Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.
The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.
Chen
H M
HM
Center for Molecular Biology in Medicine, Palo Alto VAMC, California, USA.
Boxer
L M
LM
D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D050783:Proto-Oncogene Proteins c-ets; D013539:Simian virus 40 / Q000235:genetics; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors