Interplay of SOX transcription factors and microRNAs in the brain under physiological and pathological conditions

Precise tuning of gene expression, accomplished by regulatory networks of transcription factors, epigenetic modifiers, and microRNAs, is crucial for the proper neural development and function of the brain cells. The SOX transcription factors are involved in regulating diverse cellular processes during embryonic and adult neurogenesis, such as maintaining the cell stemness, cell proliferation, cell fate decisions, and terminal differentiation into neurons and glial cells. MicroRNAs represent a class of small non-coding RNAs that play important roles in the regulation of gene expression. Together with other gene regulatory factors, microRNAs regulate different processes during neurogenesis and orchestrate the spatial and temporal expression important for neurodevelopment. The emerging data point to a complex regulatory network between SOX transcription factors and microRNAs that govern distinct cellular activities in the developing and adult brain. Deregulated SOX/microRNA interplay in signaling pathways that influence the homeostasis and plasticity in the brain has been revealed in various brain pathologies, including neurodegenerative disorders, traumatic brain injury, and cancer. Therapeutic strategies that target SOX/microRNA interplay have emerged in recent years as a promising tool to target neural tissue regeneration and enhance neurorestoration. Numerous studies have confirmed complex interactions between microRNAs and SOX-specific mRNAs regulating key features of glioblastoma. Keeping in mind the crucial roles of SOX genes and microRNAs in neural development, we focus this review on SOX/microRNAs interplay in the brain during development and adulthood in physiological and pathological conditions. Special focus was made on their interplay in brain pathologies to summarize current knowledge and highlight potential future development of molecular therapies

Quercetin and lithium chloride modulate Wnt signaling in pluripotent embryonal carcinoma NT2/D1 cells

Wnt signaling functions in numerous cellular activities such as cell fate determination, patterning, and migration in embryogenesis, apoptosis, etc. In this study, we used quercetin and lithium chloride to investigate modulations of the Wnt signaling pathway in human pluripotent embryonal carcinoma NT2/D1 cell line. First, we optimized conditions for NT2/D1 cell treatments with quercetin and lithium chloride and assessed their cytotoxic effects on the cells, cell viability and proliferation rate. Our results showed that induction of cell death by quercetin and LiCl is p53-dependent in NT2/D cells. We also examined the degree of Wnt signaling modulations by analyzing the expression of c-myc, a wellknown Wnt signaling target gene. Since the retinoic acid induction of NT2/D1 cells is good in an in vitro model system for human neural differentiation, studying Wnt signaling modulation in NT2/D1 would contribute to a better understanding of the mechanisms involved in neural stem cell maintenance and human neural development

Quercetin and lithium chloride modulate Wnt signaling in pluripotent embryonal carcinoma NT2/D1 cells

Wnt signaling functions in numerous cellular activities such as cell fate determination, patterning, and migration in embryogenesis, apoptosis, etc. In this study, we used quercetin and lithium chloride to investigate modulations of the Wnt signaling pathway in human pluripotent embryonal carcinoma NT2/D1 cell line. First, we optimized conditions for NT2/D1 cell treatments with quercetin and lithium chloride and assessed their cytotoxic effects on the cells, cell viability and proliferation rate. Our results showed that induction of cell death by quercetin and LiCl is p53-dependent in NT2/D cells. We also examined the degree of Wnt signaling modulations by analyzing the expression of c-myc, a wellknown Wnt signaling target gene. Since the retinoic acid induction of NT2/D1 cells is good in an in vitro model system for human neural differentiation, studying Wnt signaling modulation in NT2/D1 would contribute to a better understanding of the mechanisms involved in neural stem cell maintenance and human neural development. [Projekat Ministarstva nauke Republike Srbije, br. 173051

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis

The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed

PCR amplification and sequence analysis of the rat Sox3 gene

Sox3 gen je jedan od markera najranijih faza razvića nervnog sistema kičmenjaka koji je uključen u kontrolu diferencijacije nervnih prekursora. Uprkos činjenici da je genom pacova sekvenciran i javno dostupan, samo parcijalna sekvenca Sox3 gena ove vrste je bila deponovana u bazi podataka. U ovom radu smo primenom PCR-a, sekvenciranja i bioinformatičke analize generisali kompletnu kodirajuću sekvencu Sox3 gena pacova. Analiza dobijene sekvence je pokazala da Sox3 gen kodira protein od 449 amino kiselina. Uporedna analiza ortologih SOX3 proteina pacova i čoveka pokazala je visok stepen evolutivne očuvanosti. Identifikacija i karakterizacija Sox3 gena pacova doprineće boljem razumevanju njegove uloge tokom razvića nervnog sistema i omogućiće bolji uvid u evoluciju ovog gena kod vertebrata.The Sox3 gene is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Despite the completion of a rat genome sequencing project, only a partial sequence of the rat Sox3 gene has been available in the public database. Using PCR, sequencing, and bioinformatics tools, in this study we have determined the complete coding sequence of the rat Sox3 gene encoding 449 amino acids. Comparative analysis of rat and human SOX3 proteins revealed a high degree of conservation. Identification of the rat Sox3 gene sequence would help in understanding the biological roles of this gene and provide insight into evolutionary relationships with vertebrate orthologs

Does Dietary Provision of Guanidinoacetic Acid Induce Global DNA Hypomethylation in Healthy Men and Women?

