Genomics as a basis for precision medicine

Summary. Precision medicine, also known as genome-based medicine and personalized medicine, uses knowledge of the molecular basis of a disease in order to individualize treatment for each patient. The development of novel, powerful, high-throughput technologies has enabled better insight into the genomic, epigenomic, transcriptomic and proteomic landscape of many diseases, resulting in the application of personalized medicine approaches in healthcare. Research in the field of biomedicine in Serbia has followed the modern trends and has made a great contribution to the implementation of genomics in Serbian clinical practice. This is a review of the state of the art of scientific achievements and their application, which have paved the way for personalized medicine in Serbia

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

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

DOI:10.2298/ABS0703161S MUTATIONS IN THE PAH GENE: A TOOL FOR POPULATION GENETICS STUDY

Abstract – Phenylketonuria (PKU), an inborn error of metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the Serbian population, 19 different PAH mutations have been identified. We used PAH mutations as molecular markers for population genetics study. The low homozygosity value of the PAH gene (0.10) indicates that PKU in Serbia is heterogeneous, reflecting numerous migrations throughout Southeast Europe. The strategy for molecular diagnostics of PKU was designed accordingly. To elucidate the origin of the most common (L48S) PKU mutation in Serbia, we performed haplotype analysis by PCR-RFLP. Our results suggest that the L48S mutation was imported into Serbia from populations with different genetic backgrounds. Key words: Phenylketonuria, phenylalanine hydroxylase gene mutations, homozygosity value, expected heterozygosity, haplotype analysis UDC 616.441-008.6: 575.17: 577.

Regulation of <i>Cxcl12</i> promoter activity in pancreatic beta cells: a working model.

<p>(A) In the basal state, non-automodified PARP-1 (acting as a transcriptional inhibitor) and YY1 (acting as a transcriptional activator) strongly bind to the <i>Cxcl12</i> promoter, enabling its transcription (Fig. 3, 4). (B) DNA damage induced by STZ (diabetogenic-like) treatment causes PARP-1 binding to the DNA breaks, leading to a net increase in PARP-1 activity and consequently to poly(ADP-ribosyl)ation of PARP-1 and associated chromatin proteins. After 30 min of STZ treatment, DNA is moderately damaged (Fig. 5A), which results in weak poly(ADP-ribosyl)ation so that most of PARP-1 molecules remain attached to the promoter, however, as YY1 partially dissociates from the promoter (Fig. 6), <i>Cxcl12</i> transcription is still at the basal level (Fig. 5B). (C) After 6 h of STZ treatment, the DNA is severely damaged (Fig. 5A), causing intensive poly(ADP-ribosyl)ation of both PARP-1 and associated chromatin proteins and PARP-1 dissociation from the promoter, with resulting opening of the chromatin. Severe beta cell injury induces YY1 expression (Fig. 5D, E). Furthermore, the open chromatin structure enables intense YY1 binding to the promoter (Fig. 6), upregulation of <i>Cxcl12</i> expression (Fig. 5B, E) and consequent increased cell survival. Treatment with PARP-1 inhibitor (3AB) causes reduced poly(ADP-ribosyl)ation and intense PARP-1 binding to the promoter, resulting in decreased <i>Cxcl12</i> transcription (Fig. 7C).</p

The effect of PARP-1 inhibition on <i>Cxcl12</i> promoter regulation.

<p>(A) Viability assay was performed with wt cells treated with increasing concentrations of 3AB, followed by STZ treatment. (B) Immunoblot analysis was performed with anti-PARP-1, anti-caspase 3 and anti-β-actin (loading control) antibodies using cell lysates isolated from control, STZ-treated and cells pre-treated with 3AB, followed by STZ treatment. The apoptotic (89 kD) and necrotic (55 kD) PARP-1 fragment are indicated. (C) <i>Cxcl12</i> and <i>Parp-1</i> transcription after treatments with either 3AB or STZ, and after incubation with 3AB, followed by STZ treatment, was estimated by RT-qPCR. Relative mRNA levels are presented as the ratios of <i>Cxcl12/β-actin</i> and <i>Parp-1</i>/<i>β-actin.</i> (*) Mean values were significantly different from those of untreated control cells (p<0.05). (^#) Mean values were significantly different from those of STZ-treated cells (p<0.05). Results are expressed as the means±SEM from three separate experiments performed in triplicate. (D) EMSA showing binding of recombinant PARP-1 and total nuclear proteins to the <i>Cxcl12</i> promoter (lanes 2 and 3, respectively). (E) Nuclear proteins from control (C) and STZ-treated wt cells (STZ) probed with anti-ADP-ribose antibody to detect automodified PARP-1 and other ADP-ribosylated proteins.</p

PARP-1 downregulates and YY1 upregulates <i>Cxcl12</i> promoter activity.

