SUMOylation Status and Effects of SUMylation on DAX-1

DAX-1 (Dosage Sensitive Sex Reversal Adrenal Hypoplasia Congenita on the X Chromosome gene 1) is a Nuclear Hormone Receptor, which acts as a transcriptional repressor in the nucleus. DAX-1 plays an important role in development and also appears to have some influence on the progression of cancer. In an effort to better understand DAX-1 function both in normal and disease states we are examining one type of posttranslational modification, SUMOylation. SUMOylation involves the addition of the small polypeptide conjugate SUMO (Small Ubiquitin-like Modifier) to proteins, this can have a variety of effects on protein activity. To study the effects of SUMOylation on DAX-1, the overall SUMOylation status of DAX-1 in mammalian cell lines was determined. It was found that DAX-1 is SUMOylated in several cell lines, both normal and carcinoma cells. Mutations were made in putative SUMOylation sites within the DAX-1 gene and assayed for changes in gene expression and activity

Selective Activation of Estrogen Receptor-β Transcriptional Pathways by an Herbal Extract

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes

Estradiol and Selective Estrogen Receptor Modulators Differentially Regulate Target Genes with Estrogen Receptors α and β

Estrogens and selective estrogen receptor modulators (SERMs) interact with estrogen receptor (ER) α and β to activate or repress gene transcription. To understand how estrogens and SERMs exert tissue-specific effects, we performed microarray analysis to determine whether ERα or ERβ regulate different target genes in response to estrogens and SERMs. We prepared human U2OS osteosarcoma cells that are stably transfected with a tetracycline-inducible vector to express ERα or ERβ. Western blotting, immunohistochemistry, and immunoprecipitation studies confirmed that U2OS-ERα cells synthesized only ERα and that U2OS-ERβ cells expressed exclusively ERβ. U2OS-ERα and U2OS-ERβ cells were treated either with 17β-estradiol (E(2)), raloxifene, and tamoxifen for 18 h. Labeled cRNAs were hybridized with U95Av2 GeneChips (Affymetrix). A total of 228, 190, and 236 genes were significantly activated or repressed at least 1.74-fold in U2OS-ERα and U2OS-ERβ cells by E(2), raloxifene, and tamoxifen, respectively. Most genes regulated in ERα cells in response to E(2), raloxifene, and tamoxifen were distinct from those regulated in ERβ cells. Only 38 of the 228 (17%) genes were regulated by E(2) in both U2OS-ERα and U2OS-ERβ cells. Raloxifene and tamoxifen regulated only 27% of the same genes in both the ERα and ERβ cells. A subset of genes involved in bone-related activities regulated by E(2), raloxifene, and tamoxifen were also distinct. Our results demonstrate that most genes regulated by ERα are distinct from those regulated by ERβ in response to E(2) and SERMs. These results indicate that estrogens and SERMs exert tissue-specific effects by regulating unique sets of targets genes through ERα and ER

Molecular Biology of the Cell

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