TY  - CONF
AU  - Đorđević, Marija
AU  - Feuerstein-Akgoz, Clarissa
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Rajić, Jovana
AU  - Sarić, Ana
AU  - Grdović, Nevena
AU  - Dinić, Svetlana
AU  - Uskoković, Aleksandra
AU  - Mihailović, Mirjana
AU  - Gerhauser, Clarissa
AU  - Jurkowski, Tomasz
AU  - Vidaković, Melita
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6307
AB  - Introduction: The Aristaless-related homeobox (Arx) gene plays a key role in the development and maintaining pancreatic alpha cell phenotype, and as such represents an excellent target for alpha cell identity change towards insulin-producing cells. Therefore, this cell switch and increase in beta cell mass could be of potential use in diabetes management.
Methods: On the fifth day after transient transfection with dCas9-Dnmt3a3L-KRAB construct and four gRNAs targeting Arx promoter, αTC1-6 were sorted to collect GFP-positive (transfected) cells (EpiC). The mRNA-seq libraries were pooled in equimolar amounts and sequenced in a single end-setting on the Illumina NextSeq 500 High output machine with 75 bases long reads. KEGG pathway overrepresentation analysis was performed using an application on all significantly up- or downregulated genes using default settings.
Results: Directed induction of DNA methylation on the Arx gene promoter reduces its expression and causes the up-regulation of 357 genes, while 266 genes were down-regulated in EpiC compared to Mock transfected cells. The KEGG pathways analysis of biological processes confirmed several biological pathways associated with genes differentially expressed in EpiC vs. Mock transfected cells at the 5th posttransfection day (pval ≤ 0.05). As the most significant, up-regulated pathways we found Type II diabetes, Insulin secretion, Longevity regulation pathways. As significant, down-regulated pathways pop-up Fatty acid metabolism and PPAR signaling pathway.
Conclusion: Reduction of ArxmRNA level is sufficient to initiate the transdifferentiation process of alpha cells into insulin-producing cells by triggering several biological pathways tight related to insulin secretion and function.
PB  - Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade
C3  - Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia
T1  - Changes in up and down regulated gene expression after transient suppression of Arx gene in pancreatic alphaTC1-6 cell line
SP  - 151
EP  - 151
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6307
ER  - 

@conference{
author = "Đorđević, Marija and Feuerstein-Akgoz, Clarissa and Arambašić Jovanović, Jelena and Tolić, Anja and Rajić, Jovana and Sarić, Ana and Grdović, Nevena and Dinić, Svetlana and Uskoković, Aleksandra and Mihailović, Mirjana and Gerhauser, Clarissa and Jurkowski, Tomasz and Vidaković, Melita",
year = "2023",
abstract = "Introduction: The Aristaless-related homeobox (Arx) gene plays a key role in the development and maintaining pancreatic alpha cell phenotype, and as such represents an excellent target for alpha cell identity change towards insulin-producing cells. Therefore, this cell switch and increase in beta cell mass could be of potential use in diabetes management.
Methods: On the fifth day after transient transfection with dCas9-Dnmt3a3L-KRAB construct and four gRNAs targeting Arx promoter, αTC1-6 were sorted to collect GFP-positive (transfected) cells (EpiC). The mRNA-seq libraries were pooled in equimolar amounts and sequenced in a single end-setting on the Illumina NextSeq 500 High output machine with 75 bases long reads. KEGG pathway overrepresentation analysis was performed using an application on all significantly up- or downregulated genes using default settings.
Results: Directed induction of DNA methylation on the Arx gene promoter reduces its expression and causes the up-regulation of 357 genes, while 266 genes were down-regulated in EpiC compared to Mock transfected cells. The KEGG pathways analysis of biological processes confirmed several biological pathways associated with genes differentially expressed in EpiC vs. Mock transfected cells at the 5th posttransfection day (pval ≤ 0.05). As the most significant, up-regulated pathways we found Type II diabetes, Insulin secretion, Longevity regulation pathways. As significant, down-regulated pathways pop-up Fatty acid metabolism and PPAR signaling pathway.
Conclusion: Reduction of ArxmRNA level is sufficient to initiate the transdifferentiation process of alpha cells into insulin-producing cells by triggering several biological pathways tight related to insulin secretion and function.",
publisher = "Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade",
journal = "Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia",
title = "Changes in up and down regulated gene expression after transient suppression of Arx gene in pancreatic alphaTC1-6 cell line",
pages = "151-151",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6307"
}

Đorđević, M., Feuerstein-Akgoz, C., Arambašić Jovanović, J., Tolić, A., Rajić, J., Sarić, A., Grdović, N., Dinić, S., Uskoković, A., Mihailović, M., Gerhauser, C., Jurkowski, T.,& Vidaković, M.. (2023). Changes in up and down regulated gene expression after transient suppression of Arx gene in pancreatic alphaTC1-6 cell line. in Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia
Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade., 151-151.
https://hdl.handle.net/21.15107/rcub_ibiss_6307

Đorđević M, Feuerstein-Akgoz C, Arambašić Jovanović J, Tolić A, Rajić J, Sarić A, Grdović N, Dinić S, Uskoković A, Mihailović M, Gerhauser C, Jurkowski T, Vidaković M. Changes in up and down regulated gene expression after transient suppression of Arx gene in pancreatic alphaTC1-6 cell line. in Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia. 2023;:151-151.
https://hdl.handle.net/21.15107/rcub_ibiss_6307 .

Đorđević, Marija, Feuerstein-Akgoz, Clarissa, Arambašić Jovanović, Jelena, Tolić, Anja, Rajić, Jovana, Sarić, Ana, Grdović, Nevena, Dinić, Svetlana, Uskoković, Aleksandra, Mihailović, Mirjana, Gerhauser, Clarissa, Jurkowski, Tomasz, Vidaković, Melita, "Changes in up and down regulated gene expression after transient suppression of Arx gene in pancreatic alphaTC1-6 cell line" in Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia (2023):151-151,
https://hdl.handle.net/21.15107/rcub_ibiss_6307 .

TY  - DATA
AU  - Đorđević, Marija
AU  - Stepper, Peter
AU  - Feuerstein-Akgoz, Clarissa
AU  - Gerhauser, Clarissa
AU  - Paunović, Verica
AU  - Tolić, Anja
AU  - Rajić, Jovana
AU  - Dinić, Svetlana
AU  - Uskoković, Aleksandra
AU  - Grdović, Nevena
AU  - Mihailović, Mirjana
AU  - Jurkowska, Renata
AU  - Jurkowski, Tomasz
AU  - Arambašić Jovanović, Jelena
AU  - Vidaković, Melita
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5669
AB  - Figure 1. UCSC genome browser views of murine Arx gene’s DNA methylation sites in the pancreas and other tissues. If compared with other tissues, Arx gene in pancreatic tissue (young and old) exhibits low DNA methylation. CpG island was shown as a green box. Table 1. Targeted sequences for sgRNAs. Table 2. Primers used for RT-qPCR. Table 3. Primers used for HRM. Table 4. Touchdown PCR program for amplification of bisulfite converted DNA, starting at 55 °C. Table 5. Primers for NGS library preparation. Table 6. Antibodies used for Immunoblot analysis (IBA) and Immunocytochemistry (ICC). Table 7. Primers for PCR reaction after ChIP.
PB  - Frontiers Media S.A.
T2  - Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms
T1  - Supplementary Material for Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska RZ, Jurkowski TP, Jovanović JA, Vidaković M. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. Front Endocrinol (Lausanne). 2023;14:1134478.
VL  - 14
SP  - 1134478
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5669
ER  - 

@misc{
author = "Đorđević, Marija and Stepper, Peter and Feuerstein-Akgoz, Clarissa and Gerhauser, Clarissa and Paunović, Verica and Tolić, Anja and Rajić, Jovana and Dinić, Svetlana and Uskoković, Aleksandra and Grdović, Nevena and Mihailović, Mirjana and Jurkowska, Renata and Jurkowski, Tomasz and Arambašić Jovanović, Jelena and Vidaković, Melita",
year = "2023",
abstract = "Figure 1. UCSC genome browser views of murine Arx gene’s DNA methylation sites in the pancreas and other tissues. If compared with other tissues, Arx gene in pancreatic tissue (young and old) exhibits low DNA methylation. CpG island was shown as a green box. Table 1. Targeted sequences for sgRNAs. Table 2. Primers used for RT-qPCR. Table 3. Primers used for HRM. Table 4. Touchdown PCR program for amplification of bisulfite converted DNA, starting at 55 °C. Table 5. Primers for NGS library preparation. Table 6. Antibodies used for Immunoblot analysis (IBA) and Immunocytochemistry (ICC). Table 7. Primers for PCR reaction after ChIP.",
publisher = "Frontiers Media S.A.",
journal = "Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms",
title = "Supplementary Material for Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska RZ, Jurkowski TP, Jovanović JA, Vidaković M. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. Front Endocrinol (Lausanne). 2023;14:1134478.",
volume = "14",
pages = "1134478",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5669"
}

Đorđević, M., Stepper, P., Feuerstein-Akgoz, C., Gerhauser, C., Paunović, V., Tolić, A., Rajić, J., Dinić, S., Uskoković, A., Grdović, N., Mihailović, M., Jurkowska, R., Jurkowski, T., Arambašić Jovanović, J.,& Vidaković, M.. (2023). Supplementary Material for Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska RZ, Jurkowski TP, Jovanović JA, Vidaković M. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. Front Endocrinol (Lausanne). 2023;14:1134478.. in Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms
Frontiers Media S.A.., 14, 1134478.
https://hdl.handle.net/21.15107/rcub_ibiss_5669

Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska R, Jurkowski T, Arambašić Jovanović J, Vidaković M. Supplementary Material for Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska RZ, Jurkowski TP, Jovanović JA, Vidaković M. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. Front Endocrinol (Lausanne). 2023;14:1134478.. in Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms. 2023;14:1134478.
https://hdl.handle.net/21.15107/rcub_ibiss_5669 .

Đorđević, Marija, Stepper, Peter, Feuerstein-Akgoz, Clarissa, Gerhauser, Clarissa, Paunović, Verica, Tolić, Anja, Rajić, Jovana, Dinić, Svetlana, Uskoković, Aleksandra, Grdović, Nevena, Mihailović, Mirjana, Jurkowska, Renata, Jurkowski, Tomasz, Arambašić Jovanović, Jelena, Vidaković, Melita, "Supplementary Material for Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska RZ, Jurkowski TP, Jovanović JA, Vidaković M. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. Front Endocrinol (Lausanne). 2023;14:1134478." in Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms, 14 (2023):1134478,
https://hdl.handle.net/21.15107/rcub_ibiss_5669 .

TY  - JOUR
AU  - Đorđević, Marija
AU  - Stepper, Peter
AU  - Feuerstein-Akgoz, Clarissa
AU  - Gerhauser, Clarissa
AU  - Paunović, Verica
AU  - Tolić, Anja
AU  - Rajić, Jovana
AU  - Dinić, Svetlana
AU  - Uskoković, Aleksandra
AU  - Grdović, Nevena
AU  - Mihailović, Mirjana
AU  - Jurkowska, Renata
AU  - Jurkowski, Tomasz
AU  - Arambašić Jovanović, Jelena
AU  - Vidaković, Melita
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5507
AB  - 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.
PB  - Frontiers Media S.A.
T2  - Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms
T1  - EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells
VL  - 14
DO  - 10.3389/fendo.2023.1134478
SP  - 1134478
ER  - 

@article{
author = "Đorđević, Marija and Stepper, Peter and Feuerstein-Akgoz, Clarissa and Gerhauser, Clarissa and Paunović, Verica and Tolić, Anja and Rajić, Jovana and Dinić, Svetlana and Uskoković, Aleksandra and Grdović, Nevena and Mihailović, Mirjana and Jurkowska, Renata and Jurkowski, Tomasz and Arambašić Jovanović, Jelena and Vidaković, Melita",
year = "2023",
abstract = "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.",
publisher = "Frontiers Media S.A.",
journal = "Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms",
title = "EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells",
volume = "14",
doi = "10.3389/fendo.2023.1134478",
pages = "1134478"
}

Đorđević, M., Stepper, P., Feuerstein-Akgoz, C., Gerhauser, C., Paunović, V., Tolić, A., Rajić, J., Dinić, S., Uskoković, A., Grdović, N., Mihailović, M., Jurkowska, R., Jurkowski, T., Arambašić Jovanović, J.,& Vidaković, M.. (2023). EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. in Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms
Frontiers Media S.A.., 14, 1134478.
https://doi.org/10.3389/fendo.2023.1134478

Đorđević M, Stepper P, Feuerstein-Akgoz C, Gerhauser C, Paunović V, Tolić A, Rajić J, Dinić S, Uskoković A, Grdović N, Mihailović M, Jurkowska R, Jurkowski T, Arambašić Jovanović J, Vidaković M. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. in Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms. 2023;14:1134478.
doi:10.3389/fendo.2023.1134478 .

