Pro-inflammatory role of microrna-200 in vascular smooth muscle cells from diabetic mice.

OBJECTIVE: Vascular smooth muscle cells (VSMC) from type 2 diabetic db/db mice exhibit enhanced proinflammatory responses implicated in accelerated vascular complications. We examined the role of microRNA(miR)-200 family members and their target Zeb1, an E-box binding transcriptional repressor, in these events. METHODS AND RESULTS: The expression levels of miR-200b, miR-200c, and miR-429 were increased, although protein levels of Zeb1 were decreased in VSMC and aortas from db/db mice relative to control db/+ mice. Transfection of miR-200 mimics into VSMC downregulated Zeb1 by targeting its 3'-UTR, upregulated the inflammatory genes cyclooxygenase-2 and monocyte chemoattractant protein-1, and promoted monocyte binding in db/+VSMC. In contrast, miR-200 inhibitors reversed the enhanced monocyte binding of db/dbVSMC. Zeb1 gene silencing with siRNAs also increased these proinflammatory responses in db/+VSMC confirming negative regulatory role of Zeb1. Both miR-200 mimics and Zeb1 siRNAs increased cyclooxygenase-2 promoter transcriptional activity. Chromatin immunoprecipitation showed that Zeb1 occupancy at inflammatory gene promoters was reduced in VSMC from type 2 diabetic db/db mice. Furthermore, Zeb1 knockdown increased miR-200 levels demonstrating a feedback regulatory loop. CONCLUSION: Disruption of the reciprocal negative regulatory loop between miR-200 and Zeb1 under diabetic conditions enhances proinflammatory responses of VSMC implicated in vascular complications.

Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells.

Argonaute proteins are the core components of effector complexes that facilitate RNA interference (RNAi). Small interfering RNAs (siRNAs) targeted to promoter regions mediate transcriptional gene silencing (TGS) in human cells through heterochromatin formation. RNAi effector complexes have yet to be implicated in the mechanism of mammalian TGS. Here we describe the role of the human Argonaute-1 homolog (AGO1) in directing TGS at the promoters for human immunodeficiency virus-1 coreceptor CCR5 and tumor suppressor RASSF1A. AGO1 associates with RNA polymerase II (RNAPII) and is required for histone H3 Lys9 dimethylation and TGS. AGO1, TAR RNA-binding protein-2 (7TRBP2) and Polycomb protein EZH2 colocalize to the siRNA-targeted RASSF1A promoter, implicating Polycomb silencing in the mechanism of mammalian TGS. These results establish a connection between RNAi components AGO1 and TRBP2, RNAPII transcription and Polycomb-regulated control of gene expression.

Epigenetics: deciphering its role in diabetes and its chronic complications.

1. Increasing evidence suggests that epigenetic factors might regulate the complex interplay between genes and the environment, and affect human diseases, such as diabetes and its complications. 2. Clinical trials have underscored the long lasting beneficial effects of strict glycaemic control for reducing the progression of diabetic complications. They have also shown that diabetic complications, such as diabetic nephropathy, a chronic kidney disorder, can continue even after blood glucose normalization, suggesting a metabolic memory of the prior glycaemic state. 3. Dysregulation of epigenetic post-transcriptional modifications of histones in chromatin, including histone lysine methylation, has been implicated in aberrant gene regulation associated with the pathology of diabetes and its complications. Genome-wide studies have shown cell-type specific changes in histone methylation patterns under diabetic conditions. In addition, studies in vascular cells have shown long lasting changes in epigenetic modifications at key inflammatory gene promoters after prior exposure to diabetic conditions, suggesting a possible mechanism for metabolic memory. 4. Recent studies have shown roles for histone methylation, DNA methylation, as well as microRNA in diabetic nephropathy. Whether these epigenetic factors play a role in metabolic memory of diabetic kidney disease is less well understood. 5. The incidence of diabetes is growing rapidly, as also the cost of treating the resulting complications. A better understanding of metabolic memory and the potential involvement of epigenetic mechanisms in this phenomenon could enable the development of new therapeutic targets for the treatment and/or prevention of sustained diabetic complications.

Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes.

