Marija Klasić

BackgroundSerotonin (5-hydroxytryptamine, 5-HT) signaling is involved in neurodevelopment, mood regulation, energy metabolism, and other physiological processes. DNA methylation plays a significant role in modulating the expression of genes responsible for maintaining 5-HT balance, such as 5-HT transporter (SLC6A4), monoamine oxidase A (MAOA), and 5-HT receptor type 2A (HTR2A). Maternal metabolic health can influence long-term outcomes in offspring, with DNA methylation mediating these effects. We investigated associations between maternal metabolic parameters—pre-pregnancy body mass index (pBMI), gestational weight gain (GWG), and glucose tolerance status (GTS), i.e., gestational diabetes mellitus (GDM) versus normal glucose tolerance (NGT)—and cord blood methylation of SLC6A4, MAOA, and HTR2A in participants from our PlaNS birth cohort. CpG sites (15, 9, and 2 in each gene …

Hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 4 alpha (HNF4A), and forkhead box protein A2 (FOXA2) are key transcription factors that regulate a complex gene network in the liver, creating a regulatory transcriptional loop. The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes. Our in silico analysis of HNF1A, HNF4A, and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrichment of these transcription factors specifically in the liver. Our previous studies identified HNF1A as a master regulator of fucosylation, glycan branching, and galactosylation of plasma glycoproteins. Here, we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype. We used the state-of-the …

AimTo assess the frequency of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and high-risk types of human papillomavirus (HPV16 and HPV18) infections in lung adenocarcinoma samples.MethodsLung adenocarcinoma cytological smears and their DNA isolates were obtained from patients hospitalized at the Department for Lung Diseases Jordanovac, Zagreb, in 2016 and 2017. Overall, 67 lung adenocarcinoma samples were examined: 34 with epidermal growth factor receptor gene (EGFR) mutations and 33 without EGFR mutations. The EGFR mutation status and virus presence were assessed with a polymerase chain reaction, and random samples were additionally tested for EBV with Sanger sequencing. HCMV, EBV, HPV16, and HPV18 infections were evaluated in relation to EGFR mutation, smoking status, and sex. A meta-analysis of available data about HPV infection in non-small cell lung …

Epigenetic mechanisms are critical in communication between environment and genome, thus playing an important role in many diseases, including cancer. Molecular tools have been developed for targeted and specific manipulation of epigenetic modifications, including the CRISPR/dCas9 system, that enables studying interplay of different epigenetic marks and their effects on gene expression. Fusion of different epigenetic modifiers to the inactivated dCas9 allows for introduction of desired epigenetic modification in the genome region of interest. In this study, we investigated interactions between DNA methylation and histone modifications, and the effects of these epigenetic marks on the activity of two genes, ZEB1 and SNAI1, coding for transcription factors involved in epithelial to mesenchymal transition (EMT). To achieve this, we used the DNMT3A-dSpCas9 fusion for targeted DNA methylation in combination with dCas9 fusions with different histone modifying effectors domains: G9a-SET for induction of H3K9 mono and di-methylation, G9a-Y1154F-SET for H3K9 trimethylation, LSD1-SET for H3K4me1/2 demethylation, and the catalytic domain of KDM5a for H3K4me3 reduction. Multiple gRNAs were used to target the entire CpG islands of ZEB1 and SNAI1 genes in the HepG2 cell line. A synergistic effect of the G9a-dSpCas9 and DNMT3A-dSpCas9 was observed on both ZEB1 and SNAI1 gene expression, while the synergy effect on DNA methylation was observed only in SNAI1 gene promoter. Interestingly, none of the effector domains themselves were able to increase DNA methylation level. However, in combination with DNMT3A …

Background & AimsDNA methylation alterations may provide important insights into gene-environment interaction in cancer, aging, and complex diseases, such as inflammatory bowel disease (IBD). We aim first to determine whether the circulating DNA methylome in patients requiring surgery may predict Crohn’s disease (CD) recurrence following intestinal resection; and second to compare the circulating methylome seen in patients with established CD with that we had reported in a series of inception cohorts.MethodsTOPPIC was a placebo-controlled, randomized controlled trial of 6-mercaptopurine at 29 UK centers in patients with CD undergoing ileocolic resection between 2008 and 2012. Genomic DNA was extracted from whole blood samples from 229 of the 240 patients taken before intestinal surgery and analyzed using 450KHumanMethylation and Infinium Omni Express Exome arrays (Illumina, San Diego …

Tumour cells develop by accumulating changes in the genome that result in changes of main cellular processes. Aberrations of basic processes such as replication and chromatin reassembly are particularly important for genomic (in)stability. The aim of this study was to analyse the expression of genes whose products are crucial for the regulation of replication and chromatin reassembly during lymphomagenesis (DNMT1, PCNA, MCM2, CDT1, EZH2, GMNN, EP300). Non-tumour B cells were used as a control, and follicular lymphoma (FL) and the two most common groups of diffuse large B cell lymphoma (DLBCL) samples were used as a model for tumour progression. The results showed that there are significant changes in the expression of the analysed genes in lymphomagenesis, but also that these changes do not display linearity when assessed in relation to the degree of tumour aggression. Additionally, an …

