Characterization of K562 cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies.

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.

Intestinal microbiota regulates diabetes and cancer progression by IL-1ß and NOX4 dependent signaling cascades.

Diabetes changes the host microbiota, a condition known as dysbiosis. Dysbiosis is an important factor for the pathogenesis of diabetes and colorectal cancer (CRC). We aimed at identifying the microbial signature associated with diabetes and CRC; and identifying the signaling mechanism altered by dysbiosis and leading to CRC progression in diabetes. MKR mice that can spontaneously develop type 2 diabetes were used. For CRC induction, another subset of mice was treated with azoxymethane and dextran sulfate sodium. To identify the role of microbiota, microbiota-depleted mice were inoculated with fecal microbial transplant from diabetic and CRC mice. Further, a mouse group was treated with probiotics. At the end of the treatment, 16S rRNA sequencing was performed to identify microbiota in the fecal samples. Blood was collected, and colons were harvested for molecular, anatomical, and histological analysis. Our results show that diabetes is associated with a microbial signature characterized by reduction of butyrate-forming bacteria. This dysbiosis is associated with gastrointestinal complications reflected by a reduction in colon lengths. These changes are reversed upon treatment with probiotics, which rectified the observed dysbiosis. Inoculation of control mice with diabetic or cancer microbiota resulted in the development of increased number of polyps. Our data also show that inflammatory cytokines (mainly interleukin (IL)-1ß) and NADPH oxidase (NOX)4 are over-expressed in the colon tissues of diabetic mice. Collectively our data suggest that diabetes is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria leading to over-expression of IL-1ß and NOX4 leading to gastrointestinal complications and CRC.

Sulforaphane prevents and reverses allergic airways disease in mice via anti-inflammatory, antioxidant, and epigenetic mechanisms.

Sulforaphane has been investigated in human pathologies and preclinical models of airway diseases. To provide further mechanistic insights, we explored L-sulforaphane (LSF) in the ovalbumin (OVA)-induced chronic allergic airways murine model, with key hallmarks of asthma. Histological analysis indicated that LSF prevented or reversed OVA-induced epithelial thickening, collagen deposition, goblet cell metaplasia, and inflammation. Well-known antioxidant and anti-inflammatory mechanisms contribute to the beneficial effects of LSF. Fourier transform infrared microspectroscopy revealed altered composition of macromolecules, following OVA sensitization, which were restored by LSF. RNA sequencing in human peripheral blood mononuclear cells highlighted the anti-inflammatory signature of LSF. Findings indicated that LSF may alter gene expression via an epigenetic mechanism which involves regulation of protein acetylation status. LSF resulted in histone and α-tubulin hyperacetylation in vivo, and cellular and enzymatic assays indicated decreased expression and modest histone deacetylase (HDAC) inhibition activity, in comparison with the well-known pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Molecular modeling confirmed interaction of LSF and LSF metabolites with the catalytic domain of metal-dependent HDAC enzymes. More generally, this study confirmed known mechanisms and identified potential epigenetic pathways accounting for the protective effects and provide support for the potential clinical utility of LSF in allergic airways disease.

Genomic DNA methylation distinguishes subtypes of human focal cortical dysplasia.

OBJECTIVES: Focal cortical dysplasia (FCD) is a major cause of drug-resistant focal epilepsy in children, and the clinicopathological classification remains a challenging issue in daily practice. With the recent progress in DNA methylation-based classification of human brain tumors we examined whether genomic DNA methylation and gene expression analysis can be used to also distinguish human FCD subtypes. METHODS: DNA methylomes and transcriptomes were generated from massive parallel sequencing in 15 surgical FCD specimens, matched with 5 epilepsy and 6 nonepilepsy controls. RESULTS: Differential hierarchical cluster analysis of DNA methylation distinguished major FCD subtypes (ie, Ia, IIa, and IIb) from patients with temporal lobe epilepsy patients and nonepileptic controls. Targeted panel sequencing identified a novel likely pathogenic variant in DEPDC5 in a patient with FCD type IIa. However, no enrichment of differential DNA methylation or gene expression was observed in mechanistic target of rapamycin (mTOR) pathway-related genes. SIGNIFICANCE: Our studies extend the evidence for disease-specific methylation signatures toward focal epilepsies in favor of an integrated clinicopathologic and molecular classification system of FCD subtypes incorporating genomic methylation.

Vascular histone deacetylation by pharmacological HDAC inhibition.

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

Catalysis in a Cage: Condition-Dependent Speciation and Dynamics of Exchanged Cu Cations in SSZ-13 Zeolites.