Background/Aims: Guanidinoacetic acid (GAA) is an experimental dietary additive and has been reported to induce methyl depletion when provided by the diet. However, no study evaluated whether supplemental GAA affects DNA methylation, a critical epigenetic process for genome regulation. Methods: In this open-label, repeated-measure interventional trial, we evaluated the impact of 12 weeks of GAA supplementation on global DNA methylation in 14 healthy participants (8 women and 6 men, age 22.2 +/- 2.3 years, body mass index 24.8 +/- 5.7). Results: Dietary provision of GAA had no effect on global DNA methylation, with 5-methylcytosine (m5C) nonsignificantly increased by 13.4% at postadministration when averaged across participants (95% confidence interval -5.5 to 32.3; p = 0.26). Notable DNA hypomethylation (corresponding to a 5% drop in m5C) was found in 3 of 14 participants at follow-up. Conclusion: Global DNA methylation seems to be unaltered by dietary provision of 3 g of GAA per day for 12 weeks in healthy men and women

Epigenetic regulation of human SOX3 gene expression during early phases of neural differentiation of NT2/D1 cells

Sox3/SOX3 is one of the earliest neural markers in vertebrates. Together with the Sox1/SOX1 and Sox2/SOX2 genes it is implicated in the regulation of stem cell identity. In the present study, we performed the first analysis of epigenetic mechanisms (DNA methylation and histone marks) involved in the regulation of the human SOX3 gene expression during RA-induced neural differentiation of NT2/D1 cells. We show that the promoter of the human SOX3 gene is extremely hypomethylated both in undifferentiated NT2/D1 cells and during the early phases of RA-induced neural differentiation. By employing chromatin immunopre-cipitation, we analyze several histone modifications across different regions of the SOX3 gene and their dynamics following initiation of differentiation. In the same timeframe we investigate profiles of selected histone marks on the promoters of human SOX1 and SOX2 genes. We demonstrate differences in histone signatures of SOX1, SOX2 and SOX3 genes. Considering the importance of SOXB1 genes in the process of neural differentiation, the present study contributes to a better understanding of epigenetic mechanisms implicated in the regulation of pluripotency maintenance and commitment towards the neural lineage

Current Opportunities for Targeting Dysregulated Neurodevelopmental Signaling Pathways in Glioblastoma

Glioblastoma (GBM) is the most common and highly lethal type of brain tumor, with poor survival despite advances in understanding its complexity. After current standard therapeutic treatment, including tumor resection, radiotherapy and concomitant chemotherapy with temozolomide, the median overall survival of patients with this type of tumor is less than 15 months. Thus, there is an urgent need for new insights into GBM molecular characteristics and progress in targeted therapy in order to improve clinical outcomes. The literature data revealed that a number of different signaling pathways are dysregulated in GBM. In this review, we intended to summarize and discuss current literature data and therapeutic modalities focused on targeting dysregulated signaling pathways in GBM. A better understanding of opportunities for targeting signaling pathways that influences malignant behavior of GBM cells might open the way for the development of novel GBM-targeted therapies

Direct PCR amplification of the HVSI region in mitochondrial DNA from buccal cell swabs

Amplification of human mitochondrial DNA (mtDNA) has been widely used in population genetics, human evolutionary and molecular anthropology studies. mtDNA hypervariable segments I and II (HVSI and HVSII) were shown to be a suitable tool in genetic analyses due to the unique properties of mtDNA, such as the lack of recombination, maternal mode of inheritance, rapid evolutionary rate and high population-specific polymorphisms. Here we present a rapid and low-cost method for direct PCR amplification of a 330 bp fragment of HVSI from buccal cell samples. Avoiding the DNA isolation step makes this method appropriate for the analysis of a large number of samples in a short period of time. Since the transportation of samples and fieldwork conditions can affect the quality of samples and subsequent DNA analysis, we tested the effects of long-term storage of buccal cell swabs on the suitability of such samples for direct PCR amplification. We efficiently amplified a 330 bp fragment of HVSI even after the long-term storage of buccal cells at room temperature, +4°C or at -20°C, for up to eight months. All examined PCR products were successfully sequenced, regardless of sample storage time and conditions. Our results suggest that the direct PCR amplification of the HVSI region from buccal cells is a method well suited for large-scale mtDNA population studies

SOX transcription factors and glioma stem cells: Choosing between stemness and differentiation

Glioblastoma (GBM) is the most common, most aggressive and deadliest brain tumor. Recently, remarkable progress has been made towards understanding the cellular and molecular biology of gliomas. GBM tumor initiation, progression and relapse as well as resistance to treatments are associated with glioma stem cells (GSCs). GSCs exhibit a high proliferation rate and self-renewal capacity and the ability to differentiate into diverse cell types, generating a range of distinct cell types within the tumor, leading to cellular heterogeneity. GBM tumors may contain different subsets of GSCs, and some of them may adopt a quiescent state that protects them against chemotherapy and radiotherapy. GSCs enriched in recurrent gliomas acquire more aggressive and therapy-resistant properties, making them more malignant, able to rapidly spread. The impact of SOX transcription factors (TFs) on brain tumors has been extensively studied in the last decade. Almost all SOX genes are expressed in GBM, and their expression levels are associated with patient prognosis and survival. Numerous SOX TFs are involved in the maintenance of the stemness of GSCs or play a role in the initiation of GSC differentiation. The fine-tuning of SOX gene expression levels controls the balance between cell stemness and differentiation. Therefore, innovative therapies targeting SOX TFs are emerging as promising tools for combatting GBM. Combatting GBM has been a demanding and challenging goal for decades. The current therapeutic strategies have not yet provided a cure for GBM and have only resulted in a slight improvement in patient survival. Novel approaches will require the fine adjustment of multimodal therapeutic strategies that simultaneously target numerous hallmarks of cancer cells to win the battle against GBM