Abstract
Three members of the human myb gene family (c-myb, A-myb, and B-myb) encode transcriptional regulators that can bind to specific DNA sequences. High levels of c-myb expression are usually found in immature hemopoietic cells, but the B-myb is more commonly expressed in many types of cells. To understand the regulation of the activity of B-myb gene product (B-Myb), its functional domains were analyzed. Like c-Myb, B-Myb also has a transcriptional activation domain containing a cluster of acidic amino acids in the region downstream of the DNA-binding domain, which consists of three tandem repeats of 51-52 amino acids. In contrast to c-Myb, B-Myb does not contain a negative regulatory domain. Furthermore, the multiple nuclear localization signals are in at least two regions in the COOH-terminal half of B-Myb, and one of them is adjacent to a potential cdc2 kinase site. These results indicate that B-Myb contains DNA-binding and transcriptional activation domains similar to those of c-Myb, but a regulatory mechanism of B-Myb activity is quite different from that for c-Myb.
Highlights
Three members of the human myb gene family (c- that maintains the helix-turn-helix-related structure of the myb, A-myb, and B-myb) encode transcriptional reg- DNA-bindingunit of Myb [24,25,26,27]
These results indicate that thc-emyb gene prod- that B-myb maybe the c-myb equivalent of fibroblasts uct(c-Myb)’ is importantinmaintainingtheproliferative andepithelial cells
It contains three functional domains poietic cell lines [38]. These results suggest that B-myb gene product (B-Myb) has that are responsiblefor DNA binding, transcriptional acti- some differentphysiological rolesfrom c-Myb rather than the vation, andnegative regulation, respectively [20]
Summary
Effector plasmid pactl [17] lacking the cDNA sequence to be ex- Lack of Negative Autoregulation of B-Myb Activity-Both pressed, have been described. To make mutant 6, the that this negative autoregulation of c-Myb activity is a consequence of both homodimer formationof c-Myb through the leucine zipper and the inability of c-Myb dimers t o bind t o DNA.' To examine whether the B-Myb activity is negatively autoregulated by a similar mechanism, we have AccIII site (nucleotide 1872)was changed to a blunt end followed by measuredtrans-activationbyB-Myb as a function of t h e ligation This causes a frame shift so that a termination codon is introduced 6 amino acids codons past this AccIII site. DNA-binding and Transcriptional Activation Domain-To identify the functional domainsof B-Myb, a series of deletion mutants were madeand used for the transient cotransfection assay using the reporter plasmid pAlOCAT6MBS-I DNA-binding domain consistingof three tandem repeats near ing that NLSsof B-Myb are notin the DNA-binding domain, AWY T
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