Đorđević, Marija, Stepper, Peter, Feuerstein-Akgoz, Clarissa, Gerhauser, Clarissa, Paunović, Verica, Tolić, Anja, Rajić, Jovana, Dinić, Svetlana, Uskoković, Aleksandra, Grdović, Nevena, Mihailović, Mirjana, Jurkowska, Renata, Jurkowski, Tomasz, Arambašić Jovanović, Jelena, Vidaković, Melita, "EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells" in Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms, 14 (2023):1134478,
https://doi.org/10.3389/fendo.2023.1134478 . .

TY  - CONF
AU  - Tolić, Anja
AU  - Rajić, Jovana
AU  - Đorđević, Miloš
AU  - Đorđević, Marija
AU  - Uskoković, Aleksandra
AU  - Grdović, Nevena
AU  - Mihailović, Mirjana
AU  - Arambašić Jovanović, Jelena
AU  - Dinić, Svetlana
AU  - Nestorović, Nataša
AU  - Jurkowski, Tomasz
AU  - Vidaković, Melita
PY  - 2020
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4783
AB  - Since our previous work indicated interplay of Ten-eleven translocation (TET) and Poly (ADP-ribose) polymerase (PARP) proteins our aim was to further study the effects of their interaction. 
The ability of recombinant PARP-1 to poly ADP-ribosylate (PARylate) catalytic domains of TET proteins was examined in vitro. It was observed that all TETs (TET1, TET2, TET3) readily undergo PARylation. To our knowledge, this is the first report of PARP-1 PARylation of TET2 and TET3 while TET1 PARylation has been previously documented. PARylation of TET proteins was evidenced by western blot signal stretching from the position of unmodified TET. This type of signal is characteristic for PARylation since proteins are modified to varying degrees by covalently attached negatively charged PAR polymers. This slows their movement during electrophoresis and individual molecules are expected to stop at different positions resulting in signal stretching upwards.  PARylation can introduce electrostatic and topological changes in modified proteins, resulting in altered enzymatic activity. Therefore we evaluated TET1 activity in vitro using an ELISA type in-house assay, which showed that PARP-1-dependent PARylation lowers the ability of TET1 catalytic domain to oxidize 5mC to 5hmC. 
To corroborate these results in cellulo, we examined changes in DNA methylation in mouse embryonic fibroblasts (NIH3T3) compared to a PARP-1 knock out mouse embryonic fibroblast cell line (P ARP-/-). Lower methylation was observed in PARP-/- cells by immunocytochemical staining. Next, we analyzed global DNA methylation by ELISA assay and we again detected lower methylation levels in PARP-/- and also in NIH3T3 cells treated by a PARP inhibitor niraparib. Finally, DNA hydroxymethylation was assessed by immunocytochemistry and stronger signal was observed in PARP-/- cells and NIH3T3 cells treated by niraparib, compared to control NIH3T3 cells. 
In summary, inhibition of PARylation or absence of PARP-1 lead to decreased methylation and increased hydroxymethylation of DNA in cellulo. Together, our results point to the inhibitory influence that PARP-1 and PARylation exert on TET1 activity. Further studies are needed to evaluate the effects of PARylation of other TET proteins as well as to examine potential influence of other PARPs.
PB  - Cold Spring Harbor Laboratory
C3  - 2020 virtual meeting on EPIGENETICS & CHROMATIN; 2020 Sep 15-18; Virtual meeting
T1  - PARP-1 and PARylation inhibit TET1 demethylation activity
SP  - 286
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4783
ER  - 

@conference{
author = "Tolić, Anja and Rajić, Jovana and Đorđević, Miloš and Đorđević, Marija and Uskoković, Aleksandra and Grdović, Nevena and Mihailović, Mirjana and Arambašić Jovanović, Jelena and Dinić, Svetlana and Nestorović, Nataša and Jurkowski, Tomasz and Vidaković, Melita",
year = "2020",
abstract = "Since our previous work indicated interplay of Ten-eleven translocation (TET) and Poly (ADP-ribose) polymerase (PARP) proteins our aim was to further study the effects of their interaction. 
The ability of recombinant PARP-1 to poly ADP-ribosylate (PARylate) catalytic domains of TET proteins was examined in vitro. It was observed that all TETs (TET1, TET2, TET3) readily undergo PARylation. To our knowledge, this is the first report of PARP-1 PARylation of TET2 and TET3 while TET1 PARylation has been previously documented. PARylation of TET proteins was evidenced by western blot signal stretching from the position of unmodified TET. This type of signal is characteristic for PARylation since proteins are modified to varying degrees by covalently attached negatively charged PAR polymers. This slows their movement during electrophoresis and individual molecules are expected to stop at different positions resulting in signal stretching upwards.  PARylation can introduce electrostatic and topological changes in modified proteins, resulting in altered enzymatic activity. Therefore we evaluated TET1 activity in vitro using an ELISA type in-house assay, which showed that PARP-1-dependent PARylation lowers the ability of TET1 catalytic domain to oxidize 5mC to 5hmC. 
To corroborate these results in cellulo, we examined changes in DNA methylation in mouse embryonic fibroblasts (NIH3T3) compared to a PARP-1 knock out mouse embryonic fibroblast cell line (P ARP-/-). Lower methylation was observed in PARP-/- cells by immunocytochemical staining. Next, we analyzed global DNA methylation by ELISA assay and we again detected lower methylation levels in PARP-/- and also in NIH3T3 cells treated by a PARP inhibitor niraparib. Finally, DNA hydroxymethylation was assessed by immunocytochemistry and stronger signal was observed in PARP-/- cells and NIH3T3 cells treated by niraparib, compared to control NIH3T3 cells. 
In summary, inhibition of PARylation or absence of PARP-1 lead to decreased methylation and increased hydroxymethylation of DNA in cellulo. Together, our results point to the inhibitory influence that PARP-1 and PARylation exert on TET1 activity. Further studies are needed to evaluate the effects of PARylation of other TET proteins as well as to examine potential influence of other PARPs.",
publisher = "Cold Spring Harbor Laboratory",
journal = "2020 virtual meeting on EPIGENETICS & CHROMATIN; 2020 Sep 15-18; Virtual meeting",
title = "PARP-1 and PARylation inhibit TET1 demethylation activity",
pages = "286",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4783"
}

Tolić, A., Rajić, J., Đorđević, M., Đorđević, M., Uskoković, A., Grdović, N., Mihailović, M., Arambašić Jovanović, J., Dinić, S., Nestorović, N., Jurkowski, T.,& Vidaković, M.. (2020). PARP-1 and PARylation inhibit TET1 demethylation activity. in 2020 virtual meeting on EPIGENETICS & CHROMATIN; 2020 Sep 15-18; Virtual meeting
Cold Spring Harbor Laboratory., 286.
https://hdl.handle.net/21.15107/rcub_ibiss_4783

Tolić A, Rajić J, Đorđević M, Đorđević M, Uskoković A, Grdović N, Mihailović M, Arambašić Jovanović J, Dinić S, Nestorović N, Jurkowski T, Vidaković M. PARP-1 and PARylation inhibit TET1 demethylation activity. in 2020 virtual meeting on EPIGENETICS & CHROMATIN; 2020 Sep 15-18; Virtual meeting. 2020;:286.
https://hdl.handle.net/21.15107/rcub_ibiss_4783 .

