ABSTRACT
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.
Subject(s)
Cell Differentiation/drug effects , Histones/metabolism , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/metabolism , Protein Processing, Post-Translational/drug effects , Tretinoin/pharmacology , Cell Line , DNA Methylation/drug effects , Epigenesis, Genetic/drug effects , Histones/genetics , Humans , Nanog Homeobox Protein/genetics , Neurogenesis/drug effects , Octamer Transcription Factor-3/geneticsABSTRACT
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.
Subject(s)
CCAAT-Binding Factor/physiology , Pluripotent Stem Cells/metabolism , SOXB1 Transcription Factors/genetics , Cell Line, Tumor , Cell Proliferation , Embryonal Carcinoma Stem Cells , Gene Expression , Humans , Tumor Suppressor Protein p53/geneticsABSTRACT
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.
Subject(s)
Mutation , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolism , Base Sequence , Genes, Reporter , HeLa Cells , Humans , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , SOXF Transcription Factors/chemistry , Transcriptional ActivationABSTRACT
Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates. Despite the mounting evidence that Sox3/SOX3 is one of the key players in the development of the nervous system, limited data are available regarding the transcriptional regulation of its expression. This review is focused on the retinoic acid induced regulation of SOX3 gene expression, with particular emphasis on the involvement of retinoid receptors. Experiments with human embryonal carcinoma cells identified two response elements involved in retinoic acid/retinoid X receptor-dependent activation of the SOX3 gene expression: distal atypical retinoic acid-response element, consisting of two unique G-rich boxes separated by 49 bp, and proximal element comprising DR-3-like motif, composed of two imperfect hexameric half-sites. Importantly, the retinoic acid-induced SOX3 gene expression could be significantly down-regulated by a synthetic antagonist of retinoid receptors. This cell model provides a solid base for further studies on mechanism(s) underlying regulation of expression of SOX3 gene, which could improve the understanding of molecular signals that induce neurogenesis in the stem/progenitor cells both during development and in adulthood.