Karl-Heinz Klempnauer

Interaction and functional collaboration of p300 and C/EBPbeta

Transcriptional coactivators such as p300 and CREB-binding protein (CBP) function as important elements in the transcription factor network, linking individual transactivators via protein-protein interactions to the basal transcriptional machinery. We have investigated whether p300 plays a role in transactivation mediated by C/EBPbeta, a conserved member of the C/EBP family. We show that C/EBPbeta-dependent transactivation is strongly inhibited by adenovirus E1A but not by E1A mutants defective in p300 binding. Ectopic expression of p300 reverses the E1A-dependent inhibition and increases the transactivation potential of C/EBPbeta. Furthermore, we show that C/EBPbeta and p300 interact with each other and demonstrate that the sequences responsible for interaction map to the E1A binding region of p300 and the amino terminus of C/EBPbeta. Finally, we show that the minimal C/EBPbeta binding site of p300 acts as a dominant-negative inhibitor of C/EBPbeta. These observations identify p300 as a bona fide coactivator for C/EBPbeta. C/EBPbeta is highly expressed in the myelomonocytic lineage of the hematopoietic system and cooperates with Myb to activate mim-1, a gene specifically expressed during myelomonocytic differentiation. Recent evidence has shown that Myb recruits CBP (and presumably p300) as a coactivator and, in contrast to C/EBPbeta, interacts with the CREB binding site of p300-CBP. We show that p300 not only stimulates the activity of Myb and C/EBPbeta individually but also increases the synergy between them. Thus, our results reveal a novel function of p300: in addition to linking specific transcription factors to the basal transcriptional machinery, p300 also mediates the cooperation between transactivators interacting with different domains of p300.

Nucleotide sequence of the retroviral leukemia gene v-myb and its cellular progenitor c-myb: the architecture of a transduced oncogene

The oncogene (v-myb) of avian myeloblastosis virus and a large portion of its cellular homolog (c-myb) have been molecularly cloned and sequenced. The portion of c-myb we analyzed contains seven interspersed segments (or exons). Fusion of these exons creates a continuous nucleotide sequence that is remarkably similar to the sequence of v-myb and that potentially encodes a protein very similar to that specified by v-myb. Comparisons between the sequences of v-myb and c-myb indicate that transduction of c-myb to form v-myb probably resulted from an initial DNA rearrangement and the subsequent use of a spliced RNA as an intermediate.

Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors.

The retroviral oncogene v‐myb encodes a transcriptional activator which is responsible for the activation of the mim‐1 gene in myelomonocytic cells transformed by v‐myb. The mim‐1 promoter contains several myb consensus binding sites and has previously been shown to be regulated directly by v‐myb. Here we report that the mim‐1 gene is activated synergistically by v‐myb and different C/EBP transcription factors. We have cloned a chicken C/EBP‐related gene that is highly expressed in myeloid cells and identified it as the chicken homolog of C/EBP beta. A dominant‐negative variant of chicken C/EBP beta interferes with the v‐myb induced activation of the mim‐1 gene in these cells, suggesting that C/EBP beta or another C/EBP transcription factor is required for the activation of mim‐1 by v‐myb. We found that C/EBP beta and other C/EBP transcription factors confer to fibroblasts the ability to induce the mim‐1 gene in the presence of v‐myb. Finally we show that, in contrast to v‐myb, c‐myb synergizes with C/EBP transcription factors only at low concentrations of c‐myb protein. Our results suggest a role for C/EBP beta, and possibly for other C/EBP transcription factors, in v‐myb function and in myeloid‐specific gene activation.

Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by the chicken c-myb gene

Analysis of the subcellular location of the proteins encoded by the oncogenes of avian myeloblastosis virus and avian leukemia virus E26 ( p45v -myb and p135gag -myb-ets, respectively) and by the chicken c-myb gene ( p75c -myb) shows that all three proteins are located in the nucleus. In AMV-infected (but not transformed) chicken fibroblasts p45v -myb also resides in the nucleus, indicating that a nuclear location of p45v -myb in these cells is not sufficient to achieve transformation. In AMV-transformed myeloblasts a small fraction of p45v -myb occupies an additional site in the perinuclear region of the cytoplasm. If the myeloblasts are caused to differentiate to macrophages, most of p45v -myb is found in the cytoplasm. This redistribution of p45v -myb within the cell may be responsible for reversion of the transformed phenotype.

Recruitment of p300 by C/EBPβ triggers phosphorylation of p300 and modulates coactivator activity

Transcriptional coactivators such as p300 act as crucial elements in the eukaryotic gene regulation network. These proteins bind to various transcription factors which recruit them to specific gene regions whose chromatin structure subsequently is remodeled. Previously, we have shown that C/EBPβ recruits p300 by interacting with the E1A‐binding site of the coactivator. We now show that C/EBPβ not only binds to p300 but also triggers massive phosphorylation of p300. This novel activity of C/EBPβ is dependent on the E1A‐binding region of p300 as well as on several subdomains of C/EBPβ, all of which are involved in the p300–C/EBPβ interaction. We have identified several sites of C/EBPβ‐inducible phosphorylation within the C‐terminal domain of p300. Mutation of these sites substantially impairs the activity of p300 as a coactivator of C/EBPβ. Interestingly, phosphorylation of p300 is also triggered by other C/EBP family members as well as by various other transcription factors that interact with the E1A‐binding domain of p300, suggesting that this novel phosphorylation mechanism may be of general relevance.