The relationships among the macroscopic compositional parameters of a Cu-exchanged SSZ-13 zeolite catalyst, the types and numbers of Cu active sites, and activity for the selective catalytic reduction (SCR) of NOx with NH3 are established through experimental interrogation and computational analysis of materials across the catalyst composition space. Density functional theory, stochastic models, and experimental characterizations demonstrate that within the synthesis protocols applied here and across Si:Al ratios, the volumetric density of six-membered-rings (6MR) containing two Al (2Al sites) is consistent with a random Al siting in the SSZ-13 lattice subject to Löwenstein's rule. Further, exchanged Cu(II) ions first populate these 2Al sites before populating remaining unpaired, or 1Al, sites as Cu(II)OH. These sites are distinguished and enumerated ex situ through vibrational and X-ray absorption spectroscopies (XAS) and chemical titrations. In situ and operando XAS follow Cu oxidation state and coordination environment as a function of environmental conditions including low-temperature (473 K) SCR catalysis and are rationalized through first-principles thermodynamics and ab initio molecular dynamics. Experiment and theory together reveal that the Cu sites respond sensitively to exposure conditions, and in particular that Cu species are solvated and mobilized by NH3 under SCR conditions. While Cu sites are spectroscopically and chemically distinct away from these conditions, they exhibit similar turnover rates, apparent activation energies and apparent reaction orders at the SCR conditions, even on zeolite frameworks other than SSZ13.

Dynamic changes in the cardiac methylome during postnatal development.

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-ß, fibroblast growth factor, and Wnt/ß-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation.

EZH2 inhibitors promote ß-like cell regeneration in young and adult type 1 diabetes donors.

ß-cells are a type of endocrine cell found in pancreatic islets that synthesize, store and release insulin. In type 1 diabetes (T1D), T-cells of the immune system selectively destroy the insulin-producing ß-cells. Destruction of these cells leads to a lifelong dependence on exogenous insulin administration for survival. Consequently, there is an urgent need to identify novel therapies that stimulate ß-cell growth and induce ß-cell function. We and others have shown that pancreatic ductal progenitor cells are a promising source for regenerating ß-cells for T1D owing to their inherent differentiation capacity. Default transcriptional suppression is refractory to exocrine reaction and tightly controls the regenerative potential by the EZH2 methyltransferase. In the present study, we show that transient stimulation of exocrine cells, derived from juvenile and adult T1D donors to the FDA-approved EZH2 inhibitors GSK126 and Tazemetostat (Taz) influence a phenotypic shift towards a ß-like cell identity. The transition from repressed to permissive chromatin states are dependent on bivalent H3K27me3 and H3K4me3 chromatin modification. Targeting EZH2 is fundamental to ß-cell regenerative potential. Reprogrammed pancreatic ductal cells exhibit insulin production and secretion in response to a physiological glucose challenge ex vivo. These pre-clinical studies underscore the potential of small molecule inhibitors as novel modulators of ductal progenitor differentiation and a promising new approach for the restoration of ß-like cell function.

Deep sequencing reveals increased DNA methylation in chronic rat epilepsy.

Epilepsy is a frequent neurological disorder, although onset and progression of seizures remain difficult to predict in affected patients, irrespective of their epileptogenic condition. Previous studies in animal models as well as human epileptic brain tissue revealed a remarkably diverse pattern of gene expression implicating epigenetic changes to contribute to disease progression. Here we mapped for the first time global DNA methylation patterns in chronic epileptic rats and controls. Using methyl-CpG capture associated with massive parallel sequencing (Methyl-Seq) we report the genomic methylation signature of the chronic epileptic state. We observed a predominant increase, rather than loss of DNA methylation in chronic rat epilepsy. Aberrant methylation patterns were inversely correlated with gene expression changes using mRNA sequencing from same animals and tissue specimens. Administration of a ketogenic, high-fat, low-carbohydrate diet attenuated seizure progression and ameliorated DNA methylation mediated changes in gene expression. This is the first report of unsupervised clustering of an epigenetic mark being used in epilepsy research to separate epileptic from non-epileptic animals as well as from animals receiving anti-convulsive dietary treatment. We further discuss the potential impact of epigenetic changes as a pathogenic mechanism of epileptogenesis.

Pharmacological inhibition of human EZH2 can influence a regenerative ß-like cell capacity with in vitro insulin release in pancreatic ductal cells.

