A high fat, high sugar diet induces hepatic Peroxisome proliferator-activated receptor gamma coactivator 1-alpha promoter hypermethylation in male Wistar rats.

Previously we reported that a high fat, high sugar (HFHS) diet induced adiposity, hyperinsulinaemia, hyperleptinaemia, hypertriglyceridaemia and increased liver mass in male Wistar rats. In the present study, the mechanisms underlying the increased liver mass were further elucidated by assessing hepatic lipid accumulation and the expression and methylation status of key metabolic genes using histology, quantitative real-time PCR and pyrosequencing, respectively. The HFHS diet induced hepatic steatosis, increased hepatic triglycerides (1.8-fold, p < 0.001), and increased the expression of sterol regulatory element-binding transcription factor 1 (Srebf1) (2.0-fold, p < 0.001) and peroxisome proliferator-activated receptor gamma (Pparg) (1.7-fold, p = 0.017) in the liver. The expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc1a) was decreased (2.6-fold, p < 0.010), which was accompanied by hypermethylation (p = 0.018) of a conserved CpG site in the promoter of Pgc1a in HFHS fed rats compared to controls. In silico analysis identified putative binding sites for CCAAT/enhancer-binding protein beta (C/EBPß) and hepatocyte nuclear factor 1 (HNF1) within proximity to the hypermethylated CpG. As Pgc1a is a co-activator of several transcription factors regulating multiple metabolic pathways, hypermethylation of this conserved CpG site in the promoter of Pgc1a may be one possible mechanism contributing to the development of hepatic steatosis in response to a HFHS diet. However, further work is required to confirm the role of Pgc1a in steatosis.

The role of maternal DNA methylation in pregnancies complicated by gestational diabetes.

Diabetes in pregnancy is associated with adverse pregnancy outcomes and poses a serious threat to the health of mother and child. Although the pathophysiological mechanisms that underlie the association between maternal diabetes and pregnancy complications have not yet been elucidated, it has been suggested that the frequency and severity of pregnancy complications are linked to the degree of hyperglycemia. Epigenetic mechanisms reflect gene-environment interactions and have emerged as key players in metabolic adaptation to pregnancy and the development of complications. DNA methylation, the best characterized epigenetic mechanism, has been reported to be dysregulated during various pregnancy complications, including pre-eclampsia, hypertension, diabetes, early pregnancy loss and preterm birth. The identification of altered DNA methylation patterns may serve to elucidate the pathophysiological mechanisms that underlie the different types of maternal diabetes during pregnancy. This review aims to provide a summary of existing knowledge on DNA methylation patterns in pregnancies complicated by pregestational type 1 (T1DM) and type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM). Four databases, CINAHL, Scopus, PubMed and Google Scholar, were searched for studies on DNA methylation profiling in pregnancies complicated with diabetes. A total of 1985 articles were identified, of which 32 met the inclusion criteria and are included in this review. All studies profiled DNA methylation during GDM or impaired glucose tolerance (IGT), while no studies investigated T1DM or T2DM. We highlight the increased methylation of two genes, Hypoxia-inducible Factor-3α (HIF3α) and Peroxisome Proliferator-activated Receptor Gamma-coactivator-Alpha (PGC1-α), and the decreased methylation of one gene, Peroxisome Proliferator Activated Receptor Alpha (PPARα), in women with GDM compared to pregnant women with normoglycemia that were consistently methylated across diverse populations with varying pregnancy durations, and using different diagnostic criteria, methodologies and biological sources. These findings support the candidacy of these three differentially methylated genes as biomarkers for GDM. Furthermore, these genes may provide insight into the pathways that are epigenetically influenced during maternal diabetes and which should be prioritized and replicated in longitudinal studies and in larger populations to ensure their clinical applicability. Finally, we discuss the challenges and limitations of DNA methylation analysis, and the need for DNA methylation profiling to be conducted in different types of maternal diabetes in pregnancy.

A pilot investigation of genetic and epigenetic variation of FKBP5 and response to exercise intervention in African women with obesity.

