Natasa Kovacevic-Grujicic

Improved transfection efficiency of cultured human cells

We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine™ (Life Technologies) and Effectene™ (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene™ transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine™ reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.

Up‐regulation of the SOX3 gene expression by retinoic acid: characterization of the novel promoter‐response element and the retinoid receptors involved

Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates and it is implicated in the genetic cascades that direct brain formation. We have previously shown that early phases of differentiation and neural induction of NT2/D1 embryonal carcinoma cells by retinoic acid (RA) involve up‐regulation of the SOX3 gene expression. Here, we present identification of a novel positive regulatory promoter element involved in RA‐dependent activation of the SOX3 gene expression in NT2/D1 cells. This element represents a direct repeat 3‐like motif that directly interacts with retinoid X receptor (RXR) α in a sequence‐specific manner. It is capable of independently mediating the RA effect in a heterologous promoter context and its disruption caused significant reduction of RA/RXR transactivation of the SOX3 promoter. Furthermore, by using synthetic antagonists of retinoid receptors, we have shown for the first time, that RA‐induced SOX3 gene expression could be significantly down‐regulated by the synthetic antagonist of RXR. Also, this data showed that RXRs, but not RA receptors, are mediators of RA effect on the SOX3 gene up‐regulation in NT2/D1 cells. Presented data will be valuable for future investigation of SOX3 gene expression, not only in NT2/D1 model system, but also in diverse developmental, physiological and pathological settings.

Early growth response protein 1 acts as an activator of SOX18 promoter

Sex-determining region Y box 18 (Sox18/SOX18) gene is an important regulator of vascular development playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. The aim of this study was to perform comprehensive functional characterization of the human SOX18 promoter, including determination of transcription start point (tsp) and identification of control elements involved in the regulation of SOX18 gene expression, with an emphasis on angiogenesis-related transcription factors. Analyses were performed in HeLa cells, representing a tumor cell line, and in EA.hy926 cells used as an endothelial model system. We have determined unique tsp of SOX18 gene, located 172 nucleotides upstream from ATG codon. Further, we have shown that SOX18 promoter region, -726 to -89 bp relative to tsp, contains positive cis-regulatory element(s) that stimulates SOX18 promoter activity, while region -89 to + 166 represents the minimal promoter. Within this region we have recognized the presence of essential element(s), positioned from -89 to +29, which harbors cluster of three putative early growth response 1 (EGR1) binding sites. By in vitro binding assays and functional analyses we have shown that these three putative binding sites are functionally relevant and sufficient for EGR1-induced SOX18 transcription. Mutations of these binding sites significantly impaired activity of the SOX18 promoter, particularly in EA.hy926 cells, indicating the importance of these regulatory elements for SOX18 promoter activity in endothelial setting. By data presented in this study, we have established SOX18 as a novel target gene regulated by EGR1 transcription factor, thus providing the first functional link between two transcription factors previously shown to be involved in the control of angiogenesis.

ZBP-89 and Sp3 down-regulate while NF-Y up-regulates SOX18 promoter activity in HeLa cells

The aim of this study has been to identify transcription factors involved in transcriptional regulation of the human SOX18 gene expression. Structural analysis revealed that the SOX18 promoter lacks a TATA box, but is CG-rich containing many putative binding sites for transcription factors that can bind and act through GC-boxes. Alignment analysis of promoter regions between human and mouse revealed conserved putative binding sites for transcription factors NF-Y and Sp-family members. Mithramycin A treatment led to increased SOX18 expression in vivo raising the possibility that the GC-rich sequence of the human SOX18 promoter might be occupied by transcription factor(s) that acts as repressor(s). Using in vitro binding assays we have demonstrated that transcription factors Sp3, ZBP-89 and NF-Y are capable of binding to the SOX18 promoter region spanning the sequence −200 to −162 relative to ATG and that formation of complexes could be efficiently reduced by mithramycin A. Furthermore, co-transfection experiments revealed that over-expression of Sp3 and ZBP-89 down-regulate, while over-expression of NF-Y up-regulates SOX18 promoter activity in HeLa cells. The involvement of these transcription factors in the regulation of SOX18 expression in HeLa cells was further confirmed in vivo by Western blot analyses. In this paper, for the first time, we have demonstrated that Sp3, ZBP-89 and NF-Y are involved in transcriptional regulation of the human SOX18 gene expression. Presented data provide the initial information about transcriptional regulation that will help in better understanding of molecular mechanisms involved in regulation of SOX18 gene expression.

Mitochondrial DNA perspective of Serbian genetic diversity.

Although south-Slavic populations have been studied to date from various aspects, the population of Serbia, occupying the central part of the Balkan Peninsula, is still genetically understudied at least at the level of mitochondrial DNA (mtDNA) variation. We analyzed polymorphisms of the first and the second mtDNA hypervariable segments (HVS-I and HVS-II) and informative coding-region markers in 139 Serbians to shed more light on their mtDNA variability, and used available data on other Slavic and neighboring non-Slavic populations to assess their interrelations in a broader European context. The contemporary Serbian mtDNA profile is consistent with the general European maternal landscape having a substantial proportion of shared haplotypes with eastern, central, and southern European populations. Serbian population was characterized as an important link between easternmost and westernmost south-Slavic populations due to the observed lack of genetic differentiation with all other south-Slavic populations and its geographical positioning within the Balkan Peninsula. An increased heterogeneity of south Slavs, most likely mirroring turbulent demographic events within the Balkan Peninsula over time (i.e., frequent admixture and differential introgression of various gene pools), and a marked geographical stratification of Slavs to south-, east-, and west-Slavic groups, were also found. A phylogeographic analyses of 20 completely sequenced Serbian mitochondrial genomes revealed not only the presence of mtDNA lineages predominantly found within the Slavic gene pool (U4a2a*, U4a2a1, U4a2c, U4a2g, HV10), supporting a common Slavic origin, but also lineages that may have originated within the southern Europe (H5*, H5e1, H5a1v) and the Balkan Peninsula in particular (H6a2b and L2a1k).

