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Cloning and functional analysis of FLJ20420: a novel transcription factor for the BAG-1 promoter.

ABSTRACT: BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5' cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ?26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix), except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski). Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA -transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p?=?0.0006). The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p?=?0.0001). Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal expression may be involved in the oncogenesis of human malignancies such as lung cancer.

SUBMITTER: Liu H

PROVIDER: S-EPMC3342300 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Cloning and functional analysis of FLJ20420: a novel transcription factor for the BAG-1 promoter.

Liu Hongyu H Li Ying Y Li Yongwen Y Liu Baoxin B Wu Heng H Wang Jing J Wang Yuli Y Wang Min M Tang Shou-Ching SC Zhou Qinghua Q Chen Jun J

PloS one 20120502 5

BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5' cDNA library by ...[more]

PMID: 22567091

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Project description:MotivationThe identification of transcription factor (TF) binding sites and the regulatory circuitry that they define is currently an area of intense research. Data from whole-genome chromatin immunoprecipitation (ChIP-chip), whole-genome expression microarrays, and sequencing of multiple closely related genomes have all proven useful. By and large, existing methods treat the interpretation of functional data as a classification problem (between bound and unbound DNA), and the analysis of comparative data as a problem of local alignment (to recover phylogenetic footprints of presumably functional elements). Both of these approaches suffer from the inability to model and detect low-affinity binding sites, which have recently been shown to be abundant and functional.ResultsWe have developed a method that discovers functional regulatory targets of TFs by predicting the total affinity of each promoter for those factors and then comparing that affinity across orthologous promoters in closely related species. At each promoter, we consider the minimum affinity among orthologs to be the fraction of the affinity that is functional. Because we calculate the affinity of the entire promoter, our method is independent of local alignment. By comparing with functional annotation information and gene expression data in Saccharomyces cerevisiae, we have validated that this biophysically motivated use of evolutionary conservation gives rise to dramatic improvement in prediction of regulatory connectivity and factor-factor interactions compared to the use of a single genome. We propose novel biological functions for several yeast TFs, including the factors Snt2 and Stb4, for which no function has been reported. Our affinity-based approach towards comparative genomics may allow a more quantitative analysis of the principles governing the evolution of non-coding DNA.AvailabilityThe MatrixREDUCE software package is available from http://www.bussemakerlab.org/software/MatrixREDUCE.Supplementary informationSupplementary data are available at Bioinformatics online.

Dataset Information

Cloning and functional analysis of FLJ20420: a novel transcription factor for the BAG-1 promoter.

Publications

Cloning and functional analysis of FLJ20420: a novel transcription factor for the BAG-1 promoter.

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