We investigated gluteal (GSAT) and abdominal subcutaneous adipose tissue (ASAT) DNA methylation of FKBP5 in response to a 12-week intervention in African women with obesity, as well as the effect of the rs1360780 single nucleotide polymorphism (SNP) on FKBP5 methylation, gene expression and post-exercise training adaptations in obesity and metabolic related parameters. Exercise (n = 19) participants underwent 12-weeks of supervised aerobic and resistance training while controls (n = 12) continued their usual behaviours. FKBP5 methylation was measured in GSAT and ASAT using pyrosequencing. SNP and gene expression analyses were conducted using quantitative real-time PCR. Exercise training induced FKBP5 hypermethylation at two CpG dinucleotides within intron 7. When stratified based on the rs1360780 SNP, participants with the CT genotype displayed FKBP5 hypermethylation in GSAT (p < 0.05), and ASAT displayed in both CC and CT carriers. CC allele carriers displayed improved cardiorespiratory fitness, insulin sensitivity, gynoid fat mass, and waist circumference (p < 0.05) in response to exercise training, and these parameters were attenuated in women with the CT genotype. These findings provide a basis for future studies in larger cohorts, which should assess whether FKBP5 methylation and/or genetic variants such as the rs1360780 SNP could have a significant impact on responsiveness to exercise interventions.

Changes in subcutaneous adipose tissue microRNA expression in response to exercise training in obese African women.

The mechanisms that underlie exercise-induced adaptations in adipose tissue have not been elucidated, yet, accumulating studies suggest an important role for microRNAs (miRNAs). This study aimed to investigate miRNA expression in gluteal subcutaneous adipose tissue (GSAT) in response to a 12-week exercise intervention in South African women with obesity, and to assess depot-specific differences in miRNA expression in GSAT and abdominal subcutaneous adipose tissue (ASAT). In addition, the association between exercise-induced changes in miRNA expression and metabolic risk was evaluated. Women underwent 12-weeks of supervised aerobic and resistance training (n = 19) or maintained their regular physical activity during this period (n = 12). Exercise-induced miRNAs were identified in GSAT using Illumina sequencing, followed by analysis of differentially expressed miRNAs in GSAT and ASAT using quantitative real-time PCR. Associations between the changes (pre- and post-exercise training) in miRNA expression and metabolic parameters were evaluated using Spearman's correlation tests. Exercise training significantly increased the expression of miR-155-5p (1.5-fold, p = 0.045), miR-329-3p (2.1-fold, p < 0.001) and miR-377-3p (1.7-fold, p = 0.013) in GSAT, but not in ASAT. In addition, a novel miRNA, MYN0617, was identified in GSAT, with low expression in ASAT. The exercise-induced differences in miRNA expression were correlated with each other and associated with changes in high-density lipoprotein concentrations. Exercise training induced adipose-depot specific miRNA expression within subcutaneous adipose tissue depots from South African women with obesity. The significance of the association between exercise-induced miRNAs and metabolic risk warrants further investigation.

Cafeteria diet induces global and Slc27a3-specific hypomethylation in male Wistar rats.

Increased visceral adipose tissue (VAT) is associated with metabolic dysfunction, while subcutaneous adipose tissue (SAT) is considered protective. The mechanisms underlying these differences are not fully elucidated. This study aimed to investigate molecular differences in VAT and SAT of male Wistar rats fed a cafeteria diet (CD) or a standard rodent diet (STD) for three months. The expression of fatty acid metabolism genes was analysed by quantitative real-time PCR. Global and gene-specific DNA methylation was quantified using the Imprint® Methylated DNA Quantification Kit and pyrosequencing, respectively. Bodyweight, retroperitoneal fat mass, insulin resistance, leptin and triglyceride concentrations and adipocyte hypertrophy were higher in CD- compared to STD-fed rats. The expression of solute carrier family 27 member 3 (Slc27a3), a fatty acid transporter, was 9.6-fold higher in VAT and 6.3-fold lower in SAT of CD- versus STD-fed rats. Taqman probes confirmed increased Slc27a3 expression, while pyrosequencing showed Slc27a3 hypomethylation in VAT of CD- compared to STD-fed rats. The CD decreased global methylation in both VAT and SAT, although no depot differences were observed. Dysregulated fatty acid influx in VAT, in response to a CD, provides insight into the mechanisms underlying depot-differences in adipose tissue expansion during obesity and metabolic disease. Abbreviations: CD: cafeteria diet; E2F1: E2F Transcription Factor 1; EMSA: electrophoretic mobility shift assay; EGFR: epidermal growth factor receptor; GCF: GC-Rich Sequence DNA-Binding Factor; HOMA-IR: Homeostasis model for insulin resistance; NKX2-1: NK2 homeobox 1; PCR: Polymerase chain reaction; qRT-PCR: quantitative real-time PCR; RF: retroperitoneal fat; SAT: subcutaneous adipose tissue; Slc27a3: solute carrier family 27 member 3; STD: standard diet; TNFα: tumour necrosis factor alpha; TTS: transcriptional start site; T2D: Type 2 Diabetes; VAT: visceral adipose tissue; WT1 I: Wilms' tumour protein 1.

