Sundararajan Jayaraman

Histone deacetylase inhibitors and candidate gene expression: An in vivo and in vitro approach to studying chromatin remodeling in a clinical population

OBJECTIVE The emerging field of psychiatric epigenetics is constrained by the dearth of research methods feasible in living patients. With this focus, we report on two separate approaches, one in vitro and one in vivo, developed in our laboratory. METHOD In the first approach, we isolated lymphocytes from 12 subjects and cultured their cells with either 0.7 mM valproic acid (VPA), 100 nM Trichostatin A (TSA), or DMSO (control) for 24h based upon previous dose response experiments. We then measured GAD67 mRNA expression using realtime RT-PCR, total acetylated histone 3 (H3K9,K14ac) levels using Western blot analysis, and attachment of H3K9,K14ac to the GAD67 promoter using ChIP. In the second approach, we measured GAD67 mRNA and total H3K9,K14ac levels in lymphocytes from 11 schizophrenia and 7 bipolar patients before and after 4 weeks of clinical treatment with Depakote ER (VPA). RESULTS In the first approach, VPA induced a 383% increase in GAD67 mRNA, an 89% increase in total H3K9,K14ac levels, and a 482% increase in H3K9,K14ac attachment to the GAD67 promoter. TSA induced comparable changes on all measures. In the second approach, bipolar subjects had significantly higher baseline levels of H3K9,K14ac compared to subjects with schizophrenia. Subjects with clinically relevant serum levels of VPA (> or = 65 microg/mL) showed a significant increase in GAD67 mRNA expression. CONCLUSIONS Our results utilizing two separate approaches for examining chromatin remodeling in real clinical time provide possible means to investigate epigenetic events in living patients.

Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells

A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30) transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization, superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells. Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc transporter ZnT-1 gene expression.

Chromatin remodeling resets the immune system to protect against autoimmune diabetes in mice

Epigenetic alteration of the genome has been shown to provide palliative effects in mouse models of certain human autoimmune diseases. We have investigated whether chromatin remodeling could provide protection against autoimmune diabetes in NOD mice. Treatment of female mice during the transition from prediabetic to diabetic stage (18–24 weeks of age) with the well‐characterized histone deacetylase inhibitor, trichostatin A effectively reduced the incidence of diabetes. However, similar treatment of overtly diabetic mice during the same time period failed to reverse the disease. Protection against diabetes was accompanied by histone hyperacetylation in pancreas and spleen, enhanced frequency of CD4+ CD62L+ cells in the spleen, reduction in cellular infiltration of islets, restoration of normoglycemia and glucose‐induced insulin release by beta cells. Activation of splenic T lymphocytes derived from protected mice in vitro with pharmacological agents that bypass the antigen receptor or immobilized anti‐CD3 antibody resulted in enhanced expression of Ifng mRNA and protein without altering the expression of Il4, Il17, Il18, Inos and Tnfa genes nor the secretion of IL‐2, IL‐4, IL‐17 and TNF‐α proteins. Consistently, expression of the transcription factor involved in Ifng transcription, Tbet/Tbx21 but not Gata3 and Rorgt, respectively, required for the transcription of Il4 and Il17, was upregulated in activated splenocytes of protected mice. These results indicate that chromatin remodeling can lead to amelioration of diabetes by using multiple mechanisms including differential gene transcription. Thus, epigenetic modulation could be a novel therapeutic approach to block the transition from benign to frank diabetes.

Epigenetic Mechanisms of Metabolic Memory in Diabetes

Diabetes affects 28.5 million people in the US.1 In addition, several million people with diabetes are estimated to be undiagnosed, thus increasing the risk for developing long-term complications, including macrovascular diseases and end-stage renal disease in a fairly significant fraction of the population. Type 1 diabetes, an autoimmune disease that mainly afflicts children, is characterized by the loss of insulin-producing beta cells. Until now, our understanding of this insidious disease relied on classic genetic approaches including genome wide association studies and single nucleotide polymorphisms. Although 40 immune response related genes have been implicated in type 1 diabetes, we are far from understanding how these genes render susceptibility to this complex disorder.2 Type 2 diabetes is common among people >45 years of age and characterized by the lack of serum autoantibodies, progressive insulin resistance, and beta cell insufficiency. Despite the differences in the etiology, both type 1 and type 2 diabetes are associated with microvascular complications such as diabetic nephropathy, neuropathy, and retinopathy, as well as macrovascular cardiovascular diseases including atherosclerosis, hypertension, and stroke. Tight glycemic control for 3 to 5 years may prevent cardiovascular disease but not all diabetes-related endpoints, referred to as the hyperglycemic memory3 or the legacy effect.4 Although it is unclear how the external cues such as hyperglycemia is perceived as a signal for gene transcription, several intracellular events including generation of oxidative stress and advanced glycation end products, and engagement of the mitogen activated protein kinase pathway have been implicated in metabolic memory (Figure). Recent data indicated a role for epigenetic mechanisms in a preclinical model of type 1 diabetes.5 Although gene-environment interactions have been implicated in the manifestation of type 2 diabetes, compelling evidence for the involvement of epigenetic mechanisms in the initiation and transmission of the metabolic syndrome to …

