Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb.

The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets.

The -6.1-kilobase chicken lysozyme enhancer is a multifactorial complex containing several cell-type-specific elements.

In the chromatin domain of the chicken lysozyme gene of myeloid and oviduct cells, which both have the potential to activate the gene, a developmentally stable DNase I-hypersensitive site is formed around 6.1 kb upstream of the gene. This implies that this DNA region, which has previously been demonstrated to function as a transcriptional enhancer element in myeloid cells, is intimately involved in the cell-type-specific activation of the lysozyme gene locus. Deletion analysis identifies a 157-bp minimal fragment that confers the same promacrophage-specific enhancer activity as the originally described 562-bp -6.1-kb enhancer fragment. By introducing specific point mutations, we demonstrate in transient gene transfer experiments that the minimal fragment consists of at least six adjacent elements, each substantially contributing to enhancer function. The compact multifactorial enhancer complex includes a nuclear factor I (NF-I)/TGGCA binding site, homologies to AP1, and octanucleotide or enhancer core consensus motifs. Point mutation of the NF-I binding site results in the loss of NF-I binding in vitro and enhancer activity in vivo after gene transfer. Surprisingly, four overlapping oligonucleotides, each consisting of at least two elements of the -6.1-kb enhancer, confer myeloid-cell-specific enhancer activity. We found several myeloid-cell-specific DNA-binding proteins interacting with the -6.1-kb enhancer, a result consistent with that described above. Therefore, we suggest that more than a single trans-acting factor mediates the cell type specificity of the -6.1-kb enhancer.

Comparison of in vivo translation rates and messenger RNA levels of alpha2U-globulin in rat liver and Morris hepatoma 5123D.

The synthesis of the male rat hepatic protein alpha2U-globulin has been examined in Morris hepatoma 5123D and male host liver using pulse incorporation of labeled amino acids in vivo, followed by immunoprecipitation of the newly synthesized alpha2U-globulin from the soluble protein fraction of liver and hepatoma tissue. It was found that no alpha2U-globulin synthesizes alpha2U-globulin at a normal level (0.9 to 1.0% of total hepatic protein synthesis). A variety of liver-derived cell culture lines also did not have alpha2U-globulin synthesis. The level of the specific mRNA coding for alpha2U-globulin can be quantitated using in vitro translation of polyadenylate-containing RNA in a Krebs II ascites cell-free translational system, followed by immunoprecipitation of the alpha2U-globulin synthesized in vitro. Using this technique, it was found that host liver contained alpha2U-globulin mRNA at normal levels, whereas hepatoma tissue contained no detectable mRNA coding for this protein. Thus, alpha2U-globulin synthesis is deleted in the minimal-deviation hepatoma 5123D as a consequence of the inability of that tissue to produce functional mRNA coding for alpha2U-globulin. The implications for the regulation of gene expression in malignant cells are discussed.

Primary structure of the chicken c-mil protein:identification of domains shared with or absent from the retroviral v-mil protein.

The complete primary structure of the protein product of the proto-oncogene c-mil was deduced from the nucleotide sequence of chicken c-mil cDNA clones. The c-mil protein contains 647 amino acid residues and has a calculated molecular weight of 73,132. Based on sequence comparisons with proteins of known or presumed biochemical function, two domains were recognized on the c-mil protein. In the carboxyl-terminal half of the protein, a 250-amino acid segment displays significant homology to the protein kinase domains of the src oncogene protein or of protein kinase C. In the amino-terminal half, a cysteine-rich segment (Cys-X2-Cys-X9-Cys-X2-Cys-X7-Cys-X7-Cys) of the c-mil protein shares significant homology with two similar repetitive domains of protein kinase C. Of the two structural and presumably functional domains of the c-mil protein, only the kinase domain is contained within the carboxyl-terminal 379-amino acid polypeptide encoded by the transduced v-mil allele of avian oncogenic retrovirus MH2. Hence, truncation of the 5' coding region in the course of the transduction and the resulting lack of the authentic amino-terminal domain in the protein product of the transduced allele may be a critical event in changing mil function from physiologic to oncogenic.

