Unknown,Transcriptomics,Genomics,Proteomics

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Bidirectional manipulation of SF-1 (NR5A1) in NCI-H295R cells

ABSTRACT: SF-1 (NR5A1) was overexpressed (Over) or knocked down with shRNA (shRNA) in NCI-H295R human adrenocortical tumor cells and differential global gene expression analysed 48 hours later using Affymetrix GeneChip Human Gene 1.0ST arrays. Over: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of a pIRES2-AcGPF1-Nuc construct co-expressing SF-1 cDNA and GFP. For experimental control, a mutagenized pIRES2 construct, bearing the G35E mutation in SF-1 that impairs its transactivation function in vivo and in vitro was used. shRNA: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of the SureSilencing shRNA Plasmid for Human NR5A1 with GFP marker kit (SABioscience). For experimental control, mismatch constructs provided in the kit were used. In both experiments (Over and shRNA), cells were harvested, prepared, and submitted to fluorescence-activated cell sorting (FACS) in a MoFlo XDP sorter 48 hours after transfection. Viable GFP-expressing cells were pooled and resuspended in TRIzol reagent for RNA extraction. Total RNA was extracted, and RNA quality control performed using a 2100 Bioanalyzer. RNA samples were processed using the Affymetrix GeneChip WT Sense Target Labeling kit, starting with 200 ng total RNA. Four independent Over experiments and five independent shRNA experiments were performed and samples of labeled fragmented cDNA were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Based on quality control of array data (R/Bioconductor and Partek Genomics Suite), two Over arrays (one SF-1 wild type and one mutant control) and one shRNA array (mismatch control) were excluded and are not deposited here. Differential gene expression analysis was performed using the limma package in R/Bioconductor. A Benjamini-Hochberg-corrected P value cut-off of 0.05 was used to select significant differentially expressed genes in each setting (Over and shRNA). Finally, results from both analyses were combined to identify a subset of positively-regulated SF-1 target genes in these cells, ie transcripts up-regulated by SF-1 overexpression (Over) and down-regulated by SF-1 knockdown (shRNA). Four SF-1 overexpression experiments (Over; intervention = overexpression of wild type SF-1; control = overexpression of functionally-impaired mutant G35E SF-1) and five SF-1 knockdown experiments (shRNA; intervention = SF-1-specific small hairpin RNA; control = shRNA mismatch control) were performed in NCI-H295R cells. Differential gene expression in each setting was analysed using GeneChip Human Gene 1.0ST arrays. Data for arrays that met quality control are presented here: Over, 3 intervention and 3 control arrays; shRNA, 5 intervention and 4 control arrays. Total of 15 arrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Bruno Ferraz-de-Souza

PROVIDER: E-GEOD-26529 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:SF-1 (NR5A1) was overexpressed (Over) or knocked down with shRNA (shRNA) in NCI-H295R human adrenocortical tumor cells and differential global gene expression analysed 48 hours later using Affymetrix GeneChip Human Gene 1.0ST arrays. Over: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of a pIRES2-AcGPF1-Nuc construct co-expressing SF-1 cDNA and GFP. For experimental control, a mutagenized pIRES2 construct, bearing the G35E mutation in SF-1 that impairs its transactivation function in vivo and in vitro was used. shRNA: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of the SureSilencing shRNA Plasmid for Human NR5A1 with GFP marker kit (SABioscience). For experimental control, mismatch constructs provided in the kit were used. In both experiments (Over and shRNA), cells were harvested, prepared, and submitted to fluorescence-activated cell sorting (FACS) in a MoFlo XDP sorter 48 hours after transfection. Viable GFP-expressing cells were pooled and resuspended in TRIzol reagent for RNA extraction. Total RNA was extracted, and RNA quality control performed using a 2100 Bioanalyzer. RNA samples were processed using the Affymetrix GeneChip WT Sense Target Labeling kit, starting with 200 ng total RNA. Four independent Over experiments and five independent shRNA experiments were performed and samples of labeled fragmented cDNA were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Based on quality control of array data (R/Bioconductor and Partek Genomics Suite), two Over arrays (one SF-1 wild type and one mutant control) and one shRNA array (mismatch control) were excluded and are not deposited here. Differential gene expression analysis was performed using the limma package in R/Bioconductor. A Benjamini-Hochberg-corrected P value cut-off of 0.05 was used to select significant differentially expressed genes in each setting (Over and shRNA). Finally, results from both analyses were combined to identify a subset of positively-regulated SF-1 target genes in these cells, ie transcripts up-regulated by SF-1 overexpression (Over) and down-regulated by SF-1 knockdown (shRNA).

Project description:The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female -induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

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Bidirectional manipulation of SF-1 (NR5A1) in NCI-H295R cells

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