Tolić, Anja, Rajić, Jovana, Đorđević, Miloš, Đorđević, Marija, Uskoković, Aleksandra, Grdović, Nevena, Mihailović, Mirjana, Arambašić Jovanović, Jelena, Dinić, Svetlana, Nestorović, Nataša, Jurkowski, Tomasz, Vidaković, Melita, "PARP-1 and PARylation inhibit TET1 demethylation activity" in 2020 virtual meeting on EPIGENETICS & CHROMATIN; 2020 Sep 15-18; Virtual meeting (2020):286,
https://hdl.handle.net/21.15107/rcub_ibiss_4783 .

TY  - JOUR
AU  - Tolić, Anja
AU  - Rajić, Jovana
AU  - Đorđević, Marija
AU  - Đorđević, Miloš
AU  - Dinić, Svetlana
AU  - Grdović, Nevena
AU  - Arambašić Jovanović, Jelena
AU  - Mihailović, Mirjana
AU  - Poznanović, Goran
AU  - Jurkowski, Tomasz P.
AU  - Vidaković, Melita
AU  - Uskoković, Aleksandra
PY  - 2019
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3460
AB  - Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction
and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now
firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene
upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation
state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation
(TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation
process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3)
and PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary.
T2  - Archives of Biological Sciences
T1  - Enrichment of Cxcl12 promoter with TET2: a possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1
IS  - 3
VL  - 71
DO  - 10.2298/ABS190404027T
SP  - 455
EP  - 462
ER  - 

@article{
author = "Tolić, Anja and Rajić, Jovana and Đorđević, Marija and Đorđević, Miloš and Dinić, Svetlana and Grdović, Nevena and Arambašić Jovanović, Jelena and Mihailović, Mirjana and Poznanović, Goran and Jurkowski, Tomasz P. and Vidaković, Melita and Uskoković, Aleksandra",
year = "2019",
abstract = "Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction
and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now
firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene
upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation
state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation
(TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation
process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3)
and PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary.",
journal = "Archives of Biological Sciences",
title = "Enrichment of Cxcl12 promoter with TET2: a possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1",
number = "3",
volume = "71",
doi = "10.2298/ABS190404027T",
pages = "455-462"
}

Tolić, A., Rajić, J., Đorđević, M., Đorđević, M., Dinić, S., Grdović, N., Arambašić Jovanović, J., Mihailović, M., Poznanović, G., Jurkowski, T. P., Vidaković, M.,& Uskoković, A.. (2019). Enrichment of Cxcl12 promoter with TET2: a possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1. in Archives of Biological Sciences, 71(3), 455-462.
https://doi.org/10.2298/ABS190404027T

Tolić A, Rajić J, Đorđević M, Đorđević M, Dinić S, Grdović N, Arambašić Jovanović J, Mihailović M, Poznanović G, Jurkowski TP, Vidaković M, Uskoković A. Enrichment of Cxcl12 promoter with TET2: a possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1. in Archives of Biological Sciences. 2019;71(3):455-462.
doi:10.2298/ABS190404027T .

Tolić, Anja, Rajić, Jovana, Đorđević, Marija, Đorđević, Miloš, Dinić, Svetlana, Grdović, Nevena, Arambašić Jovanović, Jelena, Mihailović, Mirjana, Poznanović, Goran, Jurkowski, Tomasz P., Vidaković, Melita, Uskoković, Aleksandra, "Enrichment of Cxcl12 promoter with TET2: a possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1" in Archives of Biological Sciences, 71, no. 3 (2019):455-462,
https://doi.org/10.2298/ABS190404027T . .

TY  - CHAP
AU  - Vidaković, Melita
AU  - Tolić, Anja
AU  - Grdović, Nevena
AU  - Ravichandran, Mirunalini
AU  - Jurkowski, Tomasz P.
PY  - 2019
UR  - https://link.springer.com/referenceworkentry/10.1007%2F978-3-319-55530-0_55
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3616
AB  - Diabetes and diabetic complications, autoimmunity and inflammatory diseases, have recently become the focus of epigenetic therapy, since with epigenetic drugs it is possible to reverse aberrant gene expression profiles associated with the disease states. For diabetes, the therapy challenges depend on identifying the most appropriate molecular target and its influence on a relevant gene product. This chapter summarizes the current view on the interplay between ten-eleven translocation (TETs) and the poly(ADP-ribose) polymerase (PARPs) family of enzymes in regulating DNA methylation and how this interplay could be targeted to attenuate diabetes. This molecular interchange jigsaw puzzle is emerging as an important focus of research, and we can expect to see further advances in the elucidation of its role in diabetes as well as other pathologies. Moreover, the possibility for designating specific PARP-1 inhibitors as potential “EPI-drugs” for diabetes prevention/attenuation is also discussed. Understanding the epigenetic machinery and the differential roles of its components is essential for the development of targeted epigenetic therapies for diseases.
PB  - Springer International Publishing
T2  - Handbook of Nutrition, Diet, and Epigenetics
T1  - PARylation, DNA (De)methylation, and Diabetes
DO  - 10.1007/978-3-319-55530-0_55
SP  - 1857
EP  - 1876
ER  - 

@inbook{
author = "Vidaković, Melita and Tolić, Anja and Grdović, Nevena and Ravichandran, Mirunalini and Jurkowski, Tomasz P.",
year = "2019",
abstract = "Diabetes and diabetic complications, autoimmunity and inflammatory diseases, have recently become the focus of epigenetic therapy, since with epigenetic drugs it is possible to reverse aberrant gene expression profiles associated with the disease states. For diabetes, the therapy challenges depend on identifying the most appropriate molecular target and its influence on a relevant gene product. This chapter summarizes the current view on the interplay between ten-eleven translocation (TETs) and the poly(ADP-ribose) polymerase (PARPs) family of enzymes in regulating DNA methylation and how this interplay could be targeted to attenuate diabetes. This molecular interchange jigsaw puzzle is emerging as an important focus of research, and we can expect to see further advances in the elucidation of its role in diabetes as well as other pathologies. Moreover, the possibility for designating specific PARP-1 inhibitors as potential “EPI-drugs” for diabetes prevention/attenuation is also discussed. Understanding the epigenetic machinery and the differential roles of its components is essential for the development of targeted epigenetic therapies for diseases.",
publisher = "Springer International Publishing",
journal = "Handbook of Nutrition, Diet, and Epigenetics",
booktitle = "PARylation, DNA (De)methylation, and Diabetes",
doi = "10.1007/978-3-319-55530-0_55",
pages = "1857-1876"
}

