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

ChIP-qPCR analysis of <i>SOX3</i> regulatory regions.

<p>(A) Schematic representation of the human <i>SOX3</i> gene indicating <i>SOX3</i> upstream (left tile), <i>SOX3</i> core promoter (center tile) and <i>SOX3</i> downstream (right tile) regions analyzed by ChIP. The positions of analyzed regions, relative to TSS, are indicated. (B-F) ChIP-qPCR results for the indicated histone modification that correspond to <i>SOX3</i> regions presented above. The enrichment was calculated relative to Flag and normalized against H3 or H2B. In comparative experiments, the enrichment in undifferentiated cells was assigned the value 1 and other samples were normalized to this value. Each ChIP experiment was repeated three times (biological replicates) followed by duplicate qPCR reactions. Results are presented as the mean ± S.D., *P<0.05.</p

Human <i>SOX3</i> promoter is hypomethylated during early phases of neural differentiation of NT2/D1 cells.

<p>(A) CpG islands localization within the human <i>SOX3</i> promoter determined by MethPrimer online software. Arrows I and II indicate CpG island regions. Bellow MethPrimer graph is a schematic representation of the human <i>SOX3</i> promoter and regions analyzed using MSP and pyrosequencing (Pyro S). Individual CpGs are represented as black dots. Numbers represent end points of the analyzed promoter region relative to tss (+1). (B) Analysis of <i>SOX3</i> promoter methylation in untreated (NT2/D1) and cells treated with RA in indicated time points (2, 4 and 7 days) by MSP. Product obtained with primers corresponding to methylated (M) 2^nd CpG island within <i>SOX3</i> promoter and product obtained with primers corresponding to unmethylated (U) 2^nd CpG island within <i>SOX3</i> promoter were separated on agarose gel. (C) Quantitative analysis of <i>SOX3</i> promoter methylation in untreated (NT2/D1) and cells treated with RA in indicated time points (2, 4 and 7 days) by pyrosequencing of the 2^nd CpG island. Bars indicate mean levels of methylation of <i>SOX3</i> promoter in each time point. (D) Effects of 1μM 5-azaC treatment on the expression of SOX3 protein in NT2/D1 cells. Quantity of SOX3 protein in treated cells was calculated relative to untreated NT2/D1 cells (set at 1) and presented as the means ± S.D. of at least three independent experiments; *P<0.05. Caspase-3 expression was used as a positive control. Representative blots are shown.</p

<i>SOX2</i> is down-regulated during early phases of RA induced neural differentiation of NT2/D1 cells.

<p>(A) Real-time PCR analysis of <i>SOX2</i> gene expression in untreated and RA treated NT2/D1 cells (2, 4 and 7 days of induction). Data were normalized by the amount of <i>GAPDH</i> mRNA and presented relative to the corresponding value for untreated cells, and are means ± S.D., *P<0.05 from triplicate data. (B) Western blot analysis of SOX2 protein in whole cell lysates of untreated and NT2/D1 cells treated with RA for 2, 4 and 7 days. SOX2 protein quantities were expressed relative to untreated NT2/D1 cells (set at 1) and presented as the mean ± S.D. of at least three independent experiments, *P<0.05. α-tubulin was used as loading control. Representative blots are shown.</p

ChIP-qPCR analysis of the <i>SOX2</i> core promoter.

<p>Schematic representation of the human <i>SOX2</i> core promoter analyzed by ChIP. The positions of the region relative to TSS are indicated. (B) ChIP-qPCR results for the indicated histone modifications. The enrichment was calculated relative to Flag and normalized against H3 or H2B. In comparative experiments, the enrichment in undifferentiated cells was assigned the value 1 and other samples were normalized to this value. Each ChIP experiment was repeated three times (biological replicates) followed by duplicate qPCR reactions. Results are presented as the mean ± S.D., *P<0.05.</p

Immunocytochemical co-localization of SOX3 and OCT4 during early phases of RA-induced neural differentiation of NT2/D1 cells.

