Identification of an octamer-binding site controlling the activity of the small breast epithelial mucin gene promoter

Abstract : TABLE OF CONTENTS 1. Abstract 2. Introduction 2. Material and Methods 2.1. Cell Culture 2.2. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) 2.3. Rapid amplification of the 5'-cDNA end (RACE) 2.4. Identification of transcription factors binding sites within the 87-bp enhancer region (ENH) 2.5. Plasmids 2.6. Transient DNA transfections and luciferase assays 3. Results 3.1. Identification of the transcription initiation sites 3.2. Endogenous SBEM promoter activity in mammary and non-mammary cancer cells 3.3. Analysis of SBEM promoter activity in mammary and non-mammary cancer cells 3.4. The ENH region (-357/-270) drives a strong breast-specific promoter activity 3.5. Importance of octamer-binding transcription factors motif in the SBEM promoter activity 3.6. Oct1 and Oct2 enhance both exogenous and endogenous SBEM promoter activities 4. Discussion 5. Acknowledgement 6. References 1. ABSTRACT The human small breast epithelial mucin (SBEM) gene has been identified as being preferentially expressed in mammary epithelial cells and over-expressed in breast tumors. In this report, we have characterized the promoter of SBEM gene in order to identify sequences responsible for this strong mammary expression. A series of SBEM promoter/luciferase constructs were transiently transfected into both breast (MCF-7, BT-20) and non-breast (HeLa and HepG2) cell lines. In addition to the minimal promoter and to a repressor region, we have identified an 87-bp sequence (-357/-270) driving a strong breast-specific expression. Site-directed mutagenesis of a putative octamer-binding transcription factor binding site located within this latter region led to a strong decrease of the transcriptional activity of the SBEM promoter. Furthermore, transient over-expression of Oct1 and Oct2 not only increased SBEM promoter reporter activity, but also enhanced endogenous SBEM mRNA level. Overall, the data suggest that octamer-binding transcription factors participate in the strong expression of SBEM gene in breast tissues. Clarifying the SBEM gene regulation will help to dissect mechanisms underlying transcription of normal breast and breast cancer-associated genes.