Role of migratory inhibition factor in age-related susceptibility to radiation lung injury via NF-E2-related factor-2 and antioxidant regulation.

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.

A novel method for the detection of viable human pancreatic beta cells by flow cytometry using fluorophores that selectively detect labile zinc, mitochondrial membrane potential and protein thiols

Improvement over current methods of beta cell viability assessment is highly warranted in order to efficiently predict the viability and function of beta cells prior to transplantation into type 1 diabetes patients. Dispersed human islet cells were stained with the cell‐permeable zinc‐selective dye, FluoZin‐3‐AM, along with the mitochondrial membrane potential indicator [(tetramethylrhodamine ethylester (TMRE)] and the thiol‐binding dye, monochlorobimane (mBcl), and analyzed by flow cytometry. Islets were subjected to various experimental conditions to validate the usefulness of this method to accurately determine the viability and function of beta cells. Staining with FluoZin‐3 revealed the presence of higher amounts of chelatable zinc ions in beta cells than in lymphoid cells and fibroblasts. An intracellular zinc chelator competitively inhibited the binding of FluoZin‐3 to zinc ions. Mitochondrial depolarization or oxidative stress minimally affected the binding of mBcl and FluoZin‐3, respectively, to thiols and zinc ions. The combination of FluoZin‐3, TMRE, and mBcl was sufficient and necessary for the determination of the viability and function of beta cells. The data demonstrate the usefulness of the zinc‐specific dye and the indicators of mitochondrial function and thiol levels, to accurately estimate the beta cell viability and function. This novel flow cytometry method has implications for islet transplantation in type 1 diabetes patients.

Transcriptome Analysis of Epigenetically Modulated Genome Indicates Signature Genes in Manifestation of Type 1 Diabetes and Its Prevention in NOD Mice

Classic genetic studies implicated several genes including immune response genes in the risk of developing type 1 diabetes in humans. However, recent evidence including discordant diabetes incidence among monozygotic twins suggested a role for epigenetics in disease manifestation. NOD mice spontaneously develop type 1 diabetes like humans and serve as an excellent model system to study the mechanisms of type 1 diabetes as well as the efficacy of maneuvers to manipulate the disease. Using this preclinical model, we have recently demonstrated that pharmacological inhibition of histone deacetylases can lead to histone hyperacetylation, selective up-regulation of interferon-γ and its transactivator Tbx21/Tbet, and amelioration of autoimmune diabetes. In the current study, we show that chromatin remodeling can render splenocytes incapable of transferring diabetes into immunodeficient NOD.scid mice. To elucidate the underlying mechanisms of drug-mediated protection against type 1 diabetes, we performed global gene expression profiling of splenocytes using high throughput microarray technology. This unbiased transcriptome analysis unraveled the exaggerated expression of a novel set of closely related inflammatory genes in splenocytes of acutely diabetic mice and their repression in mice cured of diabetes by chromatin remodeling. Analysis of gene expression by qRT-PCR using RNA derived from spleens and pancreata of cured mice validated the suppression of most of these genes, indicating an inverse correlation between the high levels of these inflammatory genes and protection against diabetes in NOD mice. In addition, higher-level expression of genes involved in insulin sensitivity, erythropoiesis, hemangioblast generation, and cellular redox control was evident in spleens of cured mice, indicating their possible contribution to protection against type 1 diabetes. Taken together, these results are consistent with the involvement of epistatic mechanisms in the manifestation of autoimmune diabetes and further indicate the utility of chromatin remodeling in curing this complex autoimmune disorder.