Targeting the oncogenic TBX3:nucleolin complex to treat multiple sarcoma subtypes.

Sarcomas are diverse cancers of mesenchymal origin, with compromised clinical management caused by insufficient diagnostic biomarkers and limited treatment options. The transcription factor TBX3 is upregulated in a diverse range of sarcoma subtypes, where it plays a direct oncogenic role, and it may thus represent a novel therapeutic target. To identify versatile ways to target TBX3, we performed affinity purification coupled by mass spectrometry to identify putative TBX3 protein cofactors that regulate its oncogenic activity in sarcomas. Here we identify and validate the multifunctional phosphoprotein nucleolin as a TBX3 cofactor. We show that nucleolin is co-expressed with TBX3 in several sarcoma subtypes and their expression levels positively correlate in sarcoma patients which are associated with poor prognosis. Furthermore, we demonstrate that nucleolin and TBX3 interact in chondrosarcoma, liposarcoma and rhabdomyosarcoma cells where they act together to enhance proliferation and migration and regulate a common set of tumor suppressor genes. Importantly, the nucleolin targeting aptamer, AS1411, exhibits selective anti-cancer activity in these cells and mislocalizes TBX3 and nucleolin to the cytoplasm which correlates with the re-expression of the TBX3/nucleolin target tumor suppressors CDKN1A (p21CIP1) and CDKN2A (p14ARF). Our findings provide the first evidence that TBX3 requires nucleolin to promote features of sarcomagenesis and that disruption of the oncogenic TBX3-nucleolin interaction by AS1411 may be a novel approach for treating sarcomas.

DNA methylation of FKBP5 in South African women: associations with obesity and insulin resistance.

BACKGROUND: Disruption of the hypothalamic-pituitary-adrenal (HPA) axis, a neuroendocrine system associated with the stress response, has been hypothesized to contribute to obesity development. This may be mediated through epigenetic modulation of HPA axis-regulatory genes in response to metabolic stressors. The aim of this study was to investigate adipose tissue depot-specific DNA methylation differences in the glucocorticoid receptor (GR) and its co-chaperone, FK506-binding protein 51 kDa (FKBP5), both key modulators of the HPA axis. METHODS: Abdominal subcutaneous adipose tissue (ASAT) and gluteal subcutaneous adipose tissue (GSAT) biopsies were obtained from a sample of 27 obese and 27 normal weight urban-dwelling South African women. DNA methylation and gene expression were measured by pyrosequencing and quantitative real-time PCR, respectively. Spearman's correlation coefficients, orthogonal partial least-squares discriminant analysis and multivariable linear regression were performed to evaluate the associations between DNA methylation, messenger RNA (mRNA) expression and key indices of obesity and metabolic dysfunction. RESULTS: Two CpG dinucleotides within intron 7 of FKBP5 were hypermethylated in both ASAT and GSAT in obese compared to normal weight women, while no differences in GR methylation were observed. Higher percentage methylation of the two FKBP5 CpG sites correlated with adiposity (body mass index and waist circumference), insulin resistance (homeostasis model for insulin resistance, fasting insulin and plasma adipokines) and systemic inflammation (c-reactive protein) in both adipose depots. GR and FKBP5 mRNA levels were lower in GSAT, but not ASAT, of obese compared to normal weight women. Moreover, FKBP5 mRNA levels were inversely correlated with DNA methylation and positively associated with adiposity, metabolic and inflammatory parameters. CONCLUSIONS: These findings associate dysregulated FKBP5 methylation and mRNA expression with obesity and insulin resistance in South African women. Additional studies are required to assess the longitudinal association of FKBP5 with obesity and associated co-morbidities in large population-based samples.

