Biomedical subjects

K H Klempnauer

Publications and source records attributed to K H Klempnauer.

At least 37 records · Page 2Linked to original sources

PubMed1996

A novel function for Myc: inhibition of C/EBP-dependent gene activation.

We have investigated the effect of the v-Myc oncoprotein on gene expression in myelomonocytic cells. We find that v-Myc dramatically down-regulates the expression of myelomonocytic-specific genes, such as the chicken mim-1 and lysozyme genes, both of which are known targets for C/EBP transcription factors. We present evidence that Myc downregulates these genes by inhibiting the function of C/EBP transcription factors. Detailed examination of the inhibitory mechanism shows that amino-terminal sequences of v-Myc, but not its DNA-binding domain, are required for the suppression of C/EBP-dependent transactivation. Our findings identify a new function for Myc and reveal a novel mechanism by which Myc affects the expression of other genes.

Acetyltransferases↗

PubMed1996

Expression of B-Myb during mouse embryogenesis.

B-myb is a member of the myb family of nuclear sequence-specific DNA-binding proteins which has been highly conserved among vertebrates. B-myb has been implicated in the control of cell proliferation, particularly at the G1/S transition of the cell cycle. So far, most of the work on B-myb has been performed in immortalized cell lines. Since these cells might show aberrant behavior of genes involved in proliferation control we have begun to investigate the role of B-myb in normal cells. As a first step, we have studied the expression of B-myb during mouse development. Here, we show the B-myb is expressed at similar levels during all stages of embryogenesis. In situ hybridization reveals a tight linkage between B-myb expression and proliferative activity (as assessed by the expression of the S-phase specific histone H4 gene) in most tissues and throughout embryonic development. However, B-myb and histone H4 expression are uncoupled during spermatogenesis in the adult mouse. Histone H4 is expressed at high levels in the early spermatogenic progenitor cells but not in successive stages of sperm cell development. By contrast, the highest levels of B-myb expression are found during the intermediate stages of spermatogenesis. Furthermore, we have found that B-myb mRNA isolated from the testis differs in size from that of other tissues. The data presented here strongly support the notion that B-myb plays a general role during proliferation of most cells. Furthermore, our results raise the possibility that the function of B-myb in cells undergoing meiosis may be different from its role in cells dividing mitotically.

Animals↗

PubMed1996

Structure of the B-Myb DNA-binding domain in solution and evidence for multiple conformations in the region of repeat-2 involved in DNA binding: implications for sequence-specific DNA binding by Myb proteins.

A range of double and triple resonance heteronuclear NMR has been used to obtain nearly complete sequence-specific 15N, 13C and 1H resonance assignments for a 110-residue protein corresponding to the B-Myb DNA-binding domain (B-MybR2R3) and to determine its secondary structure in solution. The protein was found to contain two stable helices in repeat-2 (R2) and three in repeat-3 (R3), involving residues K12-K24 (R2-1), W30-H36 (R2-2), E64-V76 (R3-1), W81-L87 (R3-2) and D93-K105 (R3-3). In addition, the chemical shift and nuclear Overhauser effect data suggest that amino acids Q44-W49 near the C-terminus of R2 form an unstable or nascent helix, which could be stabilised on binding to a specific DNA target site. The two N-terminal helices in R2 and R3 occupy essentially identical positions in the two domains, consistent with the high level of sequence similarity between these regions. In contrast, the C-terminal region forming the third helix in R3 shows low sequence similarity with R2, accounting for the differences in secondary structure. In the case of B-MybR2R3, there is a clear chemical shift and line-broadening evidence for the existence of multiple conformations in the C-terminal region of R2, which is believed to form one half of the DNA-binding site. We propose that conformational instability of part of the DNA-binding motif is a way of increasing the specificity of Myb proteins for a relatively short (6-bp) DNA target site by reducing their affinity for non-specific DNA sequences compared to specific sites.

Amino Acid Sequence↗

PubMed1996

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.

Acetyltransferases↗

PubMed1995

Differential splicing of the mouse B-myb gene.

