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A transcriptional regulatory element in the coding sequence of the human Bcl ‐2 gene
Author(s) -
Lang Georgina,
Gombert Wendy M.,
Gould Hannah J.
Publication year - 2005
Publication title -
immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.297
H-Index - 133
eISSN - 1365-2567
pISSN - 0019-2805
DOI - 10.1111/j.1365-2567.2004.02073.x
Subject(s) - microbiology and biotechnology , myb , gene silencing , biology , enhancer , transfection , reporter gene , gene , regulatory sequence , coding region , gene expression , regulation of gene expression , genetics
Summary 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.