ABSTRACT
Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5’flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40 percent of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17 percent of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17β-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17β-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs.
Subject(s)
Female , Humans , Breast Neoplasms/genetics , Estradiol/genetics , Gene Expression Regulation, Neoplastic/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Response Elements/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Estradiol/metabolism , Estradiol/pharmacology , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Response Elements/drug effects , Transcription Factors/genetics , Transcription, Genetic/geneticsABSTRACT
In order to understand the role of the upstream region of the Mycobacterium leprae 18-kDa gene on the gene regulation, the region was divided into two at the -50 position from the first start codon of the gene and their effect on transcription was examined by using a LacZ transcriptional reporter gene assay. The presence of each of these two regions conferred transcription repression not only on its cognate M. lepraerae 18-kDa gene promoter, but also on a heterologous promoter such as the Mycobacterium bovis BCG hsp65 gene promoter. Moreover, it was found that these regions could confer transcription repression activity in both cases in an orientation-independent manner. Thus, these results indicate that the upstream region of the M. leprae 18-kDa gene harbors transcription repression responsive element(s) acting as an operator and can be further divided into two separately functional regions, suggesting a bipartite structure of the element(s). The identification of transcription repression activity of the upstream region in the M. leprae 18-kDa gene will contribute greatly for the understanding of the 18-kDa gene regulation mechanism, and provide also useful information for the manipulation of mycobacterium gene expression.
Subject(s)
Bacterial Proteins/genetics , Down-Regulation/genetics , Gene Expression Regulation, Bacterial , Mycobacterium leprae/genetics , Response Elements/genetics , Transcription, GeneticABSTRACT
STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.