Rat antibodies as probes for the characterization of progesterone receptor A and B proteins from laying hen oviduct cytosol.

The chicken oviduct contains two different hormone binding forms of the progesterone receptor, A and B. We have prepared rat antisera against both forms of the receptor partially purified from laying hen oviduct. The anti-progesterone receptor A antiserum reacts with both receptor forms on Western blots, while the anti-progesterone receptor B antiserum reacts mainly with the B form. Both antisera also react with the native progesterone receptor proteins as shown by sedimentation analysis of the antibody-receptor complexes. Receptors A and B are recognized on Western blots of total protein from dissolved tissue, indicating that both forms are likely to be physiological components. Epitope mapping experiments show that immunogenicity of both receptor molecules is restricted to structurally related protein domains of 28 kDa in receptor A and of 52 kDa in receptor B.

The genes for transcription factor nuclear factor I give rise to corresponding splice variants between vertebrate species.

Nuclear Factor I (NFI) proteins are DNA binding proteins functioning as transcription and replication factors. As part of a study of the diversity of the Nuclear Factor I protein family, we isolated and sequenced seven NFI cDNA clones from a chicken promacrophage library. Five of these clones are derived from the NFI-A gene, the other two from the NFI-C gene. Comparison of the deduced chicken NFI amino acid sequences with mammalian NFI sequences reveals that there are corresponding vertebrate isoforms, indicating a strong conservation of NFI structure during evolution. This finding leads to the prediction that more mammalian isoforms will be discovered corresponding to the chicken NFI variants reported here.

Prerequisites for tissue specific and position independent expression of a gene locus in transgenic mice.

The elucidation of general parameters influencing the transcriptional activation of gene loci at distinct stages of development is an essential prerequisite for a reproducibly successful gene transfer in both gene therapy protocols and biotechnology. Up to now research has focused mostly on the identification and characterization of individual cis-regulatory elements by transient transfection and in vitro assays. However, the most relevant assay system to test gene constructs designed for gene therapy protocols is the transgenic animal. In such an experimental system exogenous genes are usually integrated randomly in the chromatin. For gene constructs not fulfilling the requirements for correct gene locus activation this can lead to genomic position effects on gene expression. The consequences are highly variable expression levels and a disturbance of temporal and spatial expression patterns. Hence it is important to examine how cis-elements function in a chromatin context, and how they cooperate during the developmentally controlled activation of an entire gene locus. One among a few gene loci which are sufficiently characterized to enable such investigations is the chicken lysozyme locus. This review summarizes recent results aimed at identifying the necessary prerequisites for a reproducibly correct expression of the lysozyme locus in transgenic mice and the implications of our findings for gene transfer. The complete lysozyme locus is expressed independent of the chromosomal position and at a high level in macrophages of transgenic mice. Correct transgene regulation requires the cooperation of all cis-regulatory elements. Chromatin of the lysozymes locus in both the active and the inactive state is highly structured. Each cis-regulatory element on the chicken lysozyme locus is organized in its own unique chromatin environment, with nucleosomes specifically placed on specific sequences. The transcriptional activation of the lysozyme locus is accompanied by extensive rearrangements of its chromatin structure, which are disturbed when the transgenes are subjects to genomic position effects. Based on these results, we propose that a complete locus is resistant to genomic position effects, and that a distinct chromatin architecture of a gene locus is required for its correct activation.

Different macrophage populations develop from embryonic/fetal and adult hematopoietic tissues.

Immunohistochemical and ultrastructural studies have indicated the existence of a distinct "fetal macrophage" type, differing from monocyte-derived macrophages. In order to characterize macrophages of different ontogenetic origins on the molecular level, we examined their surface-marker and marker-gene expression patterns. We found that macrophages derived from chicken embryos express the lysozyme gene at significantly lower levels than macrophages derived from adult chicken. The same was observed when expression of the chicken lysozyme gene was analyzed in transgenic mice. In three independent mouse lines, mature macrophages derived from embryonic or fetal hematopoietic tissues expressed the transgene at drastically lower levels than macrophages derived from the bone marrow, spleen, or peritoneal cavity of adult mice. Macrophages obtained by in vitro differentiation of mouse embryonic stem cells (a process resembling early embryonic hematopoiesis) displayed the embryo-specific low transgene expression level. Experiments determining the developmental potential of myeloid precursors in culture and immunophenotypic analyses revealed differences between embryo-derived and adult myeloid progenitor populations. In summary, our results provide further evidence for the existence of dissimilar embryonic/fetal and adult macrophage types and describe the first molecular marker for their distinction.