The myb gene family consists of three members, the c-myb proto-oncogene and two myb-related genes (A-myb and B-myb), all of which encode nuclear DNA-binding proteins. Unlike c-myb, which plays a critical role in hematopoietic cells, B-myb is expressed in a large spectrum of hematopoietic as well as non-hematopoietic cells and has been implicated in the control of cell proliferation. The isolation of B-myb cDNA clones from several species has shown that B-myb shares limited homology to the so-called exon 9A of the c-myb gene. This exon is involved in differential splicing as only a subfraction of c-myb mRNA contains exon 9A sequences. The presence in the B-myb cDNA of a sequence related to the exon 9A of c-myb has prompted us to investigate whether B-myb mRNA is also spliced differentially. We here show that B-myb mRNAs containing or lacking exon 9A related sequences are present in many cell types. In contrast to c-myb, where RNA containing the exon 9A constitutes only a minor mRNA fraction, B-myb RNA containing the exon 9A related sequences is the major mRNA form. The proteins encoded by the two B-myb mRNA species are unable to activate promoters to which they bind. Curiously, both B-myb proteins differ in their ability to activate the HSP70 promoter by a myb binding-site independent mechanism; B-myb protein containing exon 9A related aminoacid sequences activates the HSP70 promoter much more potently than the B-myb protein which lacks these sequences. Our results suggest that differential splicing may be a general feature of the members of the myb family and provide first evidence for functional differences of the splice variants.

Amino Acid Sequence↗

PubMed1995

Expression of mouse c-myb during embryonic development.

Three members of the myb gene family, designated as A-myb, B-myb, and c-myb, have been described in many vertebrates. A large body of evidence indicates that the c-myb gene is essential for the development of most hematopoietic lineages. By contrast, the functions of A-myb and B-myb are less well understood. To further explore the relationship between the different myb family members we have compared the expression of A-myb and c-myb during mouse embryogenesis by Northern blotting and by in situ hybridization. In accordance with the important role of c-myb in the hematopoietic system, we detect high levels of c-myb expression in hematopoietic organs such as the fetal liver and the thymus. Surprisingly, we find that high levels of c-myb expression are not restricted to hematopoietic cells. We show that c-myb is strongly expressed in the neural retina and in epithelia of the respiratory tract. The side-by side analysis of c-myb and A-myb expression clearly shows that both genes are expressed in different, but overlapping sets of tissues. Our results suggest that the function of c-myb may not be restricted to hematopoietic cells.

Animals↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1994

The chicken A-myb protein is a transcriptional activator.

c-myb encodes a trans-activator that is essential for the proliferation of most hematopoietic precursor cells but lacks a major role in non-hematopoietic cells. The recent identification of two myb-related genes (A-myb and B-myb) in several vertebrate species has raised the possibility that these genes perform c-myb like functions in non-hematopoietic cells. Here, we report the isolation and preliminary characterization of the chicken A-myb gene. Like other members of the myb family, the A-myb protein contains an N-terminally located repeat domain that is highly homologous to the DNA-binding domain of the c-myb protein. Unlike c-myb, A-myb is expressed in fibroblasts, suggesting that it functions in non-hematopoietic cells. We demonstrate that A-myb transactivates myb-responsive reporter genes. Furthermore, when stably expressed in a chicken macrophage cell line. A-myb causes activation of several myb-inducible endogenous genes. These findings show that A-myb encodes a transactivating member of the myb family whose functional properties resemble those of the c-myb and v-myb proteins. Thus, our results support the idea that A-myb is a functional equivalent of c-myb.

Acetyltransferases↗

PubMed1993

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.

Acetyltransferases↗

PubMed1993

Methylation-sensitive DNA binding by v-myb and c-myb proteins.

