scholarly journals Isolation of Estrogen-Responsive Genes with a CpG Island Library

1998 ◽  
Vol 18 (1) ◽  
pp. 442-449 ◽  
Author(s):  
Toru Watanabe ◽  
Satoshi Inoue ◽  
Hisahiko Hiroi ◽  
Akira Orimo ◽  
Hiroyuki Kawashima ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Binding Site ◽  
Target Genes ◽  
Cpg Island ◽  
Cpg Islands ◽  
Human Cells ◽  
Receptor Protein ◽  
Upstream Region ◽  
Oxidase Subunit ◽  
Mcf 7

ABSTRACT In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochromec oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5′ upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5′ upstream region of the chicken β-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

scholarly journals DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 853
Author(s):  
Siti Aisyah Faten Mohamed Sa’dom ◽  
Sweta Raikundalia ◽  
Shaharum Shamsuddin ◽  
Wei Cun See Too ◽  
Ling Ling Few
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Binding Site ◽  
Promoter Activity ◽  
Cytosine Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Site Directed Mutagenesis ◽  
Choline Kinase ◽  
Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

scholarly journals PARP7 and Mono-ADP-Ribosylation Negatively Regulate Estrogen Receptor α Signaling in Human Breast Cancer Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 623
Author(s):  
Marit Rasmussen ◽  
Susanna Tan ◽  
Venkata S. Somisetty ◽  
David Hutin ◽  
Ninni Elise Olafsen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Protein Modification ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Transactivation Domain ◽  
Adp Ribosylation ◽  
Protein Levels ◽  
17Β Estradiol ◽  
Mcf 7

ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17β-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17β-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17β-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor’s ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.

scholarly journals Characterization of Binding Sequences for Butyrolactone Autoregulator Receptors in Streptomycetes

1999 ◽  
Vol 181 (16) ◽  
pp. 5075-5080 ◽  
Author(s):  
Hiroshi Kinoshita ◽  
Tomohiro Tsuji ◽  
Hiroomi Ipposhi ◽  
Takuya Nihira ◽  
Yasuhiro Yamada
Keyword(s):  
Transcription Start Site ◽  
Dna Sequences ◽  
Target Genes ◽  
Start Codon ◽  
Receptor Protein ◽  
Upstream Region ◽  
Start Site ◽  
Transcription Start ◽  
S1 Nuclease ◽  
Nuclease Mapping

ABSTRACT BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barBupstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barBtranslational start codon. The BARE located on the barAupstream region (BARE-3) was found 101 to 76 bp upstream of thebarA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barBtranscription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.

scholarly journals Rho GDP dissociation inhibitor α interacts with estrogen receptor α and influences estrogen responsiveness

2007 ◽  
Vol 39 (4) ◽  
pp. 249-259 ◽  
Author(s):  
Saad El Marzouk ◽  
Jennifer R Schultz-Norton ◽  
Varsha S Likhite ◽  
Ian X McLeod ◽  
John R Yates ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Negative Regulator ◽  
Guanosine Diphosphate ◽  
Estrogen Response ◽  
Endogenous Estrogen ◽  
Coregulatory Proteins ◽  
Gdp Dissociation Inhibitor ◽  
Mcf 7

AbstractEstrogen receptor α (ERα) is a ligand-activated transcription factor that regulates expression of estrogen-responsive genes. Upon binding of the ligand-occupied ERα to estrogen response elements (EREs) in DNA, the receptor interacts with a variety of coregulatory proteins to modulate transcription of target genes. We have isolated and identified a number of proteins associated with the DNA-bound ERα. One of these proteins, Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIα), is a negative regulator of the Rho family of GTP-binding proteins. In this study, we demonstrate that endogenously expressed RhoGDIα is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, and that RhoGDIα binds directly to ERα, alters the ERα–ERE interaction, and influences the ability of ERα to regulate transcription of a heterologous estrogen-responsive reporter plasmid in transient transfection assays as well as endogenous, estrogen-responsive genes in MCF-7 cells. Our studies suggest that, in addition to the activity of RhoGDIα in the cytoplasm, it also influences ERα signaling in the nucleus.

scholarly journals Regulation of estrogen receptor-alpha expression in MCF-7 cells by taxol

2004 ◽  
Vol 180 (3) ◽  
pp. 487-496 ◽  
Author(s):  
MB Martin ◽  
SV Angeloni ◽  
P Garcia-Morales ◽  
PF Sholler ◽  
MD Castro-Galache ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Progesterone Receptor ◽  
Gene Transcription ◽  
Estrogen Receptor Alpha ◽  
Receptor Protein ◽  
Subsequent Increase ◽  
Receptor Alpha ◽  
Alpha Gene ◽  
Er Alpha ◽  
Mcf 7

Results presented in this study demonstrate that treatment of MCF-7 cells with taxol resulted in induction of estrogen receptor-alpha (ER alpha) gene transcription with a subsequent increase in ER alpha mRNA; this effect was promoter specific since taxol did not affect total transcription in MCF-7 cells and lacked an effect on transcription of the human acidic ribosomal phosphoprotein protein PO, progesterone receptor, and pS2 genes. In contrast to the increase in transcription of the ER alpha gene, taxol inhibited translation of the ER alpha mRNA. This effect is also transcript specific since taxol did not alter total protein synthesis and did not affect the concentration of progesterone receptor protein in the cell. The overall result of taxol treatment was to decrease the concentration of ER alpha protein in the MCF-7 cells. Evidence is presented that the effects of taxol on ER alpha gene transcription may be mediated through the induction of p53.