The serotonin receptor 2A gene (HTR2A) is a strong candidate for the fetal programming of future behavior and metabolism. Maternal obesity and gestational diabetes mellitus (GDM) have been associated with an increased risk of metabolic and psychological problems in offspring. We tested the hypothesis that maternal metabolic status affects methylation of HTR2A in the placenta. The prospective study included 199 pairs of mothers and healthy full-term newborns. Genomic DNA was extracted from feto-placental samples and analyzed for genotypes of two polymorphisms (rs6311, rs6306) and methylation of four cytosine residues (−1665, −1439, −1421, −1224) in the HTR2A promoter region. Placental HTR2A promoter methylation was higher in male than female placentas and depended on both rs6311 and rs6306 genotypes. A higher maternal pre-gestational body mass index (pBMI) and, to a lesser extent, diagnosis of GDM were associated with reduced HTR2A promoter methylation in female but not male placentas. Higher pBMI was associated with reduced methylation both directly and indirectly through increased GDM incidence. Tobacco use during pregnancy was associated with reduced HTR2A promoter methylation in male but not female placentas. The obtained results suggest that HTR2A is a sexually dimorphic epigenetic target of intrauterine exposures. The findings may contribute to a better understanding of the early developmental origins of neurobehavioral and metabolic disorders associated with altered HTR2A function.

In hepatocellular carcinoma (HCC), as well as in various other cancers, protein glycosylation is altered and as such is associated with tumor proliferation, invasion, metastasis, angiogenesis, and multidrug resistance. Mechanisms are mostly epigenetic. Indeed, aberrant DNA methylation is one of the epigenetic modifications that is highly perturbed in cancer, resulting in changes in transcriptional activity of many key genes, thus leading to characteristic cancer behavior. One group of genes, which might be affected, are glyco-genes coding for glycosyltransferases. The aim of this study is to explore epigenetic regulation of glyco-genes using cutting-edge CRISPR/Cas9 based molecular tools for epigenome editing. In hepatocellular carcinoma model cell line HepG2, we targeted seven candidate glyco-genes using dCas9-DNMT3A and dCas9-TET1 fusions, and subsequently analyzed CpG methylation, transcriptional gene activity, and whole-cell N-glycome as a final phenotype. Transfected cells were collected at two time points (8th and 12th day following transfection). Targeted methylation of selected CpG sites in ST6GAL1, FUT8, MGAT4A, MGAT4B, MGAT5, and B4GALT1 genes induced hypermethylation at these sites (up to 40% on average, depending on the gene), which was followed by a statistically significant change in transcriptional activity of all these genes except ST6GAL1. Targeted demethylation of MGAT3 gene (up to 45% on average) was accompanied by a statistically significant change in its transcription. As a final phenotype, whole-cell protein glycosylation was analyzed and changes in several glycosylation traits in HepG2 …

Transcription factors HNF1A, HNF4A and FOXA1/2/3 regulate developmental and tissue-specific transcriptional gene networks in liver and pancreas. Alternative glycosylation affects protein structure and function, and aberrant protein glycosylation is observed in inflammation, diabetes and cancer. For instance, abnormal glucose stimulated insulin secretion in diabetes might occur through epigenetic changes in HNF1A and FOXA2, resulting in deregulation of MGAT4A, MGAT4B and MGAT5 glycosyltransferases responsible for proper glycosylaton of GLUT receptors on beta cells. In addition, HNF1A is identified by GWAS studies as a master regulator of key fucosyltransferases in liver. Following CRISPR-based manipulations, we analysed total protein glycosylation.

N-glycosylation is a frequent modification of proteins, essential for all domains of life. N-glycan biosynthesis is a dynamic, complex, non-templated process, wherein specific glycoforms are modulated by various microenvironmental cues, cellular signals and local availability of dedicated enzymes and sugar precursors. This intricate regulatory network comprises hundreds of proteins, whose activity is dependent on both sequence of implicated genes and the regulation of their expression. In this regard, variation in N-glycosylation patterns stems from either gene polymorphisms or from stable epigenetic regulation of gene expression in different individuals. Moreover, epigenome alters in response to various environmental factors, representing a direct link between environmental exposure and changes in gene expression, that are subsequently reflected through altered N-glycosylation. N-glycosylation itself has a …

