The gene for erythropoietin receptor is expressed in multipotential hematopoietic and embryonal stem cells: evidence for differentiation stage-specific regulation.

The principal regulator of erythropoiesis is the glycoprotein erythropoietin, which interacts with a specific cell surface receptor (EpoR). A study aimed at analyzing EpoR gene regulation has shown that both pluripotent embryonal stem cells and early multipotent hematopoietic cells express EpoR transcripts. Commitment to nonerythroid lineages (e.g., macrophage or lymphocytic) results in the shutdown of EpoR gene expression, whereas commitment to the erythroid lineage is concurrent with or followed by dramatic increases in EpoR transcription. To determine whether gene activity could be correlated with chromatin alterations, DNase-hypersensitive sites (HSS) were mapped. Two major HSS located in the promoter region and within the first intron of the EpoR gene are present in all embryonal stem and hematopoietic cells tested, the intensities of which correlate well with EpoR expression levels. In addition, a third major HSS also located within the first intron of the EpoR gene is uniquely present in erythroid cells that express high levels of EpoR. Transfection assays show that sequences surrounding this major HSS impart erythroid cell-specific enhancer activity to a heterologous promoter and that this activity is at least in part mediated by GATA-1. These data, together with concordant expression levels of GATA-1 and EpoR in both early multipotent hematopoietic and committed erythroid cells, support a regulatory role of the erythroid cell-specific transcription factor GATA-1 in EpoR transcription in these cells. However, the lack of significant levels of GATA-1 expression in embryonal stem cells implies an alternative regulatory mechanism of EpoR transcription in cells not committed to the hematopoietic lineage.

Evidence for the existence of two forms of RNA polymerases I and II in insect wing epidermis.

DNA-dependent RNA polymerases were solubilized from developing wings of the oak silkmoth, Antheraea pernyi, and partially purified by ion-exchange chromatography and sucrose gradient sedimentation. Four enzyme species were resolved on the basis of chromatographic behavior, divalent cation requirements, ionic strength optima, template preference and alpha-amanitin sensitivity. Each class (i.e. RNA polymerase I and II) was present in two forms termed IA, IB and IIA, IIB on the basis of their elution pattern from the column. Both class I enzymes were sensitive to high concentrations of alpha-amanitin but this may be due to general toxicity rather than specific inhibition. The intraclass variants did not differ significantly in enzymatic properties although form IIB was more sensitive to alpha-amanitin (50% inhibition at 2 . 10(-9) M) than form IIA (3 . 10(-8)M).

The influence of hemolymph-binding protein on juvenile hormone stability and distribution in Manduca sexta fat body and imaginal discs in vitro.

The wing discs and fat body of Manduca sexta larvae contain enzymes (i.e. carboxylesterase and epoxide hydratase) that can convert the C18 juvenile hormone (JH) to the acid, diol and acid diol. No evidence of oxidative degradation was noted. In vitro studies suggest that JH can be compartmentalized within the cells of the fat body where it is less accessible to degradative mechanisms. Experiments utilizing a hemolymph-binding protein fraction (BPF) in vitro with fat body and imaginal discs indicate that the BPF retards the uptake of JH by tissues and its subsequent degradation by tissue enzymes. BPF also appears to protect JH from degradation by enzymes released into the medium. By these mechanisms the insect can maintain elevated JH titers for relatively long periods. Binding protein may also keep JH in solution in the hemolymph allowing its rapid distribution throughout the insect. The data suggest that the binding protein plays a key role in maintaining juvenile hormone titers.

Interaction of the TGGCA-binding protein with upstream sequences is required for efficient transcription of mouse mammary tumor virus.

A high-affinity binding site for the TGGCA-binding protein, also known as nuclear factor I, has previously been shown to reside within the mouse mammary tumor virus (MMTV) long terminal repeat. We have introduced mutations into this binding site to test the importance of this ubiquitous nuclear protein in MMTV transcription. Mutations which abolish the binding of the TGGCA protein in vitro are shown to impair strongly glucocorticoid-induced transcription from this promoter in vivo. These data demonstrate that the TGGCA-binding protein is a multifunctional DNA-binding protein, capable of serving a transcriptional role in the case of MMTV, in addition to its known involvement in the replication of adenovirus.

The T----C substitution at -198 of the A gamma-globin gene associated with the British form of HPFH generates overlapping recognition sites for two DNA-binding proteins.

