Marija Mojsin

Antioxidant and antiproliferative activity of chokeberry juice phenolics during in vitro simulated digestion in the presence of food matrix

Chokeberry juice was subjected to in vitro gastric digestion in the presence of food matrix in order to determine the changes in polyphenol content and antioxidant activity. Addition of food matrix immediately decreased the total phenolic content, anthocyanin content, DPPH scavenging activity as well as total reducing power by 36%, 90%, 45% and 44%, respectively. After in vitro digestion, total phenolic content, anthocyanin content and reducing power are slightly elevated, but they are still lower than in initial non-digested juice. The effect of digested juice on Caco-2 cells proliferation was also studied, and the reduction of proliferative rate by approximately 25% was determined. Our results suggested that although a large proportion of chokeberry phenolics undergo transformation during digestion they are still potent as antioxidant and antiproliferative agents.

PBX1 and MEIS1 up-regulate SOX3 gene expression by direct interaction with a consensus binding site within the basal promoter region

Sox3/SOX3 [SRY (sex determining region Y)-box 3] is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. We have previously reported characterization of the SOX3 promoter and demonstrated that the general transcription factors NF-Y (nuclear factor-Y), Sp1 (specificity protein 1) and USF (upstream stimulatory factor) are involved in transcriptional regulation of SOX3 promoter activity. In the present study we provide the first evidence that the TALE (three-amino-acid loop extension) transcription factors PBX1 (pre-B-cell leukaemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue) participate in regulating human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus PBX/MEIS-binding site, which is conserved in all mammalian orthologue promoters analysed. PBX1 is present in the protein complex formed at this site with nuclear proteins from uninduced cells, whereas both PBX1 and MEIS1 proteins were detected in the complex created with extract from RA (retinoic acid)-induced NT2/D1 cells. By functional analysis we also showed that mutations of the PBX1/MEIS1-binding sites resulted in profound reduction of SOX3 promoter responsiveness to RA. Finally, we demonstrated that overexpressed PBX1 and MEIS1 increased endogenous SOX3 protein expression in both uninduced and RA-induced NT2/D1 cells. With the results of the present study, for the first time, we have established a functional link between the TALE proteins, PBX1 and MEIS1, and expression of the human SOX3 gene. This link is of particular interest since both TALE family members and members of the SOX superfamily are recognized as important developmental regulators.

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.

Crosstalk between SOXB1 proteins and WNT/β-catenin signaling in NT2/D1 cells

During early vertebrate embryogenesis, the expression of SOXB1 proteins is precisely regulated by a number of different mechanisms, including Wnt/β-catenin signaling. This is essential for controlling the balance between stemness and differentiation in embryonic stem cells. In the present study, we analyzed the molecular mechanism of LiCl action in NT2/D1 cells and examined the crosstalk between SOXB1 proteins and Wnt signaling in this model system. We have shown that LiCl increases β-catenin level, induces its translocation to the nucleus and consequently up-regulates β-catenin/Tcf-dependent transcription in NT2/D1 cells. Our results also suggest that LiCl treatment leads to increased expression of SOX2 and SOX3 proteins in NT2/D1 cells through activation of canonical Wnt signaling. Finally, we have detected a negative feedback loop between β-catenin and SOX2 expression in NT2/D1 cells. Since β-catenin and SOX2 have been linked to processes of self-renewal and pluripotency, our results have implications for future research on the maintenance of stemness and lineage commitment of embryonic stem cells.

TG-interacting Factor (TGIF) Downregulates SOX3 Gene Expression in the NT2/D1 Cell Line

SOX3 is a member of the Sox gene family implicated in brain formation and cognitive function. It is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Recently, we have established the first link between TALE (three-amino-acid loop extension) proteins, PBX1 (pre-B-cell leukemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue), and the expression of the human SOX3 gene. Here we present the evidence that TGIF (TG-interacting factor) is an additional TALE superfamily member involved in the regulation of human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus binding site that is conserved in primate orthologue promoters. Functional analysis demonstrated that mutation of the TGIF binding site resulted in the activation of SOX3 promoter. TGIF overexpression downregulates SOX3 promoter activity and decreases endogenous SOX3 protein expression in both uninduced and retinoic acid (RA)-induced NT2/D1 cells. Up to now, this is the first transcription factor identified as a negative regulator of SOX3 gene expression. The obtained results further underscore the significance of TALE proteins as important transcriptional regulators of SOX3 gene expression.

Histone modifications on the promoters of human OCT4 and NANOG genes at the onset of neural differentiation of NT2/D1 cells

Transcription factors OCT4 and NANOG are main constituents of a functional network that controls proliferation and pluripotency maintenance of stem cells as well as early lineage decisions. We investigated expression profiles of OCT4 and NANOG during the early phases of neural differentiation using NT2/D1 cells induced by retinoic acid as an in vitro model system of human neurogenesis. We demonstrated decrease in OCT4 and NANOG mRNA and protein levels following exposure to retinoic acid. Next, by employing chromatin immunoprecipitation, we investigated profiles of selected H3 and H2B histone marks deposited on the promoters of the OCT4 and NANOG genes. We found decline in H3K4me3, H2BK5ac, and H2BK120ac on both promoters, which paralleled the decrease in OCT4 and NANOG expression. Moreover, we found that the H2BK16ac mark is differentially enriched on these two promoters, pointing to differences in epigenetic regulation of OCT4 and NANOG gene expression. Finally, based on our data, we suggest that the early response of pluripotency genes OCT4 and NANOG to the differentiation-inducing stimuli is mediated by dynamic changes in chromatin marks, while DNA methylation is acquired in the later stages of neurogenesis.

Transcription factor NF-Y inhibits cell growth and decreases SOX2 expression in human embryonal carcinoma cell line NT2/D1

Transcription factor NF-Y belongs to the embryonic stem cell transcription factor circuitry due to its role in the regulation of cell proliferation. We investigated the role of NF-Y in pluripotency maintenance using NT2/D1 cells as one of the best-characterized human embryonal carcinoma cell line. We investigated the efficiency of protein transduction and analyzed the effects of forced expression of short isoform of NF-Y A-subunit (NF-YAs) on NT2/D1 cell growth and expression of SOX2. We found that protein transduction is an efficient method for NF-Y overexpression in NT2/D1 cells. Next, we analyzed the effect of NF-YAs overexpression on NT2/D1 cell viability and detected significant reduction in cell growth. The negative effect of NF-YAs overexpression on NT2/D1 cell pluripotency maintenance was confirmed by the decrease in the level of the pluripotency marker SOX2. Finally, we checked the p53 status and determined that the NF-Y-induced inhibition of NT2/D1 cell growth is p53-independent.

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.