Down-regulation of Bgp1(a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus 3 infection.

Together with the evidence that the reduced virus growth and the antiviral state induced by interferon (IFN)-gamma, occurring only in macrophages from resistant animals, correlated with the decrease of MHV3 binding to macrophage membrane proteins, we show here the expression of cellular and viral genes in resistant (A/J) and susceptible (BALB/c) mouse macrophages after IFN-gamma activation/infection. The expression of interferon response gene 47 and interferon regulatory factor 1 genes takes place after IFN-gamma activation in both macrophages, indicating their activation. The expression of the biliary glycoprotein 1(a) (Bgp1(a), the main virus receptor) decreased only in IFN-gamma-activated A/J mouse macrophages, in contrast to the expression of the Bgp2 (alternative receptor), which was not influenced by IFN-gamma activation. The synthesis of both viral mRNA and virus particles was delayed only in IFN-gamma-activated A/J mouse macrophages compared with susceptible BALB/c macrophages. Besides the evidence that IFN-gamma may modulate the expression of the Bgp1(a) isoform of carcinoembryonic antigen family, these data show that IFN-gamma, which induces resistance against MHV3 infection, may be involved in the down-regulation of the main viral receptor expression, a key step forward in our understanding of the molecular basis of resistance against virus infection.

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.

DNA binding and transcriptional activation by the Ski oncoprotein mediated by interaction with NFI.

The Ski oncoprotein has been found to bind non-specifically to DNA in association with unindentified nuclear factors. In addition, Ski has been shown to activate transcription of muscle-specific and viral promoters/enhancers. The present study was undertaken to identify Ski's DNA binding and transcriptional activation partners by identifying specific DNA binding sites. We used nuclear extracts from a v-Ski-transduced mouse L-cell line and selected Ski-bound sequences from a pool of degenerate oligonucleotides with anti-Ski monoclonal antibodies. Two sequences were identified by this technique. The first (TGGC/ANNNNNT/GCCAA) is the previously identified binding site of the nuclear factor I (NFI) family of transcription factors. The second (TCCCNNGGGA) is the binding site of Olf-1/EBF. By electophoretic mobility shift assays we find that Ski is a component of one or more NFI complexes but we fail to detect Ski in Olf-1/EBF complexes. We show that Ski binds NFI proteins and activates transcription of NFI reporters, but only in the presence of NFI. We also find that homodimerization of Ski is essential for co-activation with NFI. However, the C-terminal dimerization domain of c-Ski, which is missing in v-Ski, can be substituted by the leucine zipper domain of GCN4.

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.

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.

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.

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.

Chromosomal localization of the four genes (NFIA, B, C, and X) for the human transcription factor nuclear factor I by FISH.

Nuclear Factor I (NFI) proteins constitute a family of dimeric DNA-binding proteins with very similar, possibly identical, DNA-binding specificity. They function as cellular transcription factors and as replication factors for adenovirus DNA replication. Diversity in this protein family is generated by multiple genes, differential splicing, and heterodimerization. To determine the chromosomal position of NFI genes in the human genome, we isolated partial cDNA sequences derived from four independent genes: NFIA, NFIB, NFIC, and NFIX. Corresponding clones of genomic DNA served as probes for fluorescence in situ hybridization on human metaphase chromosomes. The NFIA and NFIB genes map to positions 1p31.2-p31.3 and 9p24.1, respectively. The NFIC and the NFIX genes were both localized to position 19p13.3 in the order centromere-NFIX-NFIC-telomere. Comparison of the position of NFI genes and JUN genes revealed a close physical linkage between members of the NFI and JUN gene families in the human genome.

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.

Evolution of gene regulation as revealed by differential regulation of the chicken lysozyme transgene and the endogenous mouse lysozyme gene in mouse macrophages.

Lysozyme gene expression is a marker for macrophage differentiation in vertebrates. We have previously shown that expression of the complete chicken lysozyme gene domain in macrophages of transgenic mice is directly correlated to the copy number of integrated transgenes. Thus, the chicken lysozyme locus in the mouse acts as an independent regulatory unit irrespective of its random position in the host genome. This finding allowed a comparative analysis of the regulation of the endogenous mouse lysozyme M gene and the chicken lysozyme transgene in the same animal. We demonstrate by transcript analysis of total tissue RNA and by in situ hybridization, that both genes are expressed in macrophages. In macrophages of the same animal the regulation of both genes in response to external signals was distinctly different: the lysozyme transgene responded to various agents influencing macrophage activation, in contrast, mouse lysozyme RNA levels remained unchanged under these conditions. Thus, as in chicken macrophages, the chicken lysozyme expression level in mouse macrophages is coupled to the macrophage activation status, while the mouse lysozyme is not. Our results suggest, that the cis-regulatory elements of lysozyme genes have evolved more rapidly than the function and expression of the trans-acting factors involved in the regulation of macrophage-specific gene activation.

Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation.

The complete chicken lysozyme gene locus is expressed copy number dependently and at a high level in macrophages of transgenic mice. Gene expression independent of genomic position can only be achieved by the concerted action of all cis regulatory elements located on the lysozyme gene domain. Position independency of expression is lost if one essential cis regulatory region is deleted. Here we compared the DNase I hypersensitive site (DHS) pattern formed on the chromatin of position independently and position dependently expressed transgenes in order to assess the influence of deletions within the gene domain on active chromatin formation. We demonstrate, that in position independently expressed transgene all DHSs are formed with the authentic relative frequency on all genes. This is not the case for position dependently expressed transgenes. Our results show that the formation of a DHS during cellular differentiation does not occur autonomously. In case essential regulatory elements of the chicken lysozyme gene domain are lacking, the efficiency of DHS formation on remaining cis regulatory elements during myeloid differentiation is reduced and influenced by the chromosomal position. Hence, no individual regulatory element on the lysozyme domain is capable of organizing the chromatin structure of the whole locus in a dominant fashion.

Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice.

The entire chicken lysozyme gene locus including all known cis-regulatory sequences and the 5' and 3' matrix attachment sites defining the borders of the DNase I sensitive chromatin domain, is expressed at a high level and independent of its chromosomal position in macrophages of transgenic mice. It was concluded that the lysozyme gene locus carries a locus control function. We analysed several cis-regulatory deletion mutants to investigate their influence on tissue specificity and level of expression. Position independent expression of the gene is lost whenever one of the upstream tissue specific enhancer regions is deleted, although tissue specific expression is usually retained. Deletion of the domain border fragments has no influence on copy number dependency of expression. However, without these regions an increased incidence of ectopic expression is observed. This suggests that the domain border fragments may help to suppress transgene expression in inappropriate tissues. We conclude, that position independent expression of the lysozyme gene is not controlled by a single specific region of the locus but is the result of the concerted action of several tissue specific upstream regulatory DNA elements with the promoter.

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.

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.

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.

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.