Regulation of the chicken lysozyme locus in transgenic mice.

The chicken lysozyme locus is transcriptionally activated during macrophage differentiation. Each cis-regulatory element has its unique activation stage during cell differentiation, whereby maximal transcriptional activity of the gene is only observed when all cis-elements are active. The complete chicken lysozyme locus is expressed position independently and at a high level in macrophages of transgenic mice. For correct transgene regulation, the cooperation of all cis-regulatory elements is required. These cis-regulatory elements specify the mode of regulation and we observe the same expression pattern of the transgene in the mouse and the endogenous gene in chicken macrophages. This indicates that the transcription factors responsible for chicken lysozyme regulation are highly conserved in evolution. The endogenous mouse lysozyme gene is regulated differently. The chromatin of the lysozyme locus is highly structured in the transcriptionally active, as well as in the inactive state. The transcriptional activation of the lysozyme locus is accompanied by extensive chromatin rearrangements, which are disturbed when one essential cis-regulatory element is deleted and the transgenes are subjects to genomic position effects. Based on these results, we propose that a distinct chromatin architecture of a gene locus is required for its correct activation.

Transcription factor nuclear factor I proteins form stable homo- and heterodimers.

Nuclear factor I (NFI) proteins constitute a large family of eukaryotic DNA binding proteins. They are involved in viral and cellular aspects of transcriptional regulation and they are capable of stimulating adenovirus initiation of replication. Using in vitro translated NFI proteins encoded by four different chicken NFI genes, we have detected homodimers as well as heterodimers for all combinations tested. The formation of heterodimers was critically dependent on cotranslation, indicating stable dimer formation in the absence of DNA. The unrestricted heterodimerization of NFI proteins adds, beside gene diversity and alternative splicing, another level of diversity to this protein family.

Identification of cis-acting elements as DNase I hypersensitive sites in lysozyme gene chromatin.

DNase I hypersensitive sites in chromatin of eukaryotic cells mark the positions of multifactorial cis-acting elements. Mapping DH sites by indirect end labeling is a convenient procedure used for identifying regulatory elements within extensive regions of chromatin and for gaining information about their functional specificity as well as their fine structure.

Differential activity of the -2.7 kb chicken lysozyme enhancer in macrophages of different ontogenic origins is regulated by C/EBP and PU.1 transcription factors.

Expression of the chicken lysozyme gene is upregulated during macrophage maturation. Recently, an additional regulatory feature was discovered: the gene is differentially expressed in macrophages of embryonic/fetal and adult origin. The lysozyme gene is only weakly expressed in mature embryo-derived macrophages, whereas there is a high level of expression in macrophages derived from adult animals. This finding provided a molecular tool to investigate the heretofore ill-defined differences between embryonic/fetal- and adult-type macrophages. We showed that the low expression in the embryo is associated with reduced activity of the myeloid-specific -2.7 kb lysozyme enhancer. Our protein-binding analyses and transfection studies demonstrated that this enhancer, in order to be fully active in activated macrophages, requires the combined action of C/EBPs, PU.1, and a third, as yet unidentified, protein binding to an AP-1-like site. Of these three, PU.1 and C/EBPs display significantly reduced nuclear DNA-binding activities in embryo-derived macrophages compared with adult-type cells. These results point to different roles of C/EBPs and PU.1 in embryonic/fetal and adult myelopoiesis.

An in vitro differentiation system for the examination of transgene activation in mouse macrophages.