Vidaković, M., Tolić, A., Grdović, N., Ravichandran, M.,& Jurkowski, T. P.. (2019). PARylation, DNA (De)methylation, and Diabetes. in Handbook of Nutrition, Diet, and Epigenetics
Springer International Publishing., 1857-1876.
https://doi.org/10.1007/978-3-319-55530-0_55

Vidaković M, Tolić A, Grdović N, Ravichandran M, Jurkowski TP. PARylation, DNA (De)methylation, and Diabetes. in Handbook of Nutrition, Diet, and Epigenetics. 2019;:1857-1876.
doi:10.1007/978-3-319-55530-0_55 .

Vidaković, Melita, Tolić, Anja, Grdović, Nevena, Ravichandran, Mirunalini, Jurkowski, Tomasz P., "PARylation, DNA (De)methylation, and Diabetes" in Handbook of Nutrition, Diet, and Epigenetics (2019):1857-1876,
https://doi.org/10.1007/978-3-319-55530-0_55 . .

TY  - CONF
AU  - Đorđević, Marija
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Grdović, Nevena
AU  - Uskoković, Aleksandra
AU  - Rajić, Jovana
AU  - Dinić, Svetlana
AU  - Jurkowski, Tomasz P.
AU  - Vidaković, Melita
PY  - 2017
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/2865
AB  - Since diabetes is characterized by impaired ability of pancreatic beta-cells to respond and/or produce insulin, new approaches for renewal and replacement of deficient beta-cells are indispensable. Generation of new insulin-producing cells from other cell types is a major aim of regenerative medicine. It has been shown that loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of alpha cells to functional insulin-producing cells. Previous experiments revealed dedifferentiation of pancreatic beta to alpha cells in response to ablation of DNA methyltransferase 1 (Dnmt1) in beta cells.
C3  - 1st Congress of Molecular Biologists of Serbia
T1  - Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_2865
ER  - 

@conference{
author = "Đorđević, Marija and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Miloš and Mihailović, Mirjana and Grdović, Nevena and Uskoković, Aleksandra and Rajić, Jovana and Dinić, Svetlana and Jurkowski, Tomasz P. and Vidaković, Melita",
year = "2017",
abstract = "Since diabetes is characterized by impaired ability of pancreatic beta-cells to respond and/or produce insulin, new approaches for renewal and replacement of deficient beta-cells are indispensable. Generation of new insulin-producing cells from other cell types is a major aim of regenerative medicine. It has been shown that loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of alpha cells to functional insulin-producing cells. Previous experiments revealed dedifferentiation of pancreatic beta to alpha cells in response to ablation of DNA methyltransferase 1 (Dnmt1) in beta cells.",
journal = "1st Congress of Molecular Biologists of Serbia",
title = "Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_2865"
}

Đorđević, M., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Mihailović, M., Grdović, N., Uskoković, A., Rajić, J., Dinić, S., Jurkowski, T. P.,& Vidaković, M.. (2017). Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation. in 1st Congress of Molecular Biologists of Serbia.
https://hdl.handle.net/21.15107/rcub_ibiss_2865

Đorđević M, Arambašić Jovanović J, Tolić A, Đorđević M, Mihailović M, Grdović N, Uskoković A, Rajić J, Dinić S, Jurkowski TP, Vidaković M. Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation. in 1st Congress of Molecular Biologists of Serbia. 2017;.
https://hdl.handle.net/21.15107/rcub_ibiss_2865 .

Đorđević, Marija, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Miloš, Mihailović, Mirjana, Grdović, Nevena, Uskoković, Aleksandra, Rajić, Jovana, Dinić, Svetlana, Jurkowski, Tomasz P., Vidaković, Melita, "Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation" in 1st Congress of Molecular Biologists of Serbia (2017),
https://hdl.handle.net/21.15107/rcub_ibiss_2865 .

TY  - CONF
AU  - Vidaković, Melita
AU  - Đorđević, Marija
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Grdović, Nevena
AU  - Uskoković, Aleksandra
AU  - Rajić, Jovana
AU  - Poznanović, Goran
AU  - Dinić, Svetlana
AU  - Jurkowski, Tomasz
PY  - 2017
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5816
AB  - Introduction and aim: Diabetes is the perfect candidate for cell replacement therapy since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells (b-cells}. We focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for a straightforward, one­-step transdifferentiation of mouse pancreatic alpha (a-cells) to b-cell by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). 
Methods: The a-cells were transiently transfected with four different Epi-CRISPR constructs and co-transfected with a single guided RNA (gRNA} or with a mix of different gRNAs all targeting different promoter regions of Arx. After 5, 8 and 12 days post-transfection, DNA and RNA were isolated and the cells were immunostained. The transdifferentiated cells were analysed for key features of bona fide cells, using qPCR to assess Arx expression, and immunostaining of insulin/glucagon and ELISA for measuring secreted insulin. 
Results: We succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA/mix gRNA. The suppression of Arx in a-cells was confirmed on days 5 and 8 post-transfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. Whether DNA methylation-mediated suppression of Arx in a-cells lead to their transdifferentiation to insulin-producing cells, will be confirmed by bisulfite sequencing. 
Conclusion: We are on the right course of developing a clear-cut technology capable of providing a perfect delivery system for increasing the number of insulin­producing cells in vitro.
PB  - Belgrade: University of Belgrade, Faculty of Biology
C3  - Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.
T1  - Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA
SP  - 10
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5816
ER  - 

