blood monocytes and lymphocytes obtained from 30 DCCT conventional treatment group subjects (case subjects: mean DCCT HbA1c level >9.1% [76 mmol/mol] and progression of retinopathy or nephropathy by EDIC year 10 of follow-up) versus 30 DCCT intensive treatment subjects (control subjects: mean DCCT HbA1c level <7.3% [56 mmol/mol] and without progression of retinopathy or nephropathy). Monocytes from case subjects had statistically greater numbers of promoter regions with enrichment in H3K9Ac (active chromatin mark) compared with control subjects (P = 0.0096). Among the patients in the two groups combined, monocyte H3K9Ac was significantly associated with the mean HbA1c level during the DCCT and EDIC (each P < 2.2E-16). Of note, the top 38 case hyperacetylated promoters (P < 0.05) included >15 genes related to the nuclear factor-κB inflammatory pathway and were enriched in genes related to diabetes complications. These results suggest an association between HbA1c level and H3K9Ac, and a possible epigenetic explanation for metabolic memory in humans.

Histone methylation patterns in the human genome, especially in euchromatin regions, have not been systematically characterized. In this study, we examined the profile of histone H3 methylation (Me) patterns at different lysines (Ks) in the coding regions of human genes by genome-wide location analyses by using chromatin immunoprecipitation linked to cDNA arrays. Specifically, we compared H3-KMe marks known to be associated with active gene expression, namely, H3-K4Me, H3-K36Me, and H3-K79Me, as well as those associated with gene repression, namely, H3-K9Me, H3-K27Me, and H4-K20Me. We further compared these to histone lysine acetylation (H3-K9/14Ac). Our results demonstrated that: first, close correlations are present between active histone marks except between H3-K36Me2 and H3-K4Me2. Notably, histone H3-K79Me2 is closely associated with H3-K4Me2 and H3-K36Me2 in the coding regions. Second, close correlations are present between histone marks associated with gene silencing such as H3-K9Me3, H3-K27Me2, and H4-K20Me2. Third, a poor correlation is observed between euchromatin marks (H3-K9/K14Ac, H3-K4Me2, H3-K36Me2, and H3-K79Me2) and heterochromatin marks (H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2). Fourth, H3-K9Me2 is neither associated with active nor repressive histone methylations. Finally, histone H3-K4Me2, H3-K4Me3, H3-K36Me2, and H3-K79Me2 are associated with hyperacetylation and active genes, whereas H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2 are associated with hypoacetylation. These data provide novel new information regarding histone KMe distribution patterns in the coding regions of human genes.

Aberrant histone lysine methylation patterns that change chromatin structure can promote dysregulated gene transcription and disease progression. Diabetic conditions such as high glucose (HG) are known to alter key pathologic pathways. However, their impact on cellular histone lysine methylation is unknown. We hypothesized that chronic HG can induce aberrant changes in histone H3 lysine 4 and lysine 9 dimethylation (H3K4me2 and H3K9me2) within target cells. Chromatin immunoprecipitation linked to microarrays (ChIP-on-chip) is currently a widely used approach for acquiring genome-wide information on histone modifications. We adopted this approach to profile and compare the variations in H3K4me2 and H3K9me2 in human gene coding and CpG island regions in THP-1 monocytes cultured in normal glucose and HG. Subsequently, we identified key relevant candidate genes displaying differential changes in H3K4me2 and H3K9me2 in HG versus normal glucose and also validated them with follow-up conventional ChIPs. Relevance to human diabetes was demonstrated by noting that H3K9me2 at the coding and promoter regions of two candidate genes was significantly greater in blood monocytes of diabetic patients relative to normal controls similar to the THP-1 data. In addition, regular mRNA profiling with cDNA arrays revealed correlations between mRNA and H3K9me2 levels. These novel results show histone methylation variations, for the first time, under diabetic conditions at a genome-wide level.

Human 3-methyladenine-DNA glycosylase (MPG protein) initiates base excision repair by severing the glycosylic bond of numerous damaged bases. In comparison, homologues of the Rad23 proteins (hHR23) and the hXPC protein are involved in the recognition of damaged bases in global genome repair, a subset of nucleotide excision repair. In this report, we show that the hHR23A and -B also interact with the MPG protein and can serve as accessory proteins for DNA damage recognition in base excision repair. Furthermore, the MPG⅐hHR23 protein complex elevates the rate of MPG protein-catalyzed excision from hypoxanthine-containing substrates. This increased excision rate is correlated with a greater binding affinity of the MPG proteinhHR23 protein complex for damaged DNA. These data suggest that the hHR23 proteins function as universal DNA damage recognition accessory proteins in both of these major excision repair pathways.