Project description:The goal of this study was to identify genomic binding sites of the NRSF/REST transcription factor under conditions of basal and increased SF-1 dosage in the H295R human adrenocortical tumor cell line. 4 samples: input DNA (2 replicates) - NRSF/REST ChIP basal SF-1 dosage - NRSF/REST ChIP increased SF-1 dosage
Project description:The goal of this study was to identify chromatin regulatory sites by FAIRE-seq under conditions of basal and increased dosage of transcription factor SF-1 in the H295R human adrenocortical tumor cell line. 4 samples: input DNA in basal SF-1 expression conditions - FAIRE-seq in basal SF-1 expression conditions - input DNA in SF-1 overexpression conditions - FAIRE-seq in SF-1 overexpression conditions
Project description:SF-1 is a nuclear receptor transcription factor playing a key role in adrenogonadal development and in adrenocortical tumorigenesis when overexpressed. NRSF/REST is a transcriptional repressor that represses expression of neuronal genes in non-neural tissues. Some data suggest that SF-1 and NRSF/REST can functionally interact in adrenocortical cancer cells. We studied gene expression profiles using Affymetrix microarrays in the H295R/TR SF-1 adrenocortical cancer cell line. In this cell line, SF-1 expression can be increased in a doxycycline-dependent manner (Mol. Endocrinol. 21: 2968–2987, 2007). The effects of a control siRNA and sRNAs specific for SF-1 and for NRSF/REST (in basal or increased SF-1 expression conditions) on gene expression were measured. In H295R/TR SF-1 cells SF-1 and NRSF/REST (in conditions of basal and increased SF-1 dosage) expression were knocked down by Amaxa nucleofection. RNA was extracted and hybridized to Human Gene 1.0 ST Affymetrix microarrays.
Project description:SF-1 is a nuclear receptor transcription factor playing a key role in adrenogonadal development and in adrenocortical tumorigenesis when overexpressed. We studied gene expression profiles using Affymetrix microarrays in the H295R/TR SF-1 adrenocortical cancer cell line, where SF-1 expression can be increased in a doxycycline-dependent manner (Mol. Endocrinol. 21: 2968–2987, 2007) H295R/TR SF-1 cells were cultured either in basal conditions or with doxycycline (Dox) added to the culture medium for 72 hours. RNA was extracted and hybridized to HG-U133 Plus 2.0 Affymetrix microarrays.
Project description:SF-1, a transcription factor belonging to the nuclear receptor superfamily, has a pivotal role for adrenogonadal development in humans and mice. A constant feature of childhood adrenocortical tumors (ACT) is SF-1 amplification and overexpression. Using an inducible cellular system, here we show that SF-1 overexpression increases human adrenocortical cell proliferation through opposing effects on cell cycle and apoptosis. SF-1 overexpression also selectively modulates steroidogenesis, reducing cortisol and aldosterone secretion. We identified a novel pro-apoptotic factor for adrenocortical cells, NOV/CCN3, whose levels are significantly reduced by SF-1 overexpression in human adrenocortical cells and are also reduced in primary adrenal tumors. Moreover, Sf-1 overexpression triggers adrenocortical hyperplasia and tumor formation in mice. These tumors express gonadal markers and activated Stat3. Our studies reveal the critical role of SF-1 gene dosage for adrenocortical tumorigenesis and constitute a rationale for the development of drugs targeting SF-1 transcriptional activity for ACT therapy. Keywords: differential expression, transcription factor Gene expression profiles were analyzed in two different H295R TR/SF-1 WT clones overexpressing SF-1 in a tetracycline-regulated fashion cultured in basal conditions or after three days of doxycycline treatment. For each condition, two biological replicates were examined. Array #22354 Clone #1 replicate 1 basal Cy3/Dox Cy5 Array #22416 Clone #1 replicate 2 basal Cy5/Dox Cy3 Array #22446 Clone #2 replicate 1 basal Cy3/Dox Cy5 Array #22447 Clone #2 replicate 2 basal Cy5/Dox Cy3