@conference{
author = "Vidaković, Melita and Đorđević, Marija and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Miloš and Mihailović, Mirjana and Grdović, Nevena and Uskoković, Aleksandra and Rajić, Jovana and Poznanović, Goran and Dinić, Svetlana and Jurkowski, Tomasz",
year = "2017",
abstract = "Introduction and aim: Diabetes is the perfect candidate for cell replacement therapy since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells (b-cells}. We focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for a straightforward, one­-step transdifferentiation of mouse pancreatic alpha (a-cells) to b-cell by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). 
Methods: The a-cells were transiently transfected with four different Epi-CRISPR constructs and co-transfected with a single guided RNA (gRNA} or with a mix of different gRNAs all targeting different promoter regions of Arx. After 5, 8 and 12 days post-transfection, DNA and RNA were isolated and the cells were immunostained. The transdifferentiated cells were analysed for key features of bona fide cells, using qPCR to assess Arx expression, and immunostaining of insulin/glucagon and ELISA for measuring secreted insulin. 
Results: We succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA/mix gRNA. The suppression of Arx in a-cells was confirmed on days 5 and 8 post-transfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. Whether DNA methylation-mediated suppression of Arx in a-cells lead to their transdifferentiation to insulin-producing cells, will be confirmed by bisulfite sequencing. 
Conclusion: We are on the right course of developing a clear-cut technology capable of providing a perfect delivery system for increasing the number of insulin­producing cells in vitro.",
publisher = "Belgrade: University of Belgrade, Faculty of Biology",
journal = "Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.",
title = "Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA",
pages = "10",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5816"
}

Vidaković, M., Đorđević, M., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Mihailović, M., Grdović, N., Uskoković, A., Rajić, J., Poznanović, G., Dinić, S.,& Jurkowski, T.. (2017). Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA. in Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.
Belgrade: University of Belgrade, Faculty of Biology., 10.
https://hdl.handle.net/21.15107/rcub_ibiss_5816

Vidaković M, Đorđević M, Arambašić Jovanović J, Tolić A, Đorđević M, Mihailović M, Grdović N, Uskoković A, Rajić J, Poznanović G, Dinić S, Jurkowski T. Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA. in Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.. 2017;:10.
https://hdl.handle.net/21.15107/rcub_ibiss_5816 .

Vidaković, Melita, Đorđević, Marija, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Miloš, Mihailović, Mirjana, Grdović, Nevena, Uskoković, Aleksandra, Rajić, Jovana, Poznanović, Goran, Dinić, Svetlana, Jurkowski, Tomasz, "Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA" in Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia. (2017):10,
https://hdl.handle.net/21.15107/rcub_ibiss_5816 .

TY  - CONF
AU  - Đorđević, Marija
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Grdović, Nevena
AU  - Uskoković, Aleksandra
AU  - Rajić, Jovana
AU  - Poznanović, Goran
AU  - Dinić, Svetlana
AU  - Jurkowski, Tomasz
AU  - Vidaković, Melita
PY  - 2017
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5813
AB  - We propose to transdifferentiate alpha to beta cells using our recently developed Epi-CRISPRs, a novel synthetic epigenetic tool. Using this methodology we are able to induce straightfor­ward, one-step cell transdifferentiation by targeted DNA meth­ylation and suppression of homeobox gene Arx that is essen­tial for maintaining pancreatic alpha cell identity. 
The Epi-CRISPR constructs with and one or four different guide RNAs for specific targeting the promoter region of Arx, were transiently transfected in alphaTCl-6 cells (alpha-cells). The suc­cess of a-cells transdifferentiation into insulin-producing cells was evaluated by measuring Arx, glucagon (Glu) and insulin (lns2) mRNA level, amount of secreted insulin and by immu­nostaining of insulin and glucagon in the cells. 
Our study will be valuable for later subsequent Epi-CRISPRs use in mouse in vivo model of diabetes and eventually as a po­tential therapy for diabetes attenuation in humans.
PB  - Institute of Biology and Immunology of Reproduction, BAS, COST Action CA16119 and Mouseprint Ltd.
C3  - Proceedings of CellFit meeting: In vitro 3-D total cell guidance and fitness; 2017 Sep 12-14; Albena Resort, Bulgaria
T1  - Transdifferentiation of pancreatic alpha to  beta cells via targeted epigenome editing by  Epi-CRISPRs-s directed DNA methylation
SP  - 80
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5813
ER  - 

@conference{
author = "Đorđević, Marija and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Miloš and Mihailović, Mirjana and Grdović, Nevena and Uskoković, Aleksandra and Rajić, Jovana and Poznanović, Goran and Dinić, Svetlana and Jurkowski, Tomasz and Vidaković, Melita",
year = "2017",
abstract = "We propose to transdifferentiate alpha to beta cells using our recently developed Epi-CRISPRs, a novel synthetic epigenetic tool. Using this methodology we are able to induce straightfor­ward, one-step cell transdifferentiation by targeted DNA meth­ylation and suppression of homeobox gene Arx that is essen­tial for maintaining pancreatic alpha cell identity. 
The Epi-CRISPR constructs with and one or four different guide RNAs for specific targeting the promoter region of Arx, were transiently transfected in alphaTCl-6 cells (alpha-cells). The suc­cess of a-cells transdifferentiation into insulin-producing cells was evaluated by measuring Arx, glucagon (Glu) and insulin (lns2) mRNA level, amount of secreted insulin and by immu­nostaining of insulin and glucagon in the cells. 
Our study will be valuable for later subsequent Epi-CRISPRs use in mouse in vivo model of diabetes and eventually as a po­tential therapy for diabetes attenuation in humans.",
publisher = "Institute of Biology and Immunology of Reproduction, BAS, COST Action CA16119 and Mouseprint Ltd.",
journal = "Proceedings of CellFit meeting: In vitro 3-D total cell guidance and fitness; 2017 Sep 12-14; Albena Resort, Bulgaria",
title = "Transdifferentiation of pancreatic alpha to  beta cells via targeted epigenome editing by  Epi-CRISPRs-s directed DNA methylation",
pages = "80",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5813"
}

Đorđević, M., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Mihailović, M., Grdović, N., Uskoković, A., Rajić, J., Poznanović, G., Dinić, S., Jurkowski, T.,& Vidaković, M.. (2017). Transdifferentiation of pancreatic alpha to  beta cells via targeted epigenome editing by  Epi-CRISPRs-s directed DNA methylation. in Proceedings of CellFit meeting: In vitro 3-D total cell guidance and fitness; 2017 Sep 12-14; Albena Resort, Bulgaria
Institute of Biology and Immunology of Reproduction, BAS, COST Action CA16119 and Mouseprint Ltd.., 80.
https://hdl.handle.net/21.15107/rcub_ibiss_5813

Đorđević M, Arambašić Jovanović J, Tolić A, Đorđević M, Mihailović M, Grdović N, Uskoković A, Rajić J, Poznanović G, Dinić S, Jurkowski T, Vidaković M. Transdifferentiation of pancreatic alpha to  beta cells via targeted epigenome editing by  Epi-CRISPRs-s directed DNA methylation. in Proceedings of CellFit meeting: In vitro 3-D total cell guidance and fitness; 2017 Sep 12-14; Albena Resort, Bulgaria. 2017;:80.
https://hdl.handle.net/21.15107/rcub_ibiss_5813 .