In an effort to study basic principles of marker gene activation during myeloid lineage development, we established an in vitro differentiation system for macrophages based on mouse embryonic stem (ES) cells. Under the influence of defined cytokines, ES cells gave rise to a cell population consisting predominantly of macrophages. We could show, that expression of the mouse lysozyme M gene is a faithful internal standard for indicating the proportion of macrophage cells in the differentiation culture. This controlled in vitro differentiation system can be used for quantitative studies on transgene activation. Undifferentiated ES cells were stably transfected with a construct carrying the chicken lysozyme gene locus, which had been shown previously to express lysozyme RNA cell type specifically and position independently in macrophages of transgenic mice. In undifferentiated transfected ES cell clones, the transgene was consistently inactive. Upon in vitro differentiation, the transgene was expressed exclusively in macrophages and its level of activity was independent of the chromosomal site of integration. The in vitro cell differentiation system presented here will be useful to study the cis- and trans-regulatory requirements of myeloid-specific gene activation and the influence of hematopoietic regulators on myelopoiesis through their effect on transfected marker gene expression.

Dynamic changes in the chromatin of the chicken lysozyme gene domain during differentiation of multipotent progenitors to macrophages.

The chicken lysozyme locus is regulated in oviduct and macrophages by a complex set of well-characterized cis-regulatory DNA elements. We determined the DNase I hypersensitive chromatin site pattern of the chromatin of the lysozyme locus in retrovirally transformed cell lines representing different stages of myelomonocytic cell differentiation. In the transformed multipotent progenitor stage and in erythroblasts, only a DNase I hypersensitive chromatin site at a silencer element located -2.4 kb upstream of the transcriptional start site is present. At the myeloblast stage DNase I hypersensitive chromatin sites are formed both at the distal enhancer located at -6.1 kb and at the promoter. Later in differentiation, at the monocytic stage, a second DNase I hypersensitive chromatin site appears at the medial enhancer located at -2.7 kb. Parallel with DNase I hypersensitive chromatin site formation at the medial enhancer, the DNase I hypersensitive chromatin site at the silencer element disappears. These chromatin rearrangements correlate with the mRNA expression of the gene that is undetectable in multipotent progenitors and maximal in a lipopolysaccharide-stimulated monocyte cell line. Our results show that the chromatin structure and the transcriptional activity of the gene are tightly coupled during commitment and maturation of the myelomonocytic lineage.

The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells. It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA. To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli. Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity. Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein. This region has been highly conserved between myb genes of different species. Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.

Chicken liver TGGCA protein purified by preparative mobility shift electrophoresis (PMSE) shows a 36.8 to 29.8 kd microheterogeneity.

The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity.

Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

Unlike in other mammalian species, the major whey protein in mouse is not alpha-lactalbumin, but a cysteine rich, acidic protein with a molecular weight of 14.0 kDa. We have deduced the amino acid sequence of this mouse acidic of whey protein from the nucleotide sequence of cloned cDNA. The positions of the half cysteines suggest that mouse whey acidic protein (WAP) is a two domain protein, very similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protein neurophysin.

Characterization and cloning of the mRNAs specific for the lactating mouse mammary gland.

We have characterized and cloned the lactation-specific mRNAs of mouse mammary glands. The group of eight milk-protein-specific mRNAs were identified (a) by size and antigenic properties of their translation products in vitro and (b) by characterization of their respective cDNA clones. Two alpha-caseins (43 kDa and 39 kDa) are encoded by mRNAs of 1600 nucleotides and two beta-caseins (26 kDa) are encoded by mRNAs of 1450 nucleotides in length. Three smaller caseins, gamma-casein (23.7 kDa), delta-casein (21 kDa) and epsilon-casein (14.5 kDa) are synthesized by mRNAs of 880, 1150 and 860 nucleotides. Beside these casein mRNAs a mammary specific 620 nucleotide mRNA codes for a novel acidic whey protein (13.7 kDa). cDNA clones corresponding to the mRNAs for the lactation-specific proteins have been isolated from a mammary-specific cDNA library. Cloned alpha-casein cDNA hybridizes to both alpha-casein specific mRNAs and cloned beta-casein cDNAs hybridize with both beta-casein specific mRNAs. By RNA blot analysis we show that the cloned cDNAs for mouse alpha-casein, beta-casein, gamma-casein and epsilon-casein and the acidic whey protein cross-hybridize with mRNAs of the rat, demonstrating partial sequence homology between the corresponding mRNAs of those species.