Defects in the developmental changes of human hemoglobin production characterized by the continued expression of fetal globin during adult life are classified as hereditary persistence of fetal hemoglobin (HPFH). Among the various molecular lesions associated with this phenotype, the non-deletion forms with point mutations in the promoter region are thought to provide mechanistic clues for gamma-globin gene regulation. The natural occurrence of four different base substitutions mapping within six nucleotides of a homopurine.homopyrimidine motif in the upstream promoter region demarcate a potential control element. To assess its importance for transcriptional activity, we compared the -202 (C----G), -198 (T----C) and -196 (C----T) HPFH mutations with the normal sequence in binding studies with nuclear proteins from erythroid and non-erythroid cells. Wildtype DNA and HPFH mutations at -202 or -196 showed only a weak protein interaction of unclear functional significance. In contrast, -198 (T----C) generated overlapping, high-affinity binding sites for two ubiquitous nuclear proteins. One cognate protein was identified as the transcription factor Sp1. The second one was termed NF-G.C as it interacted strongly with the homopolymer poly(dG).poly(dC). The generation of additional recognition sites for trans-acting factors by the -198 HPFH mutation correlated with a modest increase in promoter activity in vitro specifically with nuclear extracts from erythroid cells. The activation appears to be mediated by binding of Sp1, but it requires interaction with an erythroid-specific factor, most likely GF-1. Templates containing the -196 HPFH mutation showed a transcriptional activity identical to wildtype. This suggests that despite the topological proximity of the mutations, the HPFH phenotype may be established by different mechanisms.

Specific protein-DNA interaction at four sites flanking the chicken lysozyme gene.

Cloned DNA containing 22.2 kb of the chicken lysozyme gene region was screened with use of a nitrocellulose filter binding technique for specific recognition by nuclear DNA binding proteins from chicken oviduct cells. The analysis showed specific retention of four restriction fragments (BS1-BS4), which map approximately 6.1 and 3.9 kb upstream from the transcription start, and 2.8 and 6.2 kb downstream from the poly(A)-addition point of the lysozyme gene. The four DNA fragments mutually served as efficient competitors, indicating that only one class of proteins is involved in the recognition of all four sites. An apparent binding constant of KD = 6 X 10(-12) M was estimated for one of the binding fragments (BS1). Fine mapping of this fragment resolved two closely spaced contact areas at least 43 bp apart.

Cooperation of GATA-1 and Sp1 can result in synergistic transcriptional activation or interference.

GATA-1 is a lineage-restricted transcription factor. Virtually all erythroid-expressed genes contain GATA recognition sites in their regulatory elements. Cotransfection/transactivation assays have revealed that, although GATA-1 as the only cell-restricted transcription factor is sufficient to activate some of the erythroid-specific promoters, not all such promoters are responsive, suggesting a requirement for cooperation with other factors. To study the interaction of GATA-1 with other transactivators, we analyzed sequence motifs of the human gamma-globin promoter as response system by in vitro transcription and by transfections into erythroid K562 cells or into heterologous Drosophila SL2 cells. GATA-1 alone did not activate the promoter. However, GATA-1 exerted an effect in concert with the ubiquitous transactivator Sp1. Depending on the factor concentrations and the sequence context of the cognate binding sites, this interaction could result in synergistic transcriptional activation or in interference. GATA-1 and Sp1 did not cooperate in DNA binding when tested in vitro. This suggests that the functional cooperation is mediated by protein interactions with additional factor(s) which transmit the activator signal. The Sp1-binding CCACCC motif was found to be critical for high activity of the gamma-globin promoter. This site overlaps with a recognition sequence for members of the NFI/CTF family. NFI did not transactivate, but it interfered with Sp1-mediated stimulation and hence with Sp1/GATA-1 cooperation. These data, together with phylogenetic evidence, suggest that the CCACCC region is likely to represent a regulatory switch element.

A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position -166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Sp1.

The TGGCA-binding protein: a eukaryotic nuclear protein recognizing a symmetrical sequence on double-stranded linear DNA.

Low salt extracts of chicken oviduct nuclei contain a DNA binding protein with high affinity for specific DNA sequences in the flanking regions of the chicken lysozyme gene. Two of the three binding sites found within a total of 11 kb upstream from the promoter are located only 92 bp apart from each other. Upon comparison of the DNA binding sites, the symmetrical consensus sequence 5'- TGGCANNNTGCCA -3' can be deduced as the protein recognition site. This sequence is the central part of 23 to 25 base pairs protected by the DNA binding protein from DNAase I digestion. A homologous binding activity can be detected in nuclei from several chicken tissues and from mouse liver.

Selectable retrovirus vectors encoding Friend virus gp55 or erythropoietin induce polycythemia with different phenotypic expression and disease progression.