Ethnic and Adipose Depot Specific Associations Between DNA Methylation and Metabolic Risk.

BACKGROUND: Metabolic risk varies according to body mass index (BMI), body fat distribution and ethnicity. In recent years, epigenetics, which reflect gene-environment interactions have attracted considerable interest as mechanisms that may mediate differences in metabolic risk. The aim of this study was to investigate DNA methylation differences in abdominal and gluteal subcutaneous adipose tissues of normal-weight and obese black and white South African women. METHODS: Body composition was assessed using dual-energy x-ray absorptiometry and computerized tomography, and insulin sensitivity was measured using a frequently sampled intravenous glucose tolerance test in 54 normal-weight (BMI 18-25 kg/m2) and obese (BMI ≥ 30 kg/m2) women. Global and insulin receptor (INSR) DNA methylation was quantified in abdominal (ASAT) and gluteal (GSAT) subcutaneous adipose depots, using the Imprint methylation enzyme-linked immunosorbent assay and pyrosequencing. INSR gene expression was measured using quantitative real-time PCR. RESULTS: Global DNA methylation in GSAT varied according to BMI and ethnicity, with higher levels observed in normal-weight white compared to normal-weight black (p = 0.030) and obese white (p = 0.012) women. Pyrosequencing of 14 CpG sites within the INSR promoter also showed BMI, adipose depot and ethnic differences, although inter-individual variability prevented attainment of statistical significance. Both global and INSR methylation were correlated with body fat distribution, insulin resistance and systemic inflammation, which were dependent on ethnicity and the adipose depot. Adipose depot and ethnic differences in INSR gene expression were observed. CONCLUSION: We show small, but significant global and INSR promoter DNA methylation differences in GSAT and ASAT of normal-weight and obese black and white South African women. DNA methylation in ASAT was associated with centralization of body fat in white women, whereas in black women DNA methylation in GSAT was associated with insulin resistance and systemic inflammation. Our findings suggest that GSAT rather than ASAT may be a determinant of metabolic risk in black women and provide novel evidence that altered DNA methylation within adipose depots may contribute to ethnic differences in body fat distribution and cardiometabolic risk.

Corrigendum: Blood-Based DNA Methylation Biomarkers for Type 2 Diabetes: Potential for Clinical Applications.

[This corrects the article DOI: 10.3389/fendo.2018.00744.].

Altered microRNA expression during Impaired Glucose Tolerance and High-fat Diet Feeding.

OBJECTIVE: MicroRNAs (miRNAs) play a critical role in metabolic regulation. Recently, we identified novel miRNAs in the whole blood of South African women of mixed ethnic ancestry. The aim of this study was to investigate whether five of these novel miRNAs are expressed in serum and whether their expression is altered during metabolic dysregulation. METHODS: Expression levels of the five novel miRNAs (MYN08, MYNO22, MYN059, MYNO66 and MYNO95) were measured in the serum of women with Impaired Glucose Tolerance (IGT) and Normoglycemia (NGT) (n=24), and in the whole blood of vervet monkeys fed a high-fat or standard diet (n=16) using quantitative real-time PCR. RESULTS: Only three of the selected novel miRNAs (MYNO8, MYNO22 and MYNO66) were expressed in serum. The expression of MYN08 and MYNO22 were associated with fasting glucose and insulin concentrations, decreased during IGT and able to predict IGT. The expression of these miRNAs were similarly decreased in vervet monkeys fed a high-fat diet. In silico analysis identified a total of 291 putative messenger RNA targets for MYNO8 and MYNO22, including genes involved in gluconeogenesis, carbohydrate metabolism, glucose homeostasis and lipid transport. CONCLUSION: Two novel miRNAs, MYNO8 and MYNO22, are associated with metabolic dysregulation in South African women of mixed ethnic ancestry and with high-fat diet feeding in vervet monkeys. Furthermore, putative gene targets were enriched in biological processes involved in key aspects of glucose regulation, which strengthens the candidacy of these miRNAs as biomarkers for dysglycemia, and warranting further studies to assess their clinical applicability.