Đorđević, Marija, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Miloš, Mihailović, Mirjana, Grdović, Nevena, Uskoković, Aleksandra, Rajić, Jovana, Poznanović, Goran, Dinić, Svetlana, Jurkowski, Tomasz, Vidaković, Melita, "Transdifferentiation of pancreatic alpha to  beta cells via targeted epigenome editing by  Epi-CRISPRs-s directed DNA methylation" in Proceedings of CellFit meeting: In vitro 3-D total cell guidance and fitness; 2017 Sep 12-14; Albena Resort, Bulgaria (2017):80,
https://hdl.handle.net/21.15107/rcub_ibiss_5813 .

TY  - CONF
AU  - Đorđević, Marija
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Grdović, Nevena
AU  - Uskoković, Aleksandra
AU  - Rajić, Jovana
AU  - Dinić, Svetlana
AU  - Jurkowski, Tomasz P.
AU  - Vidaković, Melita
PY  - 2017
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/2868
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/2865
AB  - Introduction: Since diabetes is characterized by impaired ability of pancreatic betacells to respond and/or produce insulin, new approaches for renewal and replacement of deficient beta-cells are indispensable. The aim of this study is direct pancreatic alpha- to beta-cells transdifferentiation by using a new synthetic epigenetic tool, Epi-CRISPR system. Using Epi-CRISPR system we aim to introduce targeted DNA methylation and subsequent repression of genes responsible for maintaining alpha-cell identity. Methods: AlphaTC1-6 cells (α-cells) were transiently transfected with dCas9-Dnmt3a- Dnmt3L constructs and one or four different vectors containing guide RNA components for specific targeting the promoter region of aristaless-related homeobox gene (Arx). The success of α-cells transdifferentiation into insulinproducing cells was evaluated by measuring Arx and insulin mRNA level, amount of secreted insulin and by immunostaining of insulin/glucagon in the cells. Results: We observed Arx transcriptional repression in α-cell transfected with Epi- CRISPR construct that targets the Arx gene promoter inducing subsequent methylation. At fifth day post-transfection the expression of Arx was decreased in α- cells followed by consequent increase in insulin (mRNA and protein level). At the same time, the glucagon levels remained unchanged. At twelfth day posttransfection the transfected cells start to lose glucagon while still secreting insulin. Conclusion: This study is near to confirm Epi-CRISPR system functionality and to verify the concept of cell transdifferentiation through silencing of genes responsible for maintaining cell phenotype. The obtained results will be valuable for later Epi- CRISPRs use in mouse in vivo models of diabetes and eventually as a future therapy for diabetes attenuation in humans.
PB  - Belgrade: University of Belgrade, Faculty of Biology, Belgrade
C3  - 1st Congress of Molecular Biologists of Serbia
T1  - Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation
SP  - 73
EP  - 73
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_2868
ER  - 

@conference{
editor = "Brajušković, Goran, Đorđević, Ana",
author = "Đorđević, Marija and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Miloš and Mihailović, Mirjana and Grdović, Nevena and Uskoković, Aleksandra and Rajić, Jovana and Dinić, Svetlana and Jurkowski, Tomasz P. and Vidaković, Melita",
year = "2017",
abstract = "Introduction: Since diabetes is characterized by impaired ability of pancreatic betacells to respond and/or produce insulin, new approaches for renewal and replacement of deficient beta-cells are indispensable. The aim of this study is direct pancreatic alpha- to beta-cells transdifferentiation by using a new synthetic epigenetic tool, Epi-CRISPR system. Using Epi-CRISPR system we aim to introduce targeted DNA methylation and subsequent repression of genes responsible for maintaining alpha-cell identity. Methods: AlphaTC1-6 cells (α-cells) were transiently transfected with dCas9-Dnmt3a- Dnmt3L constructs and one or four different vectors containing guide RNA components for specific targeting the promoter region of aristaless-related homeobox gene (Arx). The success of α-cells transdifferentiation into insulinproducing cells was evaluated by measuring Arx and insulin mRNA level, amount of secreted insulin and by immunostaining of insulin/glucagon in the cells. Results: We observed Arx transcriptional repression in α-cell transfected with Epi- CRISPR construct that targets the Arx gene promoter inducing subsequent methylation. At fifth day post-transfection the expression of Arx was decreased in α- cells followed by consequent increase in insulin (mRNA and protein level). At the same time, the glucagon levels remained unchanged. At twelfth day posttransfection the transfected cells start to lose glucagon while still secreting insulin. Conclusion: This study is near to confirm Epi-CRISPR system functionality and to verify the concept of cell transdifferentiation through silencing of genes responsible for maintaining cell phenotype. The obtained results will be valuable for later Epi- CRISPRs use in mouse in vivo models of diabetes and eventually as a future therapy for diabetes attenuation in humans.",
publisher = "Belgrade: University of Belgrade, Faculty of Biology, Belgrade",
journal = "1st Congress of Molecular Biologists of Serbia",
title = "Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation",
pages = "73-73",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_2868"
}

Brajušković, G., Đorđević, A., Đorđević, M., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Mihailović, M., Grdović, N., Uskoković, A., Rajić, J., Dinić, S., Jurkowski, T. P.,& Vidaković, M.. (2017). Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation. in 1st Congress of Molecular Biologists of Serbia
Belgrade: University of Belgrade, Faculty of Biology, Belgrade., 73-73.
https://hdl.handle.net/21.15107/rcub_ibiss_2868

Brajušković G, Đorđević A, Đorđević M, Arambašić Jovanović J, Tolić A, Đorđević M, Mihailović M, Grdović N, Uskoković A, Rajić J, Dinić S, Jurkowski TP, Vidaković M. Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation. in 1st Congress of Molecular Biologists of Serbia. 2017;:73-73.
https://hdl.handle.net/21.15107/rcub_ibiss_2868 .

Brajušković, Goran, Đorđević, Ana, Đorđević, Marija, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Miloš, Mihailović, Mirjana, Grdović, Nevena, Uskoković, Aleksandra, Rajić, Jovana, Dinić, Svetlana, Jurkowski, Tomasz P., Vidaković, Melita, "Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation" in 1st Congress of Molecular Biologists of Serbia (2017):73-73,
https://hdl.handle.net/21.15107/rcub_ibiss_2868 .