BACKGROUND: Therapeutic replacement of pancreatic endocrine ß-cells is key to improving hyperglycaemia caused by insulin-dependent diabetes . Whilst the pool of ductal progenitors, which give rise to the endocrine cells, are active during development, neogenesis of islets is repressed in the human adult. Recent human donor studies have demonstrated the role of EZH2 inhibition in surgically isolated exocrine cells showing reactivation of insulin expression and the influence on the H3K27me3 barrier to ß-cell regeneration. However, those studies fall short on defining the cell type active in transcriptional reactivation events. This study examines the role of the regenerative capacity of human pancreatic ductal cells when stimulated with pharmacological inhibitors of the EZH2 methyltransferase. RESULTS: Human pancreatic ductal epithelial cells were stimulated with the EZH2 inhibitors GSK-126, EPZ6438, and triptolide using a 2- and 7-day protocol to determine their influence on the expression of core endocrine development marker NGN3, as well as ß-cell markers insulin, MAFA, and PDX1. Chromatin immunoprecipitation studies show a close correspondence of pharmacological EZH2 inhibition with reduced H3K27me3 content of the core genes, NGN3, MAFA and PDX1. Consistent with the reduction of H3K27me3 by pharmacological inhibition of EZH2, we observe measurable immunofluorescence staining of insulin protein and glucose-sensitive insulin response. CONCLUSION: The results of this study serve as a proof of concept for a probable source of ß-cell induction from pancreatic ductal cells that are capable of influencing insulin expression. Whilst pharmacological inhibition of EZH2 can stimulate secretion of detectable insulin from ductal progenitor cells, further studies are required to address mechanism and the identity of ductal progenitor cell targets to improve likely methods designed to reduce the burden of insulin-dependent diabetes.

High-Resolution Genomic Profiling of Liver Cancer Links Etiology With Mutation and Epigenetic Signatures.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization. METHODS: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection. RESULTS: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway. CONCLUSIONS: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

Circulating epigenomic biomarkers correspond with kidney disease susceptibility in high-risk populations with type 2 diabetes mellitus.

AIMS: To investigate epigenomic indices of diabetic kidney disease (DKD) susceptibility among high-risk populations with type 2 diabetes mellitus. METHODS: KDIGO (Kidney Disease: Improving Global Outcomes) clinical guidelines were used to classify people living with or without DKD. Differential gene methylation of DKD was then assessed in a discovery Aboriginal Diabetes Study cohort (PROPHECY, 89 people) and an external independent study from Thailand (THEPTARIN, 128 people). Corresponding mRNA levels were also measured and linked to levels of albuminuria and eGFR. RESULTS: Increased DKD risk was associated with reduced methylation and elevated gene expression in the PROPHECY discovery cohort of Aboriginal Australians and these findings were externally validated in the THEPTARIN diabetes registry of Thai people living with type 2 diabetes mellitus. CONCLUSIONS: Novel epigenomic scores can improve diagnostic performance over clinical modelling using albuminuria and GFR alone and can distinguish DKD susceptibility.

Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes.

Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome-wide methylation patterns are poorly defined. While methylation is known to alter gene expression, the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n = 39) that was subsequently replicated in a larger validation cohort (n = 296). Gene body-related regions made up more than 60% of the methylation differences and emphasized the importance of methylation sequencing. We observed differentially methylated genes associated with DN in 3 independent T1D registries originating from Denmark (n = 445), Hong Kong (n = 107), and Thailand (n = 130). Reduced DNA methylation at CTCF and Pol2B sites was tightly connected with DN pathways that include insulin signaling, lipid metabolism, and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubule cells. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding, and the activation of insulin-signaling phosphoproteins in hyperglycemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophages and vascular endothelial cells.

Diabetes mellitus and insulin resistance associated with Kabuki syndrome-A case report and literature review.

Kabuki syndrome (KS) is a genetic disorder characterized by distinctive facies, intellectual disability, and multi-organ anomalies. This case report highlights the importance of clinical recognizable phenotype in patients with diabetes. The development of diabetes should be considered an endocrine complication in KS patients.

Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor.

Type 1 diabetes (T1D) is an autoimmune disease that selectively destroys insulin-producing ß-cells in the pancreas. An unmet need in diabetes management, current therapy is focussed on transplantation. While the reprogramming of progenitor cells into functional insulin-producing ß-cells has also been proposed this remains controversial and poorly understood. The challenge is determining why default transcriptional suppression is refractory to exocrine reactivation. After the death of a 13-year-old girl with established insulin-dependent T1D, pancreatic cells were harvested in an effort to restore and understand exocrine competence. The pancreas showed classic silencing of ß-cell progenitor genes with barely detectable insulin (Ins) transcript. GSK126, a highly selective inhibitor of EZH2 methyltransferase activity influenced H3K27me3 chromatin content and transcriptional control resulting in the expression of core ß-cell markers and ductal progenitor genes. GSK126 also reinstated Ins gene expression despite absolute ß-cell destruction. These studies show the refractory nature of chromatin characterises exocrine suppression influencing ß-cell plasticity. Additional regeneration studies are warranted to determine if the approach of this n-of-1 study generalises to a broader T1D population.