Blood-Based DNA Methylation Biomarkers for Type 2 Diabetes: Potential for Clinical Applications.

Type 2 diabetes (T2D) is a leading cause of death and disability worldwide. It is a chronic metabolic disorder that develops due to an interplay of genetic, lifestyle, and environmental factors. The biological onset of the disease occurs long before clinical symptoms develop, thus the search for early diagnostic and prognostic biomarkers, which could facilitate intervention strategies to prevent or delay disease progression, has increased considerably in recent years. Epigenetic modifications represent important links between genetic, environmental and lifestyle cues and increasing evidence implicate altered epigenetic marks such as DNA methylation, the most characterized and widely studied epigenetic mechanism, in the pathogenesis of T2D. This review provides an update of the current status of DNA methylation as a biomarker for T2D. Four databases, Scopus, Pubmed, Cochrane Central, and Google Scholar were searched for studies investigating DNA methylation in blood. Thirty-seven studies were identified, and are summarized with respect to population characteristics, biological source, and method of DNA methylation quantification (global, candidate gene or genome-wide). We highlight that differential methylation of the TCF7L2, KCNQ1, ABCG1, TXNIP, PHOSPHO1, SREBF1, SLC30A8, and FTO genes in blood are reproducibly associated with T2D in different population groups. These genes should be prioritized and replicated in longitudinal studies across more populations in future studies. Finally, we discuss the limitations faced by DNA methylation studies, which include including interpatient variability, cellular heterogeneity, and lack of accounting for study confounders. These limitations and challenges must be overcome before the implementation of blood-based DNA methylation biomarkers into a clinical setting. We emphasize the need for longitudinal prospective studies to support the robustness of the current findings of this review.

The T-Box transcription factor 3 in development and cancer.

T-box factors comprise an archaic family of evolutionary conserved transcription factors that regulate patterns of gene expression essential for embryonic development. The T-box transcription factor 3 (TBX3), a member of this family, is expressed in several tissues and plays critical roles in, among other structures, the heart, mammary gland and limbs and haploinsufficiency of the human TBX3 gene is the genetic basis for the autosomal dominant disorder, ulnar-mammary syndrome. Overexpression of TBX3 on the other hand has been linked to several cancers including melanoma, breast, pancreatic, liver, lung, head and neck, ovarian, bladder carcinomas and a number of sarcoma subtypes. Furthermore, there is strong evidence that TBX3 promotes oncogenesis by impacting proliferation, tumour formation, metastasis as well as cell survival and drug resistance. More recently, TBX3 was however shown to also have tumour suppressor activity in fibrosarcomas and thus its functions in oncogenesis appear to be context dependent. Identification of the upstream regulators of TBX3 and the molecular mechanism(s) underpinning its oncogenic roles will make valuable contributions to cancer biology.

Managing sarcoma: where have we come from and where are we going?

Sarcomas are a heterogeneous group of neoplasms of mesenchymal origin. Approximately 80% arise from soft tissue and 20% originate from bone. To date more than 100 sarcoma subtypes have been identified and they vary in molecular characteristics, pathology, clinical presentation and response to treatment. While sarcomas represent <1% of adult cancers, they account for approximately 21% of paediatric malignancies and thus pose some of the greatest risks of mortality and morbidity in children and young adults. Metastases occur in one-third of all patients and approximately 10-20% of sarcomas recur locally. Surgery in combination with preoperative and postoperative therapies is the primary treatment for localized sarcoma tumours and is the most promising curative possibility. Metastasized sarcomas, on the other hand, are treated primarily with single-agent or combination chemotherapy, but this rarely leads to a complete and robust response and often becomes a palliative form of treatment. The heterogeneity of sarcomas results in variable responses to current generalized treatment strategies. In light of this and the lack of curative strategies for metastatic and unresectable sarcomas, there is a need for novel subtype-specific treatment strategies. With the more recent understanding of the molecular mechanisms underlying the pathogenesis of some of these tumours, the treatment of sarcoma subtypes with targeted therapies is a rapidly evolving field. This review discusses the current management of sarcomas as well as promising new therapies that are currently underway in clinical trials.

