The c-myb gene is implicated in the differentiation and proliferation of hematopoietic cells. Truncations of the N and/or C terminus of c-Myb, found in v-Myb, can potentiate its transforming ability. Two negative regulatory subregions, located in the C terminus, were mapped previously by using GAL4–c-Myb fusion proteins in transient transfection assays for the transcriptional activation of a GAL4-responsive reporter gene. To dissect the C terminus of c-Myb in terms of its involvement in transcriptional activation and oncogenic transformation, a series of C-terminal deletion mutants of c-Myb were analyzed. In addition, linker insertion mutants within the transactivation domain and/or heptad leucine repeat of c-Myb were examined along with those deletion mutants. In this study, we demonstrated that the removal of both of the two previously mapped negative regulatory subregions from the native form of c-Myb not only supertransactivates a Myb-responsive reporter gene but also potentiates its transforming ability in culture. However, in contrast to previous results, cells transformed by all of the mutants analyzed here except v-Myb itself exhibited the same phenotype as those transformed by c-Myb. The proliferating cells were bipotenial and differentiated into both the granulocytic and monocytic lineages. This result implies that the C terminus of c-Myb alone has no effect on the lineage determination. Finally, the transactivation activities of these mutants correlated with their transforming activities when a mim-1reporter gene was used but not when a model promoter containing five tandem Myb-binding sites was used. In particular, a very weakly transforming mutant with a linker insertion in the heptad leucine repeat superactivated the model promoter but not the mim-1reporter gene.

An alternatively spliced form of c-myb exists that encodes an additional 120 amino acids in chicken and 121 amino acids in human and mouse. These amino acids are encoded by an additional exon, termed exon 9A. This exon is not present in v-myb, and proteins containing these amino acids have never been tested for oncogenic transformation. A series of mybconstructs was therefore created in order to compare the functions of Myb proteins on the basis of their inclusion or exclusion of the amino acids encoded by exon 9A (E9A). We found that the presence of E9A resulted in a robust increase in transactivation for full-length c-Myb (CCC), as well as the singly truncated derivatives dCC and CCd, while doubly truncated Myb proteins v-Myb (dVd) and dCd did not exhibit any differences in transactivation. The increase in transactivation requires the Myb DNA-binding domain. When the leukemic transformation by the Myb proteins was tested, it was found that cells transformed by dVd resembled monoblasts, while cells transformed by CCC and its derivatives, dCd, dCC, and CCd, resembled myelomonoblasts. Despite differences in the morphology of the hematopoietic cells, the cell surface phenotypes and cell cycle profiles of transformed cells did not change for each pair of Myb proteins in the presence or absence of E9A. Thus, there was no direct correlation between the level of transcriptional activation and the strength of leukemic transformation.

Coordinate expression of MHC class II proteins and the class II-associated invariant chain (Ii) is important for proper MHC class II functioning in Ag processing and presentation. The coordinate regulation of these genes results, in part, from the sharing of transcriptional regulatory regions between MHC class II and Ii genes; the Ii has previously been shown to have an upstream enhancer closely related to the essential class II promoter elements. We report here the characterization of a second enhancer in the Ii gene, located within the first intron. This intronic enhancer is contained within a 155-bp region, enhances transcription from the Ii minimal promoter, and also contains elements that are homologous to class II promoter elements X1, X2, and Y boxes.