The retroviral oncogene v-myb and its cellular homolog c-myb encode nuclear DNA-binding phosphoproteins (v-MYB and c-MYB) that function as transcriptional regulators. v-MYB and c-MYB recognize a nucleotide sequence motif, PyAACG/TG, that is present in the promoter region of the myb-inducible mim-1 gene and is required for regulation of mim-1 expression by v-MYB and c-MYB. Since the myb-binding motif contains a CpG dinucleotide that constitutes a potential target for methylation, we have investigated whether recognition of the binding site by myb proteins is sensitive to CpG methylation of the binding motif. The results presented here demonstrate that bacterially expressed v-MYB as well as authentic v-MYB and c-MYB bind to the myb binding site in a methylation-sensitive manner. Our observations raise the interesting possibility that myb function can be regulated by methylation of myb binding sites.

Animals↗

PubMed1993

TATA-box dependent trans-activation of the human HSP70 promoter by Myb proteins.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA-binding phosphoproteins that function as transcriptional regulators. Previous studies have demonstrated the existence of two distinct transactivation mechanisms for Myb-inducible promoters. One of these mechanisms, exemplified by the chicken mim-1 promoter, involves specific binding of Myb protein to Myb binding sites located upstream of the transcriptional start site. A second mechanism, exemplified by the human HSP70 promoter, does not require the presence of specific Myb binding sites. Here, we have more closely investigated transactivation of the human HSP70 promoter by Myb proteins. Mutational analysis of the human HSP70 promoter failed to show a correlation between Myb-inducibility and the presence of a particular upstream transcription factor binding site, but instead showed that Myb-inducibility was dependent on the presence of a particular type of TATA-box. This suggests that the TATA-box is involved in transactivation by v-Myb and that only a subset of TATA-boxes confers Myb-inducibility. Activation of the HSP70 promoter is dependent on the presence of most of the carboxy terminal amino acid sequences of v-Myb, including the previously identified acidic transactivation domain and a putative leucine-zipper region. Finally, we show that the HSP70 promoter is activated in a Myb binding site independent fashion not only by v-Myb, but also by chicken c-Myb and chicken B-Myb. These observations raise the possibility that the ability to transactivate in a Myb binding-site independent manner is a common property among Myb proteins. Since chicken B-Myb has been shown previously to be unable to transactivate a Myb binding-site containing promoter, such as the mim-1 promoter (Foos et al., 1992), our results also show that both modes of transactivation are independent of each other.

Animals↗

PubMed1992

Functional antagonism between members of the myb family: B-myb inhibits v-myb-induced gene activation.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that control the expression of certain target genes in immature hematopoietic cells. Here, we report the isolation of a myb-related chicken gene, chicken B-myb. We show that expression of B-myb, unlike that of c-myb, is not restricted to hematopoietic cells, suggesting that B-myb functions in a broader spectrum of cell types than c-myb. We have identified the authentic chicken B-myb protein as a nuclear protein of approximately 110 kDa. We show that the B-myb protein specifically recognizes v-myb binding sites in vitro and that binding is mediated by an N-terminally located DNA binding domain. Although B-myb protein recognizes myb binding sites, B-myb fails to transactivate several myb-responsive gene constructs as well as the endogenous myb-responsive gene mim-1. Instead, we find that B-myb represses v-myb- and c-myb-mediated activation of the mim-1 gene, most likely by competing with other myb proteins for binding sites. Our results raise the possibility that B-myb is an inhibitory member of the myb family.

Amino Acid Sequence↗

PubMed1991

Sequence-specific DNA binding by Myc proteins.

Myc proteins have a tripartite carboxyl-terminal domain containing specific amino acid sequence motifs: a basic motif, a helix-loop-helix motif, and a leucine heptad repeat. Similar sequence motifs have been identified in several eukaryotic transcription factors and were shown to facilitate protein-DNA and protein-protein interactions. By using recombinant v-Myc proteins obtained by bacterial expression of full-length or partially deleted avian v-myc alleles, the functional relevance of these sequence motifs for Myc protein oligomerization and for DNA binding was investigated. All recombinant v-Myc proteins that have retained the carboxyl-terminal domain dimerize and specifically bind to double-stranded DNA containing the palindromic core sequence CACGTG. This and a closely related DNA sequence element have been defined previously as part of the binding sites for human transcription factors USF and TFE3, which specifically bind to the adenovirus major late promoter or the muE3 motif within the immunoglobulin heavy-chain enhancer, respectively. It is shown that a 61-amino-acid peptide sequence containing only the bipartite basic motif/helix-loop-helix domain of Myc is necessary and sufficient for dimerization and sequence-specific DNA binding of v-Myc recombinant proteins.