The T-box transcription factor TBX3 drives proliferation by direct repression of the p21(WAF1) cyclin-dependent kinase inhibitor.

BACKGROUND: TBX3, a member of the T-box family of transcription factors, is essential in development and has emerged as an important player in the oncogenic process. TBX3 is overexpressed in several cancers and has been shown to contribute directly to tumour formation, migration and invasion. However, little is known about the molecular basis for its role in development and oncogenesis because there is a paucity of information regarding its target genes. The cyclin-dependent kinase inhibitor p21(WAF1) plays a pivotal role in a myriad of processes including cell cycle arrest, senescence and apoptosis and here we provide a detailed mechanism to show that it is a direct and biologically relevant target of TBX3. RESULTS: Using a combination of luciferase reporter gene assays and in vitro and in vivo binding assays we show that TBX3 directly represses the p21(WAF1) promoter by binding a T-element close to its initiator. Furthermore, we show that the TBX3 DNA binding domain is required for the transcriptional repression of p21(WAF1) and that pseudo-phosphorylation of a serine proline motif (S190) located within this domain may play an important role in regulating this ability. Importantly, we demonstrate using knockdown and overexpression experiments that p21(WAF1) repression by TBX3 is biologically significant and required for TBX3-induced cell proliferation of chondrosarcoma cells. CONCLUSIONS: Results from this study provide a detailed mechanism of how TBX3 transcriptionally represses p21(WAF1) which adds to our understanding of how it may contribute to oncogenesis.

The T-Box factor TBX3 is important in S-phase and is regulated by c-Myc and cyclin A-CDK2.

The transcription factor, TBX3, is critical for the formation of, among other structures, the heart, limbs and mammary glands and haploinsufficiency of the human TBX3 gene result in ulnar-mammary syndrome which is characterized by hypoplasia of these structures. On the other hand, the overexpression of TBX3 is a feature of a wide range of cancers and it has been implicated in several aspects of the oncogenic process. This includes its ability to function as an immortalizing gene and to promote proliferation through actively repressing negative cell cycle regulators. Together this suggests that TBX3 levels may need to be tightly regulated during the cell cycle. Here we demonstrate that this is indeed the case and that TBX3 mRNA and protein levels peak at S-phase and that the TBX3 protein is predominantly localized to the nucleus of S-phase cells. The increased levels of TBX3 in S-phase are shown to occur transcriptionally through activation by c-Myc at E-box motifs located at -1210 and -701 bps and post-translationally by cyclin A-CDK2 phosphorylation. Importantly, when TBX3 is depleted by shRNA the cells accumulate in S-phase. These results suggest that TBX3 is required for cells to transit through S-phase and that this function may be linked to its role as a pro-proliferative factor.

Knockdown of Hop downregulates RhoC expression, and decreases pseudopodia formation and migration in cancer cell lines.

The Hsp90/Hsp70 organising protein (Hop) is a co-chaperone that mediates the interaction of Hsp90 and Hsp70 molecular chaperones during assembly of Hsp90 complexes in cells. Formation of Hsp90 complexes is a key intermediate step in the maturation and homeostasis of oncoproteins and several hormone receptors. In this paper, we demonstrate that knockdown of Hop decreased migration of Hs578T and MDA-MB-231 breast cancer cells. Hop was identified in isolated pseudopodia fractions; it colocalised with actin in lamellipodia, and co-sedimented with purified actin in vitro. Knockdown of Hop caused a decrease in the level of RhoC GTPase, and significantly inhibited pseudopodia formation in Hs578T cells. Our data suggest that Hop regulates directional cell migration by multiple unknown mechanisms.