Estrogen Receptor Alpha and Nuclear Factor Y Coordinately Regulate the Transcription of the SUMO-Conjugating <i>UBC9</i> Gene in MCF-7 Breast Cancer Cells

<div><p><i>UBC9</i> 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 <i>UBC9</i> gene, we identified and characterized its promoter and <i>cis</i>-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. <i>UBC9</i> mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of <i>UBC9</i> 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 <i>UBC9</i> promoter <i>in vivo</i>. Furthermore, <i>UBC9</i> expression was diminished by ER-α and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal <i>UBC9</i> promoter and provided evidence that ER-α and NF-Y regulate <i>UBC9</i> 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.</p>