Project description:The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) is a key regulator of adrenal and gonadal biology. We aimed to identify a novel subset of SF-1 target genes in the adrenal by performing ChIP-on-chip in NCI-H295R human adrenocortical cells using promoter tiling arrays. Analysis of ChIP-on-chip experiments with CisGenome identified 738 SF-1-binding regions that met criteria of an MA score more than 3.5 mean ± S.D. and a false discovery rate of <5%. Subsequent analysis focused on those regions that were located between 10 kb upstream and 3 kb downstream of the TSS of known genes, in keeping with the design of the Human Promoter 1.0R arrays. Using this approach, binding regions were annotated to 445 gene loci. The supplementary bed file contains all 946 SF-1 binding sites identified by analysis with CisGenome using standard settings (MA>3.0) Overall design: Unstimulated NCI-H295R cells ChIPed with Upstate Millipore 07-618 anti-SF-1 antibody vs input chromatin. Two full experiments were analysed (biological replicates): 2 SF-1 ChIP arrays, 2 input control arrays
Project description:Virus resistance relies in some plant-viral interactions on the RNA-DEPENDANT RNA POLYMERASE 6 (RDR6), a major actor of RNA silencing that acts at the post-transcriptional level. Here, we demonstrate that RDR6 also plays a role in basal defense and race-specific resistance. RDR6 and the microRNA miR472, which target the mRNAs of disease resistance genes of coiled-coil nucleotide-binding leucine-rich-repeats family (e.g. RPS5), act in cooperation to control post-transcriptionally these immune receptors. Induction of these resistance genes is primed in rdr6- and miR472-elicited mutants and this effect is associated with an enhanced basal and race-specific immunity in these backgrounds. Comparison of Col0 WT sRNA with miR472 over-expressor mutant.
Project description:The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of approximately 650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.
Project description:The goal of the study was to identify the genes which are regulated by Interleukin-2 in the CD4+ T cells of the scurfy mice during regulatory T-cell deficiency. Scurfy (Sf) mice bear a mutation in the forkhead box P3 (Foxp3) transcription factor, lack regulatory T-cells (Treg), develop multi-organ inflammation, and die prematurely. The major target organs affected are skin, lungs, and liver. Sf mice lacking the Il2 gene (Sf.Il2-/-), despite devoid of Treg, did not develop skin and lung inflammation, but the inflammation in liver, pancreas, submandibular gland and colon remained. Genome-wide microarray analysis revealed hundreds of genes were differentially regulated among Sf, Sf.Il2-/-, and B6 CD4+ T-cells but the most changes were those encoding receptors for trafficking/chemotaxis/retention and lymphokines. Our study suggests that IL-2 controls the skin and lung inflammation in Sf mice in an apparent "organ-specific" manner through two novel mechanisms: by regulating the expression of genes encoding receptors for T-cell trafficking/chemotaxis/retention and by regulating Th2 cell expansion and lymphokine production. Thus, IL-2 is a master regulator for multi-organ inflammation and an underlying etiological factor for various diseases associated with skin and lung inflammation. Methods: CD4+ T cells were purified by Fluorescence Assisted Cell Sorting from the peripheral lymph nodes of (A) three individual Scurfy (Sf; B6.Cg-Foxp3sf/J) male mice, (B) three individual Sf.Il2-/- male mice (Scurfy mice carrying a null Interleukin (IL)-2 gene (B6.129P2-Il2tm1Hor/J)) and (C) a pooled sample of lymph nodes from two B6 (C57BL/6J) mice. All the mice were 3 weeks old. Total RNA was prepared using RNeasy mini kit (Qiagen). RNA samples were converted to cRNA, labeled and hybridized to Affymetrix Mouse 430_2 chips (Mouse Genome 430 2.0 Array, Affymetrix, Santa Clara, CA) at the University of Virginia DNA Sciences Core Facility. 1. RNA from CD4+ T cells purified from pooled peripheral lymph nodes of two 3-week old B6 mice) - 1 biological replicate 2. RNA from CD4+ T-cells purified from peripheral lymph nodes of 3-week old scurfy (Sf) mice - 3 biological replicates. 3. RNA from CD4+ T cells purified from peripheral lymph nodes of Sf.Il2-/- mice - 3 biological replicates.