A transcriptional regulatory element in the coding sequence of the human Bcl-2 gene.
We investigated the protein-binding sites in a DNAse I hypersensitive site associated with bcl-2 gene expression in human B cells. We mapped this hypersensitive site to the coding sequence of exon 2 of the bcl-2 gene in the bcl-2-expressing REH B-cell line. Electrophoretic mobility shift assays (EMSAs) with extracts from REH cells revealed three previously unrecognized B-Myb-binding sites in this sequence. The protein was identified as B-Myb by using a specific antibody and EMSAs. Accordingly, the levels of B-Myb and bcl-2 proteins, and of Myb EMSA activity, were correlated over a wide range of cell lines, representing different stages of B-cell development. Transfection of REH cells with antisense B-myb down-regulated EMSA activity and the level of bcl-2, and led to the apoptosis of REH cells. Transfection of the bcl-2-non-expressing RPMI 8226 cell line with a B-Myb expression vector induced B-Myb EMSA activity and the expression of bcl-2. Reporter assays indicated that the HSS8 sequence containing the three B-Myb sites may act as an enhancer when it is linked to the bcl-2 gene promoter. Interaction of B-Myb with HSS8 may enhance bcl-2 gene expression by co-operating with positive regulatory elements (e.g. previously identified B-Myb response elements) or silencing negative response elements in the bcl-2 gene promoter.
Lang
Georgina
G
The Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, UK.
Gombert
Wendy M
WM
Gould
Hannah J
HJ
D017209:Apoptosis / Q000235:genetics / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D003850:Deoxyribonuclease I / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000276:immunology*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D019254:Genes, bcl-2; D006801:Humans; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense; D014066:Palatine Tonsil / Q000276:immunology; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D020598:Proto-Oncogene Proteins c-myb; D003850:Deoxyribonuclease I