scholarly journals Transcription factor EGR3 is involved in the estrogen-signaling pathway in breast cancer cells

2004 ◽  
Vol 32 (3) ◽  
pp. 649-661 ◽  
Author(s):  
A Inoue ◽  
Y Omoto ◽  
Y Yamaguchi ◽  
R Kiyama ◽  
SI Hayashi
Keyword(s):  
Breast Cancer ◽  
Transcription Factor ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Estrogen Receptor Alpha ◽  
Target Genes ◽  
Estrogen Signaling ◽  
Upstream Region ◽  
Custom Made ◽  
Mcf 7

Estrogen has been closely associated with the genesis and malignant progression of breast cancer. However, the molecular mechanism underlying the effects of estrogen is far from being completely clarified. We previously developed a custom-made cDNA microarray consisting of approximately 200 estrogen-responsive genes in breast cancer cells. Using this system, we found one estrogen-induced gene in various cancer cell lines, including breast cancer MCF-7 cells, which encode a zinc-finger transcription factor, EGR3 (early growth response 3). Northern blot analysis of estradiol-treated MCF-7 cells showed rapid and robust induction of Egr3, and addition of cycloheximide or ICI 182,780 suggested that Egr3 is the bona fide target for the estrogen receptor alpha (ERalpha). Using stable transformants derived from MCF-7 cells which were transfected with expression-controllable Egr3-expression vector, we demonstrated that Nab2 is one of the target genes for EGR3. Microarray analysis of the transformants revealed other candidate EGR3-induced genes. These strategies could be useful for analyzing downstream genes of ERalpha, and may contribute to elucidating the extensive signaling network of estrogen stimuli. Furthermore, a reporter assay using the upstream region of fasL probably involving escape from the immune system revealed that fasL is another target gene for EGR3. The roles of EGR3 in the physiology of breast cancer are discussed.

scholarly journals Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity

2004 ◽  
Vol 24 (5) ◽  
pp. 2103-2117 ◽  
Author(s):  
Young-Ho Lee ◽  
Hugh D. Campbell ◽  
Michael R. Stallcup
Keyword(s):  
Estrogen Receptor ◽  
Binding Site ◽  
Target Genes ◽  
Gene Promoter ◽  
Binding Activity ◽  
Actin Binding ◽  
Mouse Development ◽  
Methyltransferase Activity ◽  
Endogenous Estrogen ◽  
Coactivator Complex

ABSTRACT Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins.

scholarly journals Functional estrogen receptor signaling pathway activity in high-grade serous ovarian carcinoma as compared to estrogen receptor protein expression by immunohistochemistry

2021 ◽  
Author(s):  
Phyllis van der Ploeg ◽  
Laura A. M. van Lieshout ◽  
Anja van de Stolpe ◽  
Steven L. Bosch ◽  
Marjolein H. F. M. Lentjes-Beer ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Target Genes ◽  
Estrogen Therapy ◽  
Receptor Protein ◽  
Mrna Levels ◽  
High Grade ◽  
Ovarian Carcinomas ◽  
Pathway Activity

Abstract Purpose Anti-estrogen therapy may be used as a palliative treatment option in high-grade serous ovarian carcinomas (HGSC). However, clinical implementation is limited as the use of estrogen receptor (ER) protein expression by immunohistochemistry remains insufficient in predicting therapy response. To determine the accuracy of ER protein expression as a marker for ER signaling pathway activity, we aimed to correlate ER protein expression to functional ER signaling pathway activity in HGSC. Methods Immunohistochemical ER protein expression was visually scored using total percentages of stained tumor cells and histoscores. Subsequently, mRNA was extracted, and RT-qPCR analysis was performed. Functional ER pathway activity was assessed by a computational Bayesian model inferring ER signaling pathway activity from mRNA levels of ER-specific target genes. Results Our analysis of 29 HGSCs shows that neither total percentage of ER protein expression, nor ER histoscores are significantly correlated to ER signaling pathway activity (respectively, p = 0.473 and p = 0.606). Classification of HGSC into three groups based on ER histoscores 0–100 (n = 6), 101–200 (n = 15) and 201–300 (n = 8) resulted in comparable mean ER signaling pathway activity among the groups (p = 0.356). Several samples in the higher ER histoscore groups had low ER signaling pathway activity, indicating that nuclear ER protein expression is not sufficient to describe transcriptional ER activation. Conclusion Positive immunohistochemical ER staining is not always indicative of an active ER signaling pathway and is, therefore, a poor predictor of anti-estrogen response. Further research is needed to prove the predictive value of ER signaling pathway activity regarding anti-estrogen sensitivity in HGSC patients.

scholarly journals The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Defeng Xu ◽  
Tzu-Hua Lin ◽  
Chiuan-Ren Yeh ◽  
Max A. Cheng ◽  
Lu-Min Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Estrogen Receptor ◽  
Cancer Cells ◽  
Target Genes ◽  
Ovarian Cancer Cells ◽  
Ovarian Cancer Risk ◽  
Ovarian Cancers ◽  
Practical Strategy ◽  
Er Positive ◽  
Mcf 7

Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers.

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