Epigenetic evidence of an Ac/Dc axis by VPA and SAHA.

BACKGROUND: Valproic acid (VPA) is one of the most commonly used anti-epileptic drugs with pharmacological actions on GABA and blocking voltage-gated ion channels. VPA also inhibits histone deacetylase (HDAC) activity. Suberoylanilide hydroxamic acid is also a member of a larger class of compounds that inhibit HDACs. At the time of this article, there are 123 active international clinical trials for VPA (also known as valproate, convulex, divalproex, and depakote) and SAHA (vorinostat, zolinza). While it is well known that VPA and SAHA influence the accumulation of acetylated lysine residues on histones, their true epigenetic complexity remains poorly understood. RESULTS: Primary human cells were exposed to VPA and SAHA to understand the extent of histone acetylation (H3K9/14ac) using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Because histone acetylation is often associated with modification of lysine methylation, we also examined H3K4me3 and H3K9me3. To assess the influence of the HDAC inhibitors on gene expression, we used RNA sequencing (RNA-seq). ChIP-seq reveals a distribution of histone modifications that is robust and more broadly regulated than previously anticipated by VPA and SAHA. Histone acetylation is a characteristic of the pharmacological inhibitors that influenced gene expression. Surprisingly, we observed histone deacetylation by VPA stimulation is a predominant signature following SAHA exposure and thus defines an acetylation/deacetylation (Ac/Dc) axis. ChIP-seq reveals regionalisation of histone acetylation by VPA and broader deacetylation by SAHA. Independent experiments confirm H3K9/14 deacetylation of NFκB target genes by SAHA. CONCLUSIONS: The results provide an important framework for understanding the Ac/Dc axis by highlighting a broader complexity of histone modifications by the most established and efficacious anti-epileptic medication in this class, VPA and comparison with the broad spectrum HDAC inhibitor, SAHA.

DNA methylation status correlates with adult ß-cell regeneration capacity.

The role of DNA methylation in ß-cell neogenesis is poorly understood. We report that during the process of induced cell reprogramming, methylation content of the Ngn3 and Sox11 genes are diminished. These findings emphasise DNA methylation is a barrier in ß-cell regeneration in adulthood, a well described pathophysiological phenomenon of major significance in explaining ß-cell deficiency in diabetes in the adult pancreas.

Valproic acid influences the expression of genes implicated with hyperglycaemia-induced complement and coagulation pathways.

Because the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is interest in understanding the mechanisms of hepatic cell activation under hyperglycaemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycaemia stimulates major changes in chromatin organization and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA) is able to reverse some of these metabolic changes. In this study, we have used RNA-sequencing (RNA-seq) to investigate how VPA influences gene expression in hepatocytes. Interesting, we observed that VPA attenuates hyperglycaemia-induced activation of complement and coagulation cascade genes. We also observe that many of the gene activation events coincide with changes to histone acetylation at the promoter of these genes indicating that epigenetic regulation is involved in VPA action.

Prolonged Honeymoon Period in a Thai Patient with Adult-Onset Type 1 Diabetes Mellitus.

OBJECTIVE: To describe a usual case of adult-onset T1DM with prolonged honeymoon period for more than 5 years. METHODS: Repeated mixed meal stimulation tests for a period of 6-12 months together with monitoring pancreatic autoantibodies and laboratory data were followed following the onset of diagnosis. RESULTS: We report a 24-year-old Thai patient with T1DM with sustained remission without antidiabetic medication for more than 5 years while maintaining low-carbohydrate intake and regular exercise. Repeated mixed meal stimulation tests for a period of 6-12 months revealed preserved beta-cell functions. Interestingly, repeated pancreatic autoantibodies at 5 years after diagnosis still showed positive anti-GAD, anti-IA2, and anti-ZnT8. CONCLUSION: Restored beta-cell function with complete insulin withdrawal in new-onset T1DM has been reported in very few cases with some common factors as in our patient (low-carbohydrate intake with regular exercise). Delaying autoimmune activity by reducing metabolic load in newly diagnosed T1DM might play a role in maintaining the honeymoon period and could lead to an innovative therapeutic option in new-onset T1DM.