TET-mediated DNA hydroxymethylation is negatively influenced by the PARP-dependent PARylation

Background Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA—the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP–TET interplay. Results Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. Conclusions According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management. 1 Introductio

PARP-1 and YY1 Are Important Novel Regulators of CXCL12 Gene Transcription in Rat Pancreatic Beta Cells

Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12) transcription. The roles of poly(ADP-ribose) polymerase-1 (PARP-1) and transcription factor Yin Yang 1 (YY1) in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ)-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the functional interplay of these proteins could finely balance Cxcl12 transcription

Relationship between serum tumor necrosis factor receptor-2 concentration and periodontal destruction in patients with type 2 diabetes: Cross-sectional study

Introduction: The role of tumor necrosis factor-α (TNFα) is well documented in pathogenesis of chronic periodontitis (CP) and type 2 diabetes (T2D). Considering short half-life of TNFα, tumor necrosis factor receptor-2 (TNFR2) is used as prosperous surrogate marker of TNFα activity. Objective The aim was to detect TNFR2 serum concentration and correlate it with periodontal destruction in patients with diagnosed T2D and nondiabetics. Methods The study included 85 patients divided into three groups: T2D + CP (group T2D, n = 34); nondiabetics + CP (Group PD, n = 27); and healthy controls (group HC, n = 24). T2D was diagnosed according to WHO criteria (2013) and periodontitis was diagnosed using International Workshop for a Classification of Periodontal Diseases and Conditions criteria (1999). TNFR2 level was measured by enzyme-linked immunosorbent assay (ELISA). Results There was no difference in TNFR2 level among the groups (Kruskal-Wallis, p = 0.482). Significant correlation (Pearson's correlation coefficient) was observed between clinical attachment loss (CAL) and TNFR2 concentration in PD group (rp = -0.460, p = 0.016). In T2D group, correlations were observed between TNFR2 concentration and CaL (rp = 0.363, p = 0.005) and periodontal inflamed surface area (PISA) (rp = 0.345, p = 0.046) and periodontalepithelial surface area (PESA) (rp = 0.578, p = 0.000). Conclusion Higher concentration of TNFR2 was associated with higher CAL, PESA, and PISA scores in T2D group. Contrary to that, nondiabetics with higher values of CAL exhibited lower concentration of TNFR2, presenting potential protective effect on periodontal destruction. These results imply that diabetes may alter TNFR2 secretion originated from periodontium.Uvod: Uloga faktora nekroze tumora-alfa (TNFα) dokazana je u patogenezi hronične parodontopatije (HP) i dijabetesa melitusa tipa 2 (DM tip 2). S obzirom na to da je poluživot TNFα veoma kratak, receptor 2 faktora nekroze tumora (TNFR2) koristi se kao marker aktivnosti TNFα. Cilj rada Cilj ovog rada je određivanje koncentracije TNFR2 u serumu i koreliranje sa parametrima destrukcije parodoncijuma kod zdravih i ispitanika sa dijagnostikovanim DM tip 2. Metode rada U studiju je uključeno 85 pacijenata podeljenih u tri grupe: DM tip 2 + HP (DM grupa, n = 34), zdravi ispitanici + HP (PD grupa, n = 27) i zdrave kontrole (ZK grupa, n = 24). Dijagnoza DM tip 2 postavljena je na osnovu kriterijuma SZO (2013), dok je dijagnoza HP postavljena na osnovu kriterijuma Internacionalne radionice za klasifikaciju stanja i oboljenja parodoncijuma (1999). Koncentracija TNFR2 merena je ELISA metodom. Rezultati Koncentracija serumskog TNFR2 nije se razlikovala među grupama (Kraskal-Volis, p = 0,482). Postoji značajna korelacija (Pirson) između nivoa pripojnog epitela (NPE) i koncentracije TNFR2 u PD grupi (rp = -0,460, p = 0,016). U DM tip 2 grupi, statistički značajna korelacija uočena je između koncentracije TNFR2 i NPE (rp = 0,363, p = 0,005), kao i parametara uticaja inflamacije iz parodoncijuma na sistemsko zdravlje - PISA (rp = 0,345, p = 0,046) i PESA (rp = 0,578, p = 0,000). Zaključak Kod pacijenata sa dijabetesom veće koncentracije TNFR2 odgovaraju većim vrednostima NPE, PESA i PISA. Nasuprot tome, kod sistemski zdravih ispitanika sa HP veće vrednosti NPE su povezane sa manjim koncentracijama TNFR2, što bi moglo govoriti o potencijalnoj zaštitnoj ulozi ovog citokina na destrukciju parodoncijuma. Rezultati govore da dijabetes može uticati na sekreciju TNFR2 iz parodoncijuma

Body mass index, gestational weight gain and fatty acid concentrations during pregnancy: the Generation R Study

Obesity during pregnancy may be correlated with an adverse nutritional status affecting pregnancy and offspring outcomes. We examined the associations of prepregnancy body mass index and gestational weight gain with plasma fatty acid concentrations in mid-pregnancy. This study was embedded in a population-based prospective cohort study among 5636 women. We obtained prepregnancy body mass index and maximum weight gain during pregnancy by questionnaires. We measured concentrations of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), n-3 polyunsaturated fatty acid (n-3 PUFA) and n-6 polyunsaturated fatty acid (n-6 PUFA) at a median gestational age of 20.5 (95 % range 17.1–24.9) weeks. We used multivariate linear regression models. As compared to normal weight women, obese women had higher total SFA concentrations [difference: 0.10 standard deviation (SD) (95 % Confidence Interval (CI) 0, 0.19)] and lower total n-3 PUFA concentrations [difference: − 0.11 SD (95 % CI − 0.20, − 0.02)]. As compared to women with sufficient gestational weight gain, those with excessive gestational weight gain had higher SFA concentrations [difference: 0.16 SD (95 % CI 0.08, 0.25)], MUFA concentrations [difference: 0.16 SD (95 % CI 0.08, 0.24)] and n-6 PUFA concentrations [difference: 0.12 SD (95 % CI 0.04, 0.21)]. These results were not materially affected by adjustment for maternal characteristics. Our results suggest that obesity and excessive weight gain during pregnancy are associated with an adverse fatty acids profile. Further studies are needed to assess causality and direction of the observed associations. Electronic supplementary material The online version of this article (doi:10.1007/s10654-015-0106-6) contains supplementary material, which is available to authorized users