Diabetic patients continue to develop inflammation and vascular complications even after achieving glycemic control. This poorly understood "metabolic memory" phenomenon poses major challenges in treating diabetes. Recent studies demonstrate a link between epigenetic changes such as chromatin histone lysine methylation and gene expression. We hypothesized that H3 lysine-9 tri-methylation (H3K9me3), a key repressive and relatively stable epigenetic chromatin mark, may be involved in metabolic memory. This was tested in vascular smooth muscle cells (VSMC) derived from type 2 diabetic db/db mice. These cells exhibit a persistent atherogenic and inflammatory phenotype even after culture in vitro. ChIP assays showed that H3K9me3 levels were significantly decreased at the promoters of key inflammatory genes in cultured db/db VSMC relative to control db/+ cells. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase Suv39h1 were also reduced in db/db VSMC. Furthermore, db/db VSMC were hypersensitive to TNF-alpha inflammatory stimulus, which induced dramatic and sustained decreases in promoter H3K9me3 and Suv39h1 occupancy. Recruitment of corepressor HP1alpha was also reduced under these conditions in db/db cells. Overexpression of SUV39H1 in db/db VSMC reversed this diabetic phenotype. Conversely, gene silencing of SUV39H1 with shRNAs in normal human VSMC (HVSMC) increased inflammatory genes. HVSMC cultured in high glucose also showed increased inflammatory gene expression and decreased H3K9me3 at their promoters. These results demonstrate protective roles for H3K9me3 and Suv39h1 against the preactivated state of diabetic VSMC. Dysregulation of epigenetic histone modifications may be a major underlying mechanism for metabolic memory and sustained proinflammatory phenotype of diabetic cells.

The role of epigenetics in the pathology of diabetic complications.

Diabetes is associated with significantly accelerated rates of several debilitating microvascular complications such as nephropathy, retinopathy, and neuropathy, and macrovascular complications such as atherosclerosis and stroke. While several studies have been devoted to the evaluation of genetic factors related to type 1 and type 2 diabetes and associated complications, much less is known about epigenetic changes that occur without alterations in the DNA sequence. Environmental factors and nutrition have been implicated in diabetes and can also affect epigenetic states. Exciting research has shown that epigenetic changes in chromatin can affect gene transcription in response to environmental stimuli, and changes in key chromatin histone methylation patterns have been noted under diabetic conditions. Reports also suggest that epigenetics may be involved in the phenomenon of metabolic memory observed in clinic trials and animal studies. Further exploration into epigenetic mechanisms can yield new insights into the pathogenesis of diabetes and its complications and uncover potential therapeutic targets and treatment options to prevent the continued development of diabetic complications even after glucose control has been achieved.

Role of the lysine-specific demethylase 1 in the proinflammatory phenotype of vascular smooth muscle cells of diabetic mice.

Insulin resistance and type 2 diabetes are major risk factors for vascular complications. Vascular smooth muscle cells (VSMCs) derived from db/db mice, an established mouse model of type 2 diabetes, displayed enhanced inflammatory gene expression and proatherogenic responses. We examined the hypothesis that aberrant epigenetic chromatin events may the underlying mechanism for this persistent dysfunctional behavior and "memory" of the diabetic cells. Chromatin immunoprecipitation assays showed that levels of histone H3 lysine 4 dimethylation (H3K4me2), a key chromatin mark associated with active gene expression, were significantly elevated at the promoters of the inflammatory genes monocyte chemoattractant protein-1 and interleukin-6 in db/db VSMCs relative to db/+ cells. Tumor necrosis factor-alpha-induced inflammatory gene expression, H3K4me2 levels, and recruitment of RNA polymerase II at the gene promoters were also enhanced in db/db VSMCs, demonstrating the formation of open chromatin poised for transcriptional activation in diabetes. On the other hand, protein levels of lysine-specific demethylase1 (LSD1), which negatively regulates H3K4 methylation and its occupancy at these gene promoters, were significantly reduced in db/db VSMCs. High glucose (25 mmol/L) treatment of human VSMCs also increased inflammatory genes with parallel increases in promoter H3K4me2 levels and reduced LSD1 recruitment. LSD1 gene silencing with small interfering RNAs significantly increased inflammatory gene expression and enhanced VSMC-monocyte binding in nondiabetic VSMCs. In contrast, overexpression of LSD1 in diabetic db/db VSMCs inhibited their enhanced inflammatory gene expression. These results demonstrate novel functional roles for LSD1 and H3K4 methylation in VSMCs and inflammation. Dysregulation of their actions may be a major mechanism for vascular inflammation and metabolic memory associated with diabetic complications.

Role of Src tyrosine kinase in the atherogenic effects of the 12/15-lipoxygenase pathway in vascular smooth muscle cells.