<p>Immunocytochemical detection of SOX3 and OCT4 in untreated NT2/D1 cells (A1-A4) and NT2/D1 cells treated with RA for 2 (B1-B4), 4 (C1-C4), and 7 (D1-D4) days. Cells with high level of SOX3/low level of OCT4 expression are designated by white arrowheads in panels B1-B4. Cells with low level of SOX3/high level of OCT4 expression are designated by yellow arrowheads in panels B1-B4. Cells that are highly imunopositive for both markers are designated by white arrows in panels C1-C4. Cell nuclei were stained with DAPI (A3, B3, C3, D3). Scale bar: 50 μm.</p

ChIP-qPCR analysis of the <i>SOX1</i> core promoter.

<p>(A) Schematic representation of the human <i>SOX1</i> core promoter analyzed by ChIP. The positions of the region relative to TSS are indicated. (B) ChIP-qPCR results for the indicated histone modifications. The enrichment was calculated relative to Flag and normalized against H3 or H2B. In comparative experiments, the enrichment in undifferentiated cells was assigned the value 1 and other samples were normalized to this value. Each ChIP experiment was repeated three times (biological replicates) followed by duplicate qPCR reactions. Results are presented as the mean ± S.D., *P<0.05.</p

UCSC genome browser tracks showing histone modifications around <i>SOX3</i> locus in H1-hESCs.

<p>Shown from top to bottom is layered H2A.Z mark; enhancer- and promoter-associated H3K4me1, H3K4me2 and H3K4me3 marks; active marks H3K9ac, H3K9me3 and H3K27ac; repressive mark H3K27me3; marks associated with coding regions H3K36me3, H3K79me2 and H4K20me1. Arrows in the first track depict direction of transcription.</p

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

<div><p><i>Sox3/SOX3</i> is one of the earliest neural markers in vertebrates. Together with the <i>Sox1/SOX1</i> and <i>Sox2/SOX2</i> 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 <i>SOX3</i> gene expression during RA-induced neural differentiation of NT2/D1 cells. We show that the promoter of the human <i>SOX3</i> gene is extremely hypomethylated both in undifferentiated NT2/D1 cells and during the early phases of RA-induced neural differentiation. By employing chromatin immunoprecipitation, we analyze several histone modifications across different regions of the <i>SOX3</i> gene and their dynamics following initiation of differentiation. In the same timeframe we investigate profiles of selected histone marks on the promoters of human S<i>OX1</i> and <i>SOX2</i> genes. We demonstrate differences in histone signatures of <i>SOX1</i>, <i>SOX2</i> and <i>SOX3</i> genes. Considering the importance of <i>SOXB1</i> 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.</p></div

The influence of combined oral contraceptives containing drospirenone on hypothalamic-pituitary-adrenocortical axis activity and glucocorticoid receptor expression and function in women with polycystic ovary syndrome

OBJECTIVE: Most women with PCOS have increased adrenal androgen production, enhanced peripheral metabolism of cortisol and elevation in urinary excretion of its metabolites. Increased cortisol clearance in PCOS is followed by a compensatory overdrive of the hypothalamic-pituitary-adrenocortical (HPA) axis. We hypothesized that oral contraceptives containing ethinylestradiol and drospirenone (EE-DRSP) could modulate glucocorticoid receptor (GR) expression and function and thus affect HPA axis activity in PCOS patients. DESIGN: We analyzed 12 women with PCOS (age 24.17 +/- 4.88 years; body mass index 22.05 +/- 3.97 kg/m(2)) treated for 12 months with EE-DRSP and 20 BMI matched controls. In all subjects testosterone, dehydroepiandrosterone sulfate (DHEAS), sex hormone binding globulin (SHBG), cortisol (basal and after dexamethasone), concentrations of GR protein, phospo-GR211 protein, number of GR per cell (B-max) and its equilibrium dissociation constant (K-D) were measured. RESULTS: Before treatment, increased concentrations of testosterone and DHEAS (p<0.001, respectively), unaltered basal cortisol and an increased sensitivity (p<0.05) of the HPA axis to dexamethasone were observed in PCOS women in comparison to controls. After treatment, testosterone (p<0.01), DHEAS (p<0.05) and cortisol suppression after dexamethasone (p<0.01) were decreased in PCOS women. There were no changes in GR protein concentration, GR phosphorylation nor in the receptor functional parameters B-max and K-D in women with PCOS before and after the therapy, and in comparison to controls. CONCLUSIONS: Prolonged treatment with EE-DRSP in PCOS women decreased serum androgens and increased cortisol in the presence of decreased sensitivity of the HPA axis and did not exert changes in GR expression and function.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}11141009