The Friend spleen focus-forming virus induces a massive expansion of erythroid progenitor cells resulting in polycythemia and splenomegaly. The pathogenic agent is the membrane glycoprotein gp55, encoded by the env gene. Recent evidence indicates that gp55 binds to and activates the erythropoietin (Epo) receptor. It is not clear, however, whether gp55 completely mimics the natural receptor ligand (Epo). To directly compare both effectors, we constructed selectable retroviral vectors which carry either the env or the Epo gene. The selection marker allowed for clonal analysis of infected cells. After infection of DBA/2J mice, the spleen weight, hematological indices, and Epo titer of peripheral blood were monitored. Although both viruses induced an acute erythrocytosis, there were significant differences in disease phenotype and progression. The Epo virus caused an enhanced increase of hematocrit and erythrocytes, whereas with the env virus the pool of late progenitors (CFU-erythroid) was dramatically expanded, resulting in a more severe splenomegaly. The distribution of cytologically recognizable erythroid precursors was shifted towards immature cell types by the env vector compared with Epo. These data suggest that Epo and gp55 differentially affect proliferation and differentiation. Gp55 appears to promote proliferation over differentiation, whereas Epo preferentially drives differentiation.

The TGGCA protein binds to the MMTV-LTR, the adenovirus origin of replication, and the BK virus enhancer.

TGGCA-binding proteins are nuclear proteins with high affinity for double-stranded DNA homologous to the prototype recognition sequence 5'YTGGCANNNTGCCAR 3'. Their ubiquitous tissue distribution in higher vertebrates characterizes them as a class of highly conserved proteins which may exert a basic function. To obtain clues to this function, specific binding sites were mapped on three viral genomes. Recognition sites were identified in the enhancer region of the BK virus, in the LTR of the mouse mammary tumor virus, and in the origin of replication of adenovirus 12. The TGGCA-binding protein from HeLa cells appears to be identical to nuclear factor I described by others, which stimulates initiation of adenovirus DNA replication in vitro. However, data from MMTV, BKV, and from cellular genes suggest that this specific protein-DNA interaction may also be involved in the control of gene activity.

The malignant histiocytosis sarcoma virus, a recombinant of Harvey murine sarcoma virus and Friend mink cell focus-forming virus, has acquired myeloid transformation specificity by alterations in the long terminal repeat.

The malignant histiocytosis sarcoma virus (MHSV), in contrast to other viruses with the ras oncogene, induces acute histiocytosis in newborn and adult mice. Molecular structure and function studies were initiated to determine the basis of its unique macrophage-transforming potential. Characterization of the genomic structure showed that the virus evolved by recombination of the Harvey murine sarcoma virus (Ha-MuSV) and a virus of the Friend-mink cell focus-forming virus family. Structural analysis of MHSV showed two regions of the genome that are basically different from the Ha-MuSV: (i) the ras gene, which is altered by a point mutation in codon 181 leading to a Cys----Ser substitution of the p21 protein, and (ii) the U3 region of the long terminal repeat, which is largely derived from F-MCFV and contains a deletion of one direct repeat as well as a duplication of an altered enhancer-like region. Biological studies of Ha-MuSV, MHSV, and recombinants between the two viruses show that the U3 region of the MHSV long terminal repeat is essential for the malignancy and specificity of the disease. A contributing role of the ras point mutation in determining macrophage specificity, however, cannot be excluded.

DNase I-hypersensitive sites in the chromatin structure of the lysozyme gene in steroid hormone target and non-target cells.

The pattern of DNase I-hypersensitive sites in the chromatin domain of the lysozyme gene was investigated in several organs and cell-types of the chicken. In the cluster of hypersensitive chromatin sites framing the gene, different classes of sites could be discerned: A subset was common to essentially all cells examined except for erythrocytes. Thus several highly nuclease susceptible structures exist around the gene even in its repressed state. Beside the promoter region a second site 6.1 kb upstream of the transcriptional start site of the gene strictly correlates with the transcriptionally active or potentially active state of the gene in both, oviduct cells and macrophages. A final class of sites is specific for the particular lysozyme expressing tissue and the presence of its members distinguish whether the gene is steroid regulated or in a steroid independent active mode.

Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24-nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site-dependent manner.

Alternative sets of DNase I-hypersensitive sites characterize the various functional states of the chicken lysozyme gene.

The structural organization of chromatin is thought to determine the state of differentiation and activity of eukaryotic genes. Local interruptions of the regular nucleosomal array, the so-called DNase-hypersensitive sites, may indicate regions of the genome which play a critical part in regulation of differential gene activity. We present here two new observations on the chromatin structure of the chicken lysozyme gene, which strongly support a regulatory function for these sites. First, different sets of DNase I-hypersensitive sites have been found upstream from the promoter, depending on whether the gene is constitutively expressed (cultured macrophages) or in the steroid hormone-controlled state (oviduct). It seems, therefore, that diverse modes of regulation of the same gene are associated with discrete patterns of DNase I hypersensitivity. Second, one of the DNase I-hypersensitive sites in the oviduct chromatin disappears and reappears on steroid hormone withdrawal and secondary induction. These reversible changes in a narrow chromatin region reflect the transition from the potentially active to the active state of the lysozyme gene.