The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNAbinding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly,-revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products. When the sequences of the MBS-I and MBS-II sites were compared, 11 of 19 base pairs (bp) were identical. MBS-I is a high-affinity site and was shown to be a c-Myb-dependent enhancer element. MBS-II is a low-affinity site, and tandem repeats of the sequence containing this site induce cMyb-dependent transcriptional repression (unpublished results). Here we report the identification of three functional domains of c-Myb: a DNA-binding domain, a transcriptional activation domain, and a negative regulatory domain. The functional differences between c-Myb and v-Myb are discussed in the light of the structures of these functional domains.MATERIALS AND METHODS Plasmid Construction. The effector plasmids pact-c-myb, in which the 5' regulatory region of the chicken cytoplasmic 13-actin gene is linked to the mouse c-myb gene, and pactl, which was constructed by deletion of the c-myb sequence from pact-c-myb, have been described (14). The reporter plasmids pMFcolCAT6MBS-I and pMFcolCAT6SV-II contain six tandem repeats of the MBS-I and the SV-II sequence, respectively, in the BamHI site of the plasmid pMFcolCAT, in which the bacterial chloramphenicol acetyltransferase (CAT) gene is linked to the mouse a2(I)-collagen promoter. The SV-II sequence contains the MBS-II site and corresponds to positions 184-218 in the SV40 genome. All plasmids designed to express mutant c-Myb proteins in cultured cells were generated from the plasmid pact-c-myb. To make the CT1, CT5, CT6, and CT7 mutants, termination codons were introduced at nucleotides 1537, 616, 463, and 307, respectively, by site-specific mutagenesis as described by Kunkel et al. (15). Nucleotide numbers are as in ref. 5. To obtain the NT1, NT3, and NT4 mutants, the sequence recognized by the restriction enzyme Nco I was introduced at nucleotides 147, 2%, and 455, respectively, by site-specific mutagenesis, and the regions between the introduced Nco I sites and the Nco I site at nucleotide 36 that overlaps the normal c-myb initiation codon were deleted. In-frame deletion mutants ADB, ANR, and ATA were construc...
Asymmetric divisions that produce two distinct cells play fundamental roles in generating different cell types during development. In the Drosophila central nervous system, neural stem cells called neuroblasts divide unequally into another neuroblast and a ganglion mother cell which is subsequently cleaved into neurons. Correct gene expression of ganglion mother cells requires the transcription factor Prospero. Here we demonstrate the asymmetric segregation of Prospero on neuroblast division. Prospero synthesized in neuroblasts is retained in the cytoplasm and at mitosis is exclusively partitioned to ganglion mother cells, in which it is translocated to the nucleus. Differential segregation of Prospero was also found in the endoderm. We have identified a region in Prospero that is responsible for this event. The region shares a common motif with Numb, which also shows unequal segregation. We propose that asymmetric segregation of transcription factors is an intrinsic mechanism for establishing asymmetry in gene expression between sibling cells.
How complex networks of activators and repressors lead to exquisitely specific cell type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In the R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict, and combinatorial activation to induce cell type-specific expression. Further, Dve levels are finely tuned to yield cell type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell fate specification.
The morphology of axon terminals changes with differentiation into mature synapses. A molecule that might regulate this process was identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The SIF proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated SIF protein resulted in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, SIF proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases.
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