Abstract
UBC9 encodes a protein that conjugates small ubiquitin-related modifier (SUMO) to target proteins thereby changing their functions. Recently, it was noted that UBC9 expression and activity play a role in breast tumorigenesis and response to anticancer drugs. However, the underlying mechanism is poorly understood. To investigate the transcriptional regulation of the UBC9 gene, we identified and characterized its promoter and cis-elements. Promoter activity was tested using luciferase reporter assays. The binding of transcription factors to the promoter was detected by chromatin immunoprecipitation (ChIP), and their functional role was confirmed by siRNA knockdown. UBC9 mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of UBC9 mRNA and protein was found in MCF-7 breast cancer cells treated with 17β-estradiol (E2). Analysis of various deletion mutants revealed a 137 bp fragment upstream of the transcription initiation site to be sufficient for reporter gene transcription. Mutations of putative estrogen receptor α (ER-α) (one imperfect estrogen response element, ERE) and/or nuclear factor Y (NF-Y) binding sites (two CCAAT boxes) markedly reduced promoter activity. Similar results were obtained in ER-negative MDA-MB-231 cells except that the ERE mutation did not affect promoter activity. Additionally, promoter activity was stimulated upon E2 treatment and overexpression of ER-α or NF-YA in MCF-7 cells. ChIP confirmed direct binding of both transcription factors to the UBC9 promoter in vivo. Furthermore, UBC9 expression was diminished by ER-α and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal UBC9 promoter and provided evidence that ER-α and NF-Y regulate UBC9 expression on the transcriptional level in response to E2 in MCF-7 cells. These findings may contribute to a better understanding of the regulation of UBC9 in ER-positive breast cancer and be useful for the development of cancer therapies targeting UBC9.
Highlights
Reversible attachment of small ubiquitin-related modifiers (SUMO) is an important post-translational protein modification in eukaryotic cells [1,2]
In order to evaluate UBC9 gene expression we investigated UBC9 mRNA and protein expression in ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells using real-time reverse transcription PCR (RT-PCR) and Western blot analyses
In the present study we investigated the transcriptional regulation of the human UBC9 gene in MCF-7 and MDAMB-231 breast cancer cells by cloning and functional characterization of its promoter
Summary
Reversible attachment of small ubiquitin-related modifiers (SUMO) is an important post-translational protein modification in eukaryotic cells [1,2]. Mammals typically express three SUMO variants (SUMO 1-3), which are conjugated to substrates through an enzymatic cascade involving the sequential action of the E1 SAE1/SAE2 activating enzyme, the E2 conjugating enzyme UBC9 and several E3 ligases such as the protein inhibitors of activated STAT (PIAS) family proteins that confer substrate specificity [2]. This regulation is dynamic, because it is a highly reversible process due to several SUMOspecific isopeptidases that remove SUMO from targets [3,4]. Variants in the UBC9 gene have been shown to be associated with a decreased efficacy of DNA double–strand break repair [27], breast tumor grade [28] and risk of grade 1 breast cancer [29]
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