Comparison of promoter regions of SOX3, SOX14 and SOX18 orthologs in mammals

SOX proteins constitute a large family of diverse and well conserved transcription factors implicated in the control of various developmental processes. Previously we have cloned and characterized human SOX3, SOX14 and SOX18 genes and performed functional characterization of their promoter regions. To better understand organization and function of SOX3, SOX14 and SOX18 promoters and to determine evolutionary conserved regulatory regions, we performed comparative genomic analyses of orthologous genes promoters. Mammalian orthologs of the human SOX3, SOX14 and SOX18 genes show high sequence identity in their promoter regions, particularly within basal promoters of the respective human genes. Binding sites for transcription factors NF-Y, Sp1 and USF1, previously shown to play critical roles in transcriptional regulation of these human genes, are highly conserved in sequence and position among diverse mammalian species. Conservation of binding sites might indicate their highly significant roles in maintaining the transcriptional regulation of these genes among different species.

Comparative Analysis of SOX3 Protein Orthologs: Expansion of Homopolymeric Amino Acid Tracts During Vertebrate Evolution

To understand more fully the structure and evolution of the SOX3 protein, we comparatively analyzed its orthologs in vertebrates. Since complex disorders are associated with human SOX3 polyalanine expansions, our investigation focused on both compositional and evolutionary analysis of various homopolymeric amino acid tracts observed in SOX3 orthologs. Our analysis revealed that the observed homopolymeric alanine, glycine, and proline tracts are mammal-specific, except for one polyglycine tract present in birds. Since it is likely that the SOX3 protein acquired additional roles in brain development in Eutheria, we might speculate that development of novel brain functions during the course of evolution was affected, at least in part, by such structural–functional changes in the SOX3 protein.

Cyclic AMP response element binding (CREB) protein acts as a positive regulator of SOX3 gene expression in NT2/D1 cells

SOX3 is one of the earliest neural markers in vertebrates, playing the role in specifying neuronal fate. In this study we have established first functional link between CREB and human SOX3 gene which both have important roles in the nervous system throughout development and in the adulthood. Here we demonstrate both in vitro and in vivo that CREB binds to CRE half-site located -195 to -191 within the human SOX3 promoter. Overexpression studies with CREB or its dominant-negative inhibitor A-CREB indicate that this transcription factor acts as a positive regulator of basal SOX3 gene expression in NT2/D1 cells. This is further confirmed by mutational analysis where mutation of CREB binding site results in reduction of SOX3 promoter activity. Our results point at CREB as a positive regulator of SOX3 gene transcription in NT2/D1 cells, while its contribution to RA induction of SOX3 promoter is not prominent. [BMB Reports 2014; 47(4): 197-202]

Construction and functional analysis of novel dominant-negative mutant of human SOX18 protein

SOX18 transcription factor plays important roles in a range of biological processes such as vasculogenesis, hair follicle development, lymphangiogenesis, atherosclerosis, and angiogenesis. In this paper we present the generation of a novel SOX18 dominant-negative mutant (SOX18DN) encoding truncated SOX18 protein that lacks a trans-activation domain. We show that both wild-type SOX18 (SOX18wt) and truncated human SOX18 proteins are able to bind to their consensus sequence in vitro. Functional analysis confirmed that SOX18wt has potent trans-activation properties, while SOX18DN displays dominant-negative effect. We believe that these SOX18wt and SOX18DN expression constructs could be successfully used for further characterization of the function of this protein.

Mitochondrial super-haplogroup U diversity in Serbians

Abstract Background: Available mitochondrial (mtDNA) data demonstrate genetic differentiation among South Slavs inhabiting the Balkan Peninsula. However, their resolution is insufficient to elucidate the female-specific aspects of the genetic history of South Slavs, including the genetic impact of various migrations which were rather common within the Balkans, a region having a turbulent demographic history. Aim: The aim was to thoroughly study complete mitogenomes of Serbians, a population linking westward and eastward South Slavs. Subjects and methods: Forty-six predominantly Serbian super-haplogroup U complete mitogenomes were analysed phylogenetically against ∼4000 available complete mtDNAs of modern and ancient Western Eurasians. Results: Serbians share a number of U mtDNA lineages with Southern, Eastern-Central and North-Western Europeans. Putative Balkan-specific lineages (e.g. U1a1c2, U4c1b1, U5b3j, K1a4l and K1a13a1) and lineages shared among Serbians (South Slavs) and West and East Slavs were detected (e.g. U2e1b1, U2e2a1d, U4a2a, U4a2c, U4a2g1, U4d2b and U5b1a1). Conclusion: The exceptional diversity of maternal lineages found in Serbians may be associated with the genetic impact of both autochthonous pre-Slavic Balkan populations whose mtDNA gene pool was affected by migrations of various populations over time (e.g. Bronze Age pastoralists) and Slavic and Germanic newcomers in the early Middle Ages.