Base Sequence↗

PubMed1991

Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb-responsive genes during myeloid differentiation. To identify such myb-regulated genes, and to explore the mechanisms by which v-myb affects their expression, we have established a conditional expression system for v-myb. We have converted the v-myb protein to an estrogen-inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell-line causes estrogen-dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen-dependent v-myb expression system to identify a novel v-myb regulated gene.

Amino Acid Sequence↗

PubMed1990

Characterization of the v-myb DNA binding domain.

The transforming protein encoded by the v-myb oncogene is a sequence-specific DNA-binding protein that is thought to be involved in the regulation of gene expression. The N-terminal region of the v-myb protein is composed of two highly conserved tandem repeat sequences of unknown function. It has been speculated that the N-terminal v-myb repeats might be crucial for DNA-binding, since N-terminal deletions destroy the DNA-binding activity of the v-myb protein. Here, we have studied the v-myb DNA-binding domain in more detail. Our results show that the N-terminal region of the v-myb protein is sufficient for specific DNA-binding. Dissection of this region suggests that both repeats are required for DNA-binding, but that both repeats play different roles in v-myb protein DNA interaction. We also show that the myb repeats of a drosophila melanogaster homolog of c-myb function as sequence-specific DNA-binding domain. Our results support the view that specific sequence-recognition, mediated by the conserved myb repeats, is a general feature of myb-related proteins.

Animals↗

PubMed1989

Activation of transcription by v-myb: evidence for two different mechanisms.

The retroviral oncogene v-myb encodes a nuclear, sequence-specific DNA-binding protein. To investigate the possibility that v-myb encodes a transcriptional regulator, we used a transient cotransfection assay to explore the potential of v-myb to influence the expression of other genes. We found that expression of a chicken lysozyme promoter/CAT gene construct was activated by v-myb in the presence of myb-specific binding sites. Action was not observed with a truncated v-myb protein lacking its DNA-binding domain. We also observed that expression of a hybrid human HSP70 promoter/CAT gene, lacking myb-specific binding sites, was activated by v-myb. However, in this case, the truncated v-myb protein, which lacked its DNA-binding domain, also activated HSP70/CAT expression, indicating that trans-activation of this gene construct was independent of the sequence-specific DNA-binding activity of the v-myb protein. These observations suggest that v-myb encodes a trans-activator and that activation of gene expression occurs by two different mechanisms, one of which involves specific binding of v-myb protein to the regulated gene.

Animals↗

PubMed1989

Association of v-myc protein with chromatin.

The subnuclear distribution of proteins encoded by v-myb and v-myc was analysed in a cell-line of AMV-transformed chicken myeloblasts superinfected by the myc-containing retrovirus MC29. p45v-myb and p110gag-myc, co-expressed in these cells, were released in similar fashion when nuclei were treated with salt or DNAase. Analysis of nucleoprotein complexes extracted from nuclease-treated nuclei shows that p45v-myb and p110gag-myc are associated with a chromatin fraction of enhanced nuclease sensitivity. v-myb and v-myc proteins thus share the same subnuclear location and apparently interact directly with the cellular DNA.

Animals↗

PubMed1988

Viral myb oncogene encodes a sequence-specific DNA-binding activity.

The retroviral oncogene v-myb and its cellular progenitor c-myb encode nuclear DNA-binding proteins. Myb genes have been identified in a broad range of species, including vertebrates, the fruit fly Drosophila melanogaster and the plant Zea mays. The localization of the DNA-binding domain of the v-MYB protein to the highly conserved amino-terminal region suggests that the MYB/DNA interaction is important for MYB function. We show here that v-MYB specifically recognizes the nucleotide sequence pyAACG/TG. So like other nuclear transforming proteins, v-MYB seems to be a member of the class of sequence-specific DNA-binding factors presumably involved in gene regulation.

Base Sequence↗