Assessment of beta cell viability.

This unit contains detailed protocols for the simultaneous identification of the human pancreatic β cells and determination of their viability by flow cytometry. The enumeration of β cells is based on the ability of the cell‐permeable form of the zinc‐selective dye, FluoZin‐3‐AM, to bind intracellular labile zinc stored at higher levels in these cells than any other types of cells in the body. Although staining of intracellular labile zinc by FluoZin‐3‐AM is dependent on the metabolic activity of β cells, co‐staining with a mitochondrial transmembrane potential indicator allows the accurate determination of viability. Simultaneous measurement of intracellular antioxidant thiols is also compatible with the detection of β cells containing metabolically active mitochondria. The method for assessing the mitochondrial functionality by flow cytometry described herein is simple to perform and sufficient to detect the viability of β cells in human islet preparations. Curr. Protoc. Cytom. 55:6.27.1‐6.27.16.

Transfusion of Nonobese Diabetic Mice with Allogeneic Newborn Blood Ameliorates Autoimmune Diabetes and Modifies the Expression of Selected Immune Response Genes

Although allogeneic bone marrow transplantation has been shown to prevent autoimmune diabetes in heavily irradiated nonobese diabetic (NOD) mice, a similar procedure is not suitable for the treatment of patients with type 1 diabetes because of associated severe side effects. Therefore, we evaluated whether mouse newborn blood (NBB), equivalent to human umbilical cord blood, could be used for diabetes prevention without recipient preconditioning. To test this hypothesis, unconditioned, prediabetic female NOD mice were given a single injection of whole NBB derived from the allogeneic diabetes-resistant mouse strain C57BL/6. Transfusion of allogeneic NBB but not adult blood prevented diabetes incidence in a majority of treated mice for a prolonged period of time. This was accompanied by the release of insulin in response to a challenge with glucose. Invasive cellular infiltration of islets was also substantially reduced in these mice. Although NBB transfusion induced a low level of hematopoietic microchimerism, it did not strictly correlate with amelioration of diabetes. Induction of genes implicated in diabetes, such as Il18, Tnfa, and Inos but not Il4, Il17 or Ifng, was repressed in splenocytes derived from protected mice. Notably, expression of the transcription factor Tbet/Tbx21 but not Gata3 or Rorgt was upregulated in protected mice. These data indicate that allogeneic NBB transfusion can prevent diabetes in NOD mice associated with modulation of selected cytokine genes implicated in diabetes manifestation. The data presented in this study provide the proof of principle for the utility of allogeneic umbilical cord blood transfusion to treat patients with autoimmune diabetes.

The epigenetic drug Trichostatin A ameliorates experimental autoimmune encephalomyelitis via T cell tolerance induction and impaired influx of T cells into the spinal cord

Multiple sclerosis is a T cell mediated chronic demyelinating disease of the central nervous system. Although currently available therapies reduce relapses, they do not facilitate tolerization of myelin antigen-specific T lymphocytes to ensure prolonged protection against multiple sclerosis. Here, we show that treatment of NOD mice with the histone deacetylase inhibitor, Trichostatin A affords robust protection against myelin peptide induced experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Protection was accompanied by histone hyperacetylation, and reduced inflammation and axonal damage in the spinal cord. Drug treatment diminished the generation of CD4+ memory T cells and induced tolerance in CD4+ T cells recognizing the immunizing myelin peptide. During the early immunization period, CD4+ T cells producing GM-CSF+IFN-γ, GM-CSF+IL-17A, as well as those expressing both IL-17A+IFN-γ (double-producers) were detected in the secondary lymphoid organs followed by the appearance of cells producing IFN-γ and GM-CSF. On the other hand, IFN-γ producing Th1 cells appear first in the spinal cord followed by cells producing IL-17A and GM-CSF. Treatment with Trichostatin A substantially reduced the frequencies of all T cells secreting various lymphokines both in the periphery and in the spinal cord. These data indicate that epigenetic modifications induced by histone hyperacetylation facilitates T cell tolerance induction in the periphery leading to reduced migration of T cells to the spinal cord and mitigation of neuronal damage and improved clinical outcome. These results suggest that epigenetic modulation of the genome may similarly offer benefits to multiple sclerosis patients via abrogating the function of encephalitogenic T lymphocytes without exerting severe side effects associated with currently used disease-modifying therapies.