The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb

Avian myeloblastosis virus (AMV) is an oncogenic retrovirus that rapidly causes myeloblastic leukemia in chickens and transforms myeloid cells in culture. AMV carries an oncogene, v-myb, that is derived from a cellular gene, c-myb, found in the genomes of vertebrate species. We constructed a plasmid vector that allows expression of a portion of the coding region for v-myb in a procaryotic host. We then used the myb-encoded protein produced in bacteria to immunize rabbits. The antisera obtained permitted identification of the proteins encoded by both v-myb and chicken c-myb. The molecular weights of the products of v-myb and c-myb (45,000 and 75,000 respectively) indicate that the v-myb protein is an appreciably truncated version of the c-myb protein.

Transformation suppressor protein Pdcd4 interferes with JNK-mediated phosphorylation of c-Jun and recruitment of the coactivator p300 by c-Jun

The transformation suppressor gene Pdcd4 (programmed cell death gene 4) inhibits the tumor-promoter mediated transformation of mouse keratinocytes and has recently been implicated as a potential tumor suppressor gene in the development of human lung cancer. Biochemical analysis has suggested that the Pdcd4 protein is involved in protein translation as well as in nuclear events. Recent work has shown that Pdcd4 suppresses the transactivation of AP-1 responsive promoters by c-Jun, suggesting that the transformation-suppressor activity of Pdcd4 might be due, at least in part, to the inhibition of c-Jun activity. Here, we have addressed how Pdcd4 inhibits c-Jun. We show that Pdcd4 interferes with the phosphorylation of c-Jun by Jun N-terminal kinase (JNK). The inhibition of c-Jun phosphorylation by Pdcd4 appears not to be due to a general suppression of JNK activity, our data rather suggest that Pdcd4 interacts with c-Jun and thereby blocks phosphorylation of c-Jun. In addition to affecting c-Jun phosphorylation, Pdcd4 blocks the recruitment of the coactivator p300 by c-Jun. Taken together, our results strongly suggest that Pdcd4 is directly involved in the regulation of c-Jun activity.

Mouse A-myb encodes a trans-activator and is expressed in mitotically active cells of the developing central nervous system, adult testis and B lymphocytes.

C‐myb encodes a transcriptional activator that is essential for the development of the hematopoietic system but appears to lack major roles in non‐hematopoietic cells. The identification of two conserved myb‐related genes, designated A‐myb and B‐myb, has raised the possibility that these genes are functional equivalents of c‐myb in non‐hematopoietic cells. Here, we report the isolation and preliminary characterization of the mouse A‐myb gene. Mouse A‐myb maps to the proximal region of chromosome 1 and encodes a transcriptional activator with properties similar to those of the c‐myb and v‐myb proteins. During embryo‐genesis A‐myb is predominantly expressed in several regions of the developing central nervous system (CNS) and the urogenital ridge. Expression in the CNS is confined to the neural tube, the hindbrain, the neural retina and the olfactory epithelium, and coincides with the presence of proliferating immature neuronal precursor cells. In the adult mouse, A‐myb is expressed during the early stages of sperm cell differentiation and in B lymphocytes located in germinal centers of the spleen. Taken together, these results suggest a role for A‐myb in the proliferation and/or differentiation of neurogenic, spermatogenic and B‐lymphoid cells.

Phosphorylation and activation of B-Myb by cyclin A–Cdk2

BACKGROUND Cyclins and their catalytic partners, the cyclin-dependent kinases (Cdks), function as key regulators of the eukaryotic cell cycle. Specific cyclin-Cdk complexes are active at successive stages during the cell cycle and control cell-cycle progression by phosphorylating specific target proteins, most of which have not yet been identified. B-Myb, a conserved member of the Myb oncoprotein family, is a sequence-specific DNA-binding protein expressed in virtually all proliferating mammalian cells. Increasing evidence suggests that B-Myb plays an important role during the late G1 and early S phases of the cell cycle. In this study, we have examined the regulation of B-Myb activity by cyclin-Cdks. RESULTS We found that the transcriptional transactivation potential of B-Myb was repressed by a regulatory domain located at the carboxyl terminus of the protein. Coexpression of B-Myb and cyclin A relieved this repression by phosphorylation of B-Myb in its carboxy-terminal region. Tryptic phosphopeptide mapping revealed that endogenous B-Myb was phosphorylated in cells undergoing S phase. CONCLUSIONS This work provides evidence for a link between the Myb oncoprotein family and the cell-cycle machinery. We have shown that the carboxyl terminus of B-Myb acts as a cell-cycle sensor that regulates the transactivation function of B-Myb. Moreover, our studies have identified B-Myb as a target of cyclin A-Cdk2 and have indicated that B-Myb activity is regulated by phosphorylation mediated by cyclin A-Cdk2.

Interaction of C/EBPbeta and v-Myb is required for synergistic activation of the mim-1 gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which activates the myelomonocyte-specific mim-1 gene, a natural myb target gene, by cooperating with members of the C/EBP transcription factor family. The finding that v-Myb, together with C/EBP, is sufficient to activate the mim-1 gene in heterologous cell types has implicated Myb and C/EBP as a bipartite molecular switch, which regulates the expression of myelomonocyte-specific genes. To understand the relationship between v-Myb and C/EBP in more detail, we have examined the molecular basis of the activation of the mim-1 promoter by v-Myb and C/EBPbeta, a member of the C/EBP transcription factor family highly expressed in myelomonocytic cells. We have identified a composite Myb and C/EBP response element which mediates synergistic activation of the mim-1 promoter by both factors and consists of closely spaced Myb- and C/EBP-binding sites. In vitro and in vivo protein-binding studies indicate that v-Myb and C/EBPbeta interact with each other via their DNA-binding domains. We show that this interaction is essential for the synergistic activation of the mim-1 promoter by v-Myb and C/EBPbeta. Our work therefore identifies C/EBPbeta as an interaction partner of v-Myb involved in myelomonocyte gene expression.