Presentation number: MG 3
EFFECTS OF SIMULTANEOUS MANIPULATION OF DNA METHYLATION AND HISTONE MODIFICATIONS ON EXPRESSION OF GENES INVOLVED IN EPITHELIAL-MESENCHYMAL TRANSITION USING CRISPR/dCas9 TOOLS
Nika Foglar1, Goran Josipović2, Vlatka Zoldoš1
1Faculty of Science, University of Zagreb, Department of Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia
CRISPR/dCas9 molecular tools have enabled targeted manipulation of the epigenome and therefore, investigation of the direct effect of epigenetic modifications on gene expression. However, the interplay of different epigenetic modifications in the regulation of transcriptional gene activity is still poorly understood. Regarding the vast number of different chromatin modifications, it is not clear how specific modifications play together in the complex process of gene regulation. Herein, we studied the potentially synergistic effect of DNA methylation and different histone modifications on the transcriptional activity of two candidate genes, ZEB1 and SNAI1, coding for the key transcriptional factors regulating the epithelial to mesenchymal transition (EMT). For this purpose, we used the CRISPR/dCas9 system to manipulate DNA methylation, using DNMT3A-dSpCas9 fusion, and different histone modifications using dCas9 fused effector domains: G9a-SET, LSD1-SET domains, and the catalytic domain of KDM5a. In HepG2 cell line, the CpG islands of ZEB1 and SNAI1 were targeted with DNMT3A-dSpCas9 using multiple gRNAs, simultaneously covering the entire islands. The combinatorial use of DNMT3A-dSpCas9 with G9a-dSpCas9 was monitored for 70 days, and a synergistic effect on the expression of both genes was observed, which remained for a prolonged period on SNAI1. None of the effector domains for manipulation of histone modifications could itself increase DNA methylation. However, the G9a-SET domain combined with DNMT3A-dSpCas9 showed a stronger effect on DNA methylation compared to control where the inactive G9a-SET domain was used. A change in the expression of downstream EMT markers E-CAD and CRB3 following epigenetic manipulation was also observed. When DNMT3A-dSpCas9 was combined with LSD1-dSpCas9, no clear effect was observed on either of the two genes, while for the combination with KDM5a-dSpCas9, the effect was short-term. Changes in several epigenetic modifications simultaneously can lead to a synergistic effect on gene expression, depending on the gene locus. Ongoing research will unravel if introducing DNMT3L into cells can bridge DNA methylation and histone modifications.
Key words: CRISPR/dCas9 technology, DNA methylation, histone modifications, epithelial to mesenchymal transition
Presentation number: MG 4
CRISPR/dCas9 MOLECULAR TOOLS REVEAL THE REGULATION OF FUT8, MGAT4A, MGAT4B, MGAT5, MGAT3, AND B4GALT1 GENES BY CpG METHYLATION
Ana Vujić1, Marija Klasić1, Vedrana Vičić Bočkor1, Vlatka Zoldoš1, Maja Pučić Baković2, Gordan Lauc2,3
1University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Genos Ltd., Zagreb, Croatia, 3University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Biochemistry and Molecular Biology, Zagreb, Croatia
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 glycome were observed. These results suggest that alterations in CpG methylation lead to differential expression of glycosyltransferases, thus leading to aberrant protein glycosylation in HCC.
Key words: epigenome editing, gene regulation, CRISPR/dCas9, DNA methylation, protein glycosylation
Presentation number: MG 5
MANIPULATION OF HNF1A, HNF4A AND FOXA2 USING CRISPR-BASED MOLECULAR TOOLS SUGGEST THEIR ROLE IN REGULATION OF PROTEIN GLYCOSYLATION IN LIVER AND PANCREATIC CELL MODELS
Vedrana Vičić Bočkor1, Marija Klasić1, Goran Josipović2, Ana Vujić1, Nika Foglar1, Samira Smajlović1, Toma Keser3, Gordan Lauc2, Aleksandar Vojta1, Vlatka Zoldoš1
1University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia, 3University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
Transcription factors HNF1A, HNF4A and FOXA1/2/3 regulate developmental and tissue-specific transcriptional gene networks in liver and pancreas. These genes are involved in many metabolic processes as well as in acute inflammation by regulating proteins such as fibrinogen, C-reactive protein and receptor interleukin 1. 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 change 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. In order to investigate possible effects of HNF1A, HNF4A and FOXA2 expression on downstream glycogenes we used CRISPR/dCas9-based tools for manipulation of their transcriptional activity in human model cell lines for liver (HepG2) and pancreas (1.1B4). Following CRISPR-based manipulations, we analysed total protein glycosylation. Promoters of HNF1A, HNF4A and FOXA2 were targeted with KRAB-dCas9 (for silencing) and/or VPR-dCas9 (for activation) using specific sgRNAs. Silencing of HNF1A, HNF4A and FOXA2 in HepG2 cells resulted in downregulation of ST6GAL1 and FUT6 and upregulation of B4GALT1, FUK, FUT3, FUT5, FUT8 and MGAT5A, with concomitant change in glycosylation. Increase of antennary fucosylated, core fucosylated, agalactosylated and asialylated glycans and a decrease in galactosylated and sialylated structures were observed. In 1.1B4 cell line, silencing of HNF1A, HNF4A and FOXA2 led to overexpression of FUT5 and FUT6 and reduced expression of FUK and FUT8 but these changes were not reflected in the glycan profile. The HNF1A and HNF4A activation in 1.1B4 cells resulted in overexpression of FUT3, FUT5 and FUT6 and reduced expression of MGAT4A. This change led to an increase in agalactosylated, asialylated and oligomanose glycans and a decrease of sialylated, galactosylated and core fucosylated glycans. Our results indicate that HNF1A, HNF4A and FOXA2 regulate, at least partly, protein glycosylation in the pancreatic and liver cell models.