TY  - CONF
AU  - Vidaković, Melita
AU  - Đorđević, Marija
AU  - Rajić, Jovana
AU  - Arambašić Jovanović, Jelena
AU  - Grdović, Nevena
AU  - Tolić, Anja
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Uskoković, Aleksandra
AU  - Poznanović, Goran
AU  - Dinić, Svetlana
AU  - Jurkowski, Tomasz
PY  - 2017
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5805
AB  - Diabetes is the perfect candidate for cell replacement therapy, since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells. Our research is focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for straightfonward, one-step mouse pancreatic alpha (a-cells) to beta cell transdifferentiation by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). Up to now, we succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA or mix gRNA. The suppression of Arx in a-cells was confirmed on day 5 and 8 posttransfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. DNA methylation-mediated suppression of Ao( in a-cells leads to their transdifferentiation to insulin-producing beta cells will be confirmed by bisulfite sequencing (undergoing experiments).
Furthermore, we are also investigating an epithelial-mesenchymal transition (ETM), the mechanism which underlies the progressive decline in organ functioning in diabetes, such as the development of kidney and liver fibrosis. ETM is a process of reprogramming epithelial cells from a fully differentiated epithelial state to a more mesenchymal state. Our aim is to analyse the DNA methylation profile and gene expression of either epithelial (E-cadherin) or mesenchymal markers (a-smooth muscle actin and fibronectin) whose differential methylation and gene suppression could lead to more epithelial-like or mesenchyme-like phenotype. This will be accomplished using an in vitro model system based on epithelial cells treated with TGF-pi and 2. The obtained data should enable ETM reversal and stop fibrosis in diabetes and other pathologies using different compounds that act as DNA methylating/demethylating agents or using Epi-CRISPRs-based targeted DNA methylation/demethylation in future.
We are on the way to develop a clear-cut technology able to provide a perfect delivery system for increase of insulin-producing cells in vitro. This system will allow for targeted gene silencing via increased DNA methylation of gene of interest. In addition, we are able to test if any compound used for treatment in different pathological conditions affects the DNA methylation profile of the examined cells. On the other hand, there is a great need for chemical compounds able to act as DNA hypo or hypermethylated agents
PB  - COST Action CA1611
C3  - NutRedOx COST Action CA16112 & the 6'" NutriOx Atelier 20: Preventing Age-Related Diseases with Redo Active Compounds: a taste of controversory?: 2017 Sep 27-29; Strasbourg, France
T1  - Cellular Reprogramming via Epi-CRISPRs-lnduced Targeted DNA Methylation
SP  - 44
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5805
ER  - 

@conference{
author = "Vidaković, Melita and Đorđević, Marija and Rajić, Jovana and Arambašić Jovanović, Jelena and Grdović, Nevena and Tolić, Anja and Đorđević, Miloš and Mihailović, Mirjana and Uskoković, Aleksandra and Poznanović, Goran and Dinić, Svetlana and Jurkowski, Tomasz",
year = "2017",
abstract = "Diabetes is the perfect candidate for cell replacement therapy, since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells. Our research is focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for straightfonward, one-step mouse pancreatic alpha (a-cells) to beta cell transdifferentiation by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). Up to now, we succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA or mix gRNA. The suppression of Arx in a-cells was confirmed on day 5 and 8 posttransfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. DNA methylation-mediated suppression of Ao( in a-cells leads to their transdifferentiation to insulin-producing beta cells will be confirmed by bisulfite sequencing (undergoing experiments).
Furthermore, we are also investigating an epithelial-mesenchymal transition (ETM), the mechanism which underlies the progressive decline in organ functioning in diabetes, such as the development of kidney and liver fibrosis. ETM is a process of reprogramming epithelial cells from a fully differentiated epithelial state to a more mesenchymal state. Our aim is to analyse the DNA methylation profile and gene expression of either epithelial (E-cadherin) or mesenchymal markers (a-smooth muscle actin and fibronectin) whose differential methylation and gene suppression could lead to more epithelial-like or mesenchyme-like phenotype. This will be accomplished using an in vitro model system based on epithelial cells treated with TGF-pi and 2. The obtained data should enable ETM reversal and stop fibrosis in diabetes and other pathologies using different compounds that act as DNA methylating/demethylating agents or using Epi-CRISPRs-based targeted DNA methylation/demethylation in future.
We are on the way to develop a clear-cut technology able to provide a perfect delivery system for increase of insulin-producing cells in vitro. This system will allow for targeted gene silencing via increased DNA methylation of gene of interest. In addition, we are able to test if any compound used for treatment in different pathological conditions affects the DNA methylation profile of the examined cells. On the other hand, there is a great need for chemical compounds able to act as DNA hypo or hypermethylated agents",
publisher = "COST Action CA1611",
journal = "NutRedOx COST Action CA16112 & the 6'" NutriOx Atelier 20: Preventing Age-Related Diseases with Redo Active Compounds: a taste of controversory?: 2017 Sep 27-29; Strasbourg, France",
title = "Cellular Reprogramming via Epi-CRISPRs-lnduced Targeted DNA Methylation",
pages = "44",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5805"
}

Vidaković, M., Đorđević, M., Rajić, J., Arambašić Jovanović, J., Grdović, N., Tolić, A., Đorđević, M., Mihailović, M., Uskoković, A., Poznanović, G., Dinić, S.,& Jurkowski, T.. (2017). Cellular Reprogramming via Epi-CRISPRs-lnduced Targeted DNA Methylation. in NutRedOx COST Action CA16112 & the 6'" NutriOx Atelier 20: Preventing Age-Related Diseases with Redo Active Compounds: a taste of controversory?: 2017 Sep 27-29; Strasbourg, France
COST Action CA1611., 44.
https://hdl.handle.net/21.15107/rcub_ibiss_5805

Vidaković M, Đorđević M, Rajić J, Arambašić Jovanović J, Grdović N, Tolić A, Đorđević M, Mihailović M, Uskoković A, Poznanović G, Dinić S, Jurkowski T. Cellular Reprogramming via Epi-CRISPRs-lnduced Targeted DNA Methylation. in NutRedOx COST Action CA16112 & the 6'" NutriOx Atelier 20: Preventing Age-Related Diseases with Redo Active Compounds: a taste of controversory?: 2017 Sep 27-29; Strasbourg, France. 2017;:44.
https://hdl.handle.net/21.15107/rcub_ibiss_5805 .

Vidaković, Melita, Đorđević, Marija, Rajić, Jovana, Arambašić Jovanović, Jelena, Grdović, Nevena, Tolić, Anja, Đorđević, Miloš, Mihailović, Mirjana, Uskoković, Aleksandra, Poznanović, Goran, Dinić, Svetlana, Jurkowski, Tomasz, "Cellular Reprogramming via Epi-CRISPRs-lnduced Targeted DNA Methylation" in NutRedOx COST Action CA16112 & the 6'" NutriOx Atelier 20: Preventing Age-Related Diseases with Redo Active Compounds: a taste of controversory?: 2017 Sep 27-29; Strasbourg, France (2017):44,
https://hdl.handle.net/21.15107/rcub_ibiss_5805 .