OBJECTIVE: The 12/15-Lipoxygenase (12/15-LO) and its metabolite 12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] mediate proatherogenic responses in vascular smooth muscle cells (VSMCs). We examined the role of the nonreceptor tyrosine kinase Src in the signaling and epigenetic chromatin mechanisms involved in these processes. METHODS AND RESULTS: Rat VSMCs (RVSMCs) were stimulated with 12(S)-HETE (0.1 micromol/L) in the presence or absence of the Src inhibitor PP2 (10 micromol/L). Src activation and downstream signaling events including inflammatory gene expression and chromatin histone H3-Lys-9/14 acetylation were examined by immunoblotting, RT-PCR, and chromatin immunoprecipitation assays, respectively. 12(S)-HETE significantly activated Src, focal adhesion kinase, Akt, p38MAPK, and CREB. Expression of monocyte chemoattractant protein-1 and interleukin-6 genes and histone H3-Lys-9/14 acetylation on their promoters were also increased by 12(S)-HETE. PP2 inhibited these responses as well as 12(S)-HETE-induced VSMC migration. Furthermore, dominant negative mutants of Src, CREB, and a histone acetyltransferase p300 significantly blocked 12(S)-HETE-induced inflammatory gene expression. In addition, growth factor induced Src signaling and downstream events including H3-Lys-9/14 acetylation and migration were significantly attenuated in VSMCs derived from 12/15-LO(-/-) mice relative to WT. CONCLUSIONS: Src kinase signaling plays a central role in the proatherogenic responses mediated by 12/15-LO and its oxidized lipid metabolite 12(S)-HETE in VSMCs.

Role of the mod(mdg4) common region in homolog segregation in Drosophila male meiosis.

Homologous chromosomes must pair and establish stable connections during prophase I of meiosis to segregate reliably from each other at anaphase I. In most organisms, the stable connections, called chiasmata, arise from crossovers. In Drosophila males, homologs pair and segregate without crossing over. Chiasmata are replaced by a homolog conjunction complex that includes the Stromalin in Meiosis (SNM) and Modifier of Mdg4 in Meiosis (MNM) proteins. MNM is one of 31 alternative splice products of mod(mdg4), all of which share a common 402-amino-acid N terminus and differ at their C termini. Previous data demonstrated that an MNM-specific exon is required for homolog conjunction, but did not address whether the N-terminal common region, which includes a BTB domain that can mediate coalescence of protein-DNA complexes, is also required. Here we describe a mutation in the common region of mod(mdg4), Z3-3401, that causes qualitatively similar phenotypes as the MNM-specific alleles but disrupts X-Y segregation much more drastically than autosomal segregation. The mutant MNM protein in Z3-3401 is expressed throughout prophase I in spermatocytes but the protein is confined to the cytoplasm, suggesting that the Z3-3401 mutation disrupts a signal required for nuclear localization or retention. Z3-3401 fails to complement a large battery of lethal and semilethal alleles in the common region for meiotic nondisjunction, including an allele containing an amino acid substitution at a conserved residue in the BTB/POZ domain, consistent with a general requirement for the mod(mdg4) common region in homolog segregation.

Epigenetic mechanisms.

The incidence of diabetes and related complications like nephropathy is growing rapidly and has become a major health care issue. Changes in the environment and nutritional habits have been implicated as major players. Furthermore, it is becoming increasingly clear that epigenetic factors may modulate the connections between genes and the environment. While diabetes in itself is treatable to a large extent, it is still associated with significantly increased risk for complications including chronic kidney and cardiovascular diseases. Current treatments have added preventative approaches so as to avoid future diabetic complications. Unfortunately, diabetic patients are often plagued with the continued development of various complications even after achieving glucose control. This has been suggested to be attributable to a mysterious phenomenon termed 'metabolic memory' of the prior glycemic state. Recent studies have suggested that epigenetic changes to chromatin can affect gene expression in response to various stimuli, and changes in key biochemical pathways and epigenetic histone and DNA methylation patterns in chromatin have been observed in a diabetic milieu. These accumulating data suggest that metabolic or hyperglycemic memory may be due to epigenetic changes in specific target tissues altering gene expression without changing the genetic code itself. While the genetics of diabetes has long been the focus of scientific research, much less is known about the role of epigenetics and the related molecular pathways that might affect the development of diabetes and the associated complications. Further studies of epigenetic mechanisms are therefore timely and could provide valuable new insights into the pathology of diabetic complications and also uncover much needed new therapeutic targets.

Epigenetics of diabetic complications.

Type 1 and Type 2 diabetes are complex diseases associated with multiple complications, and both genetic and environmental factors have been implicated in these pathologies. While numerous studies have provided a wealth of knowledge regarding the genetics of diabetes, the mechanistic pathways leading to diabetes and its complications remain only partly understood. Studying the role of epigenetics in diabetic complications can provide valuable new insights to clarify the interplay between genes and the environment. DNA methylation and histone modifications in nuclear chromatin can generate epigenetic information as another layer of gene transcriptional regulation sensitive to environmental signals. Recent evidence shows that key biochemical pathways and epigenetic chromatin histone methylation patterns are altered in target cells under diabetic conditions and might also be involved in the metabolic memory phenomenon noted in clinical trials and animal studies. New therapeutic targets and treatment options could be uncovered from an in-depth study of the epigenetic mechanisms that might perpetuate diabetic complications despite glycemic control.

Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1.

OBJECTIVE: Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice. RESEARCH DESIGN AND METHODS: We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays. RESULTS: We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2. CONCLUSIONS: These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells.

To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to alpha-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.