Key words: epigenetics, CRISPR/dCas9, glycogenes, protein glycosylation
Presentation number: MG 6
VIRTUAL EPIGENETICS IN FORENSIC: AN EXAMPLE OF METOPIC SUTURE PREVALENCE IN MODERN CROATIAN POPULATION
Tina Bareša1, Željana Bašić1, Ivan Jerković1, Krešimir Dolić2, Marija Ćavar Borić2, Danijela Budimir Mršić2, Mislav Čavka3, Elvira Krešić3, Ivana Kružić1
1University of Split, University Department of Forensic Sciences, Split, Croatia, 2University Hospital Center Split, Department of Interventional and Diagnostic Radiology, Split, Croatia, 3University Hospital Center Zagreb, Department of Interventional and Diagnostic Radiology, Zagreb, Croatia
Aim was to determine the prevalence of metopic suture in the modern Croatian population. A total of 458 MSCT images were analyzed (250 from the University hospital Split and 208 from the University hospital Zagreb) using OsiriX 12.0 imaging software. The sample consisted of 240 females (median age 67; range 18-93) and 218 males (median age 68; range 20-91). The metopic suture was scored in 3D volume rendering view as absent or present. The persisting metopic suture was scored complete if the suture was aligned nasion to bregma. The incomplete metopic suture was scored as only nasal, only parietal, or both nasal and parietal but not connected. All the analyzed metopic sutures were scored as complete. Of the total population analyzed, persistent metopic suture had 3.1%, 6 of 240 females (2.5%), and 8 of 218 males (3.7%). There is no statistical significance (χ2 = 0.53, p=0.47) between sexes. Frequencies of metopic suture vary across populations, the highest prevalence was reported in the modern Indian population (16%), modern Dutch (11.5%), Italian (10.7%), and the lowest among modern American blacks (2.2%). The relatively small frequency in the Croatian population shows that metopic suture could be a useful forensic tool application in human individualization and identification, and it can be useful in ancestry estimation as well as in positive identification and clinical environment. This study was founded by Croatian Science Foundation UIP-2020-02-7331 “CT for ID”.
Key words: MSCT imaging, virtual databases, metopic suture, population affiliation, Croatia
Presentation number: MG 7
USE OF CRISPR/DCAS9-BASED MODULAR SYSTEM DEMONSTRATES ANTAGONISTIC AND SYNERGISTIC EFFECTS OF EPIGENETIC MANIPULATIONS ON GENE EXPRESSION
Goran Josipović1,2, Vanja Tadić1, Marija Klasić1, Vladimir Zanki3, Ivona Bečeheli1, Felicia Chung4, Akram Ghantous4, Toma Keser5, Josip Madunić1, Maria Bošković1, Gordan Lauc2,5, Zdenko Herceg4, Aleksandar Vojta1, Vlatka Zoldoš1
1Faculty of Science, University of Zagreb, Department of Biology, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia, 3Faculty of Science, University of Zagreb, Department of Chemistry, Zagreb, Croatia, 4International Agency for Research on Cancer (IARC), Lyon, France, 5Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
The development of the CRISPR/Cas9 system has enabled a shift from its primary role as a genome-editing tool to its application in targeted alteration of the eukaryotic epigenome. By linking various epigenetic effector domains to the catalytically inactive nuclease Cas9 (dCas9), the active fusions are obtained that can be targeted to a genome site of interest by a specific guide RNA (gRNA) molecule. The effect of epigenetic change on gene expression enables understanding the significance of the specific epigenetic marks in complex gene expression regulation. To further enhance CRISPR/dCas9 system for epigenome editing we have developed a fully modular and upgradeable system where various domains can easily be fused to dCas9: DNMT3A-dCas9 fusion for addition and TET1-dCas9 fusion for removing methyl group to/from CpG dinucleotides, as well as dCas9-VPR and dCas9-KRAB fusions for direct activation and silencing of gene transcriptional activity. We also enabled the use of two Cas9 orthologs from the species Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) for fusion with different effector domains which allowed both, antagonistic and synergistic manipulations at different loci. By further expanding the modular system to increase the number of gRNA molecules, to a maximum of six different ones, we have enabled to target dCas9 fusions to a larger genome region. We were able to confirm that our modular system works efficiently by simultaneous targeting the candidate gene pairs BACH2 – HNF1A and IL6ST – MGAT3 in HEK293 cells with DNMT3A-dSpCas9 and TET1-dSaCas9. Induced methylation and demethylation of the individual genes within the pairs were accompanied by a change in the level of gene transcription. In addition, we were able to demonstrate that changes in methylation and gene expression levels of gene pair HNF1A – MGAT3 have an effect on the glycan phenotype in BG1 cells. Also, by simultaneous targeting the HNF1A locus using TET1-dSaCas9 and VPR-dSpCas9 fusions, we showed a synergistic effect on gene expression which was maintained up to 30 days after transient cell transfection. Furthermore, by upgrading our system, we reduced the off-target effect of dCas9 fusions.
Key words: CRISPR/dCas9 system, Cas9 orthologs, epigenome editing, direct regulation of gene expression, CRISPR/dCas9 off-target effect