<p>(A) Constructs used in transfection experiments: pMDICluc – control plasmid; the luciferase gene was driven by the CMV promoter; pCXCL12luc – reporter construct; the luciferase gene under the control of the <i>Cxcl12</i> promoter; pECV PARP – PARP-1 cDNA expression construct; pcDNA3.1FLAGYY1– expression vector containing a YY1 expression unit. pMDICluc and pCXCL12luc constructs were used for transfection of (B) Rin-5F wt and clone #1 cells and (C) NIH3T3 (PARP^+/+) and NIH3T3 (PARP^−/−) mouse embryonic fibroblasts. Activity of the <i>Cxcl12</i> promoter was expressed relative to the activity of the control CMV promoter. (D) Transfection of NIH3T3 cells with pCXCL12luc and combined pCXCL12luc/pcDNA3.1FLAGYY1 constructs. Overexpression (OE) of YY1 was confirmed by immunoblot analysis with anti-YY1 antibody (figure inset): lane 1– NIH3T3 cell lysate; lane 2– NIH3T3 cell lysate after pcDNA3.1FLAGYY1 transfection. Transfection with pCXCL12luc or with the combination of pCXCL12luc/pECV PARP was performed in (E) NIH3T3 (PARP^+/+) cells and (F) NIH3T3 (PARP^−/−) cells. PARP-1 overexpression (OE) was verified by immunoblot analysis (figure insets): lane 1– NIH3T3 cell lysate; lane 2– NIH3T3 cell lysate after pECV PARP transfection. Statistical significance (*) p<0.05. All results are expressed as the means±SEM, obtained from three separate experiments performed in triplicate.</p

Increasing time of STZ treatment caused changes in <i>Cxcl12, Parp-1</i> and <i>Yy1</i> transcription and protein expression in Rin-5F wt cells.

<p>(A) DNA damage was determined by the Comet assay (the tail moment was the parameter of DNA damage). Transcription of <i>Cxcl12</i> (B), <i>Parp-1</i> (C) and <i>Yy1</i> (D) after increasing times of exposure to STZ was estimated by RT-qPCR. Relative mRNA levels are presented as the ratios of <i>Cxcl12/β-actin</i>, <i>Parp-1</i>/<i>β-actin</i> and <i>Yy1/β-actin.</i> (*) Mean values were significantly different from those of untreated control cells (p<0.05). Results are expressed as the means±SEM from three separate experiments performed in triplicate. (E) Immunoblot analysis was performed with anti-CXCL12, anti-PARP-1, anti-YY1 and anti-β-actin (loading control) antibodies using cell lysates isolated from control and STZ treated cells at defined time points.</p

STZ-induced changes in <i>Cxcl12</i> promoter regulation.

<p>ChIP analysis was used to investigate PARP-1 and YY1 binding affinity toward the <i>Cxcl12</i> promoter during the early (0.5 h) and late stage of oxidative stress (6 h). Immunoprecipitation was performed with anti-PARP-1 and anti-YY1 antibodies. The controls in immunoprecipitation and PCR are shown in Fig. 3.</p

Analysis of the 739 bp <i>Cxcl12</i> promoter for transcription factor binding sites.

<p>(A) Putative YY1 sites are enclosed in an oval; Sp1 binding sites are underlined; three identified published PARP-1 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059679#pone.0059679-Vidakovic1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059679#pone.0059679-Akiyama1" target="_blank">[22]</a> are marked by rectangles; the TATA-like and Int elements and Kozak sequence are indicated. Expression of YY1 and PARP-1 in the Rin-5F cell line was verified by immunoblot analysis (figure inset). (B) A schematic diagram of the three promoter fragments used in ChIP analysis; each promoter fragment contained at least one putative PARP-1 and YY1 motif, represented by a rectangle and yin-yang symbol, respectively.</p

Overexpressed CXCL12 promotes better survival of injured pancreatic beta cells.

<p>(A) Viability assay performed on wt and clone #1 cells after treatment with increasing STZ concentrations; mean values for clone #1 were significantly different (*) from those for wt cells treated with the same STZ concentration (p<0.05). Increased presence of CXCL12 protein in the cell culture medium was verified by immunoblot analysis with anti-CXCL12 antibody (figure inset): lane 1– wt cells; lane 2– clone #1 cells. (B) Relative mRNA levels determined by real-time PCR and presented as ratios of <i>ratCxcl12/β-actin, humanCXCL12/β-actin</i> and <i>ratCxcr4/β-actin.</i> Mean values of clone #1 were significantly different (*) from those of wt cells (p<0.05). (C) Assessment of DNA damage by the Comet assay in wt cells and clone #1 after STZ treatment. The mean values of the tail moment (the parameter of DNA damage), of STZ-treated cells were significantly different (*) from those of untreated control cells (p<0.05); the mean values of the tail moment of the STZ-treated clone #1 cells were significantly different (#) from those of STZ-treated wt cells (p<0.05). All results are expressed as the means±SEM from three separate experiments performed in triplicate.</p