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ER alpha coregulator peptide screening profiling by MARCoNI (Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction) assay from human embryonic kidney HEK293 cells after tranfected with ER alpha or Q375 mutations

ABSTRACT: ER alpha coregulator peptide screening profiling by MARCoNI (Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction) assay from human embryonic kidney HEK293 cells after tranfected with ER alpha or Q375 mutations

PROVIDER: PRJNA648396 | ENA |

REPOSITORIES: ENA

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Project description:Estrogen receptorï? beta (ER beta) has potent anti-proliferative and anti-inflammatory properties, suggesting that ER beta-selective agonists might be a new class of therapeutic and chemopreventative agents. To understand how ER beta regulates genes, we identified genes regulated by the unliganded and liganded forms of ER alpha and ER beta in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ER alpha, whereas many genes were regulated by estradiol (E2). These results demonstrate ER alpha requires a ligand to regulate a single class of genes. In contrast, ER beta regulated three classes of genes. Class I genes were regulated primarily by unliganded ER beta. Class II genes were regulated only with E2, whereas Class III genes were regulated by both unliganded ER beta and E2. There were 453 Class I genes, 258 Class II genes and 83 Class III genes. To explore the mechanism whereby ER beta regulates different classes of genes ChIP-seq was performed to identify ER beta binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in Class I genes, whereas ERE, NFKB1 and SP1 sites were more enriched in class II genes. ER beta bound to all three classes of genes demonstrating that ER beta binding is not responsible for differential regulation of genes by unliganded and liganded ER beta. The coactivator, NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ER beta regulate three classes of genes by interacting with different transcription factors and coactivators. U2OS cell lines are stably transfected with a doxycycline-inducible ER alpha or beta. 18 samples are analyzed with triplicate for each of the 6 conditions. The 6 conditions include ER alpha transfected without doxycycline added, ER alpha transfected with doxycycline added, ER alpha transfected with doxycycline added and E2 treatment, ER beta transfected without doxycycline added, ER beta transfected with doxycycline added, and ER beta transfected with doxycycline added and E2 treatment.

Project description:To better characterize group IE like human breast cancer based on the gene profiles of estrogen actions through estrogen receptor alpha (ER alpha), we identified an ER alpha transcriptional regulatory network for cell cycle in silico. We used two datasets from cell line (Data 1) and clinical samples (Data 2), respectively. Analyses on Data 1 via trajectory clustering and Pathway-Express confirmed the significant estrogen effect on up-regulating cell cycle activities. The gene expression relationships between ER alpha and cell cycle genes were re-identified in Data 2 by three statistical methods – Galton-Pearson’s correlation coefficient, Student’s t-test and the coefficient of intrinsic dependence. They were mostly (56.09%)(46/82) re-confirmed by literature search. E2F1 was found to be the major ER alpha target in regulating cell cycle gene expressions (83.72%)(36/43) via suppressive mode. However, enhanced cell cycle progression via up-regulating some cell cycle genes was predicted in silico possibly involving E2F2, in part. Both tumorigenic and tumor suppressing activities indicated by this network were predicted. This network clearly provides a robust way for uncovering estrogen actions in an ER(+) subtype specific manner. Experiment Overall Design: Two clinical datasets were used in this study. One, the 37 clinical arrays (abbreviated as 37A) consist of 26 A for patients positive in estrogen receptor alpha (ER) and in progesterone receptor (PR) immunohistochemical stain (IHC) and 11A for patients negative in ER IHC. This dataset was designated as Data 2. The 31 clinical arrays (31A) consist of 20A for patients positive in ER status but negative in PR status and 11A which are the same as in 37A. This dataset was used for data comparison. All the signals from the mRNA profile of each sample in the experiments were normalized using the internal control RNA- Stratagene's human common reference RNA via statistical method 'rank consistant lowess. Finally, those ratios were transformed by Log2.

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ER alpha coregulator peptide screening profiling by MARCoNI (Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction) assay from human embryonic kidney HEK293 cells after tranfected with ER alpha or Q375 mutations

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