Serotonin (5-HT) regulates glucose homeostasis during pregnancy by promoting proliferation1 and activity2 of insulin-producing β-cells. Dysregulation of these mechanisms can lead to maternal hyperglycemia and gestational diabetes mellitus (GDM). GDM is more common in women carrying a male fetus, 3 but the mechanisms by which fetal sex affects glucose homeostasis are not clear. Here we investigated the relationship between methylation of the gene encoding the 5-HT transporter (SLC6A4), an important regulator of extracellular 5-HT levels, and glucose levels in maternal and cord blood, considering the possible influence of fetal sex. The study included 129 mother-infant dyads from the ongoing Placental and Neonatal Serotonin (PlaNS) cohort study in Zagreb, Croatia. Maternal glucose levels in the 2nd trimester of pregnancy were obtained from medical records, fetal glucose levels were measured in cord blood. SLC6A4 gene methylation was quantified in maternal and cord blood cells by bisulfite pyrosequencing. In pregnancies with a female fetus (n= 50), maternal glucose levels correlated negatively with maternal SLC6A4 methylation (r=-0.43, p< 0.01) and positively with cord blood glucose (r= 0.34, p< 0.01) levels, while cord blood SLC6A4 methylation did not correlate with maternal or cord blood glucose levels. In pregnancies with a male fetus (n= 79), maternal glucose levels did not correlate with either maternal or cord blood SLC6A4 methylation or with cord blood glucose levels. However, a negative correlation was observed between cord blood glucose and SLC6A4 methylation levels (r=-0.40, p< 0.01). The results show a fetal …

Serotonin receptor type 2A (HTR2A), a widely distributed Gproteincoupled receptor for multifunctional signaling molecule serotonin, has been implicated in many physiological processes and its dysregulation has been associated with a number of mental health and metabolic conditions. Different lines of evidence indicate that epigenetic modifications of the HTR2A gene play a role in molecular mechanisms through which early life environment influences development and lifelong health outcomes. The promoter region of the HTR2A gene contains a number of partially methylated cytosines that have been shown to modulate the gene's transcriptional activity. Here we investigated placental and cord blood HTR2A methylation levels in relation to fetal sex and genotype as well as maternal obesity and gestational diabetes mellitus. The study was performed on motherinfant dyads enrolled at the Clinical Hospital Center Zagreb as a part of our ongoing birth cohort study PlaNS (Placental and Neonatal Serotonin). All newborns were healthy, of normal birth weight and born at term by planned Csection. Cord blood samples were obtained via umbilical venipuncture and placental tissue samples were isolated from the fetal part of the placenta. DNA methylation levels were quantified at four CpG loci within the HTR2A promoter region using bisulfite pyrosequencing. In addition, samples were genotyped for two polymorphisms (rs6311, rs6306) in the respective gene region. The four targeted CpG cytosines were methylated to different degree in the cord blood and placental tissue. rs6311 and rs6306 genotypes as well as fetal sex were found to be significant …

Alternative glycosylation of immunoglobulin G (IgG) affects its effector functions during the immune response. IgG glycosylation is altered in many diseases, but also during a healthy life of an individual. Currently, there is limited knowledge of factors that alter IgG glycosylation in the healthy state and factors involved in specific IgG glycosylation patterns associated with pathophysiology. Genetic background plays an important role, but epigenetic mechanisms also contribute to the alteration of IgG glycosylation patterns in healthy life and in disease. It is known that the expression of many glycosyltransferases is regulated by DNA methylation and by microRNA (miRNA) molecules, but the involvement of other epigenetic mechanisms, such as histone modifications, in the regulation of glycosylation-related genes (glycogenes) is still poorly understood. Recent studies have identified several differentially …

Tumorske stanice nastaju nakupljanjem promjena u genomu normalnih stanica pa stoga zadržavaju dio karakteristika onih stanica iz kojih su i nastale. Iako više nisu pod kontrolom organizma, sustava ili tkiva u kojem se tumor razvija, tumorske stanice zadržavaju mogućnost komunikacije sa stanicama iz svog mikrookoliša i ta komunikacija može utjecati na promjene u njihovim osnovnim procesima poput replikacije i ponovne uspostave kromatina. Cilj ovog istraživanja bio je analizirati ekspresiju gena MCM2, PCNA i EZH2 čiji produkti su ključni za regulaciju replikacije i ponovne uspostave kromatina i procijeniti prisutnost proteina koje ti geni kodiraju u normalnim i tumorskim B-stanicama. Korišteni su uzorci germinativnog centra tonzila (normalne Bstanice), folikularnog limfoma (indolentnog tumora porijeklom B-stanica) i dvije najčešće skupine difuznog B-velikostaničnog limfoma (agresivnog tumora B-stanica). Metode su uključivale lančanu reakciju polimerazom u stvarnome vremenu i imunohistokemijsko bojenje. Rezultati su pokazali da dolazi do značajnog smanjenja ekspresije proteina MCM2, PCNA i EZH2 u folikularnom limfomu u odnosu na normalne B-stanice, ali i do značajnog porasta njihove ekspresije u difuznom B-velikostaničnom limfomu u odnosu na folikularni limfom. Ovakvi rezultati sugeriraju da su za stupanj agresivnosti i razinu progresije limfoma značajne promjene osnovnih staničnih procesa, no da one ne pokazuju nužno i linearnost u odnosu na porast stupnja agresije tumora.