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P Charnay

Publications and source records attributed to P Charnay.

At least 73 records · Page 4Linked to original sources
PubMed1990

The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.

Amino Acid Sequence↗
PubMed1989

Structure, chromosome mapping and regulation of the mouse zinc-finger gene Krox-24; evidence for a common regulatory pathway for immediate-early serum-response genes.

The structure of Krox-24, a mouse zinc-finger-encoding gene that is transiently activated during G0/G1 transition, has been established. Krox-24 is located on mouse chromosome 18, bands C-D. The gene product, as anticipated for a putative DNA-binding protein, is localized within the cell nucleus. The Krox-24 5'-flanking region contains a series of serum response elements (SREs) similar to the SRE observed upstream of the c-fos proto-oncogene. These elements can substitute for the c-fos SRE, their effect is cumulative and they bind the same cellular factor, the serum response factor (SRF), as the c-fos SRE. This suggests that the SRE and its cognate protein are likely to be involved in the regulation of Krox-24 and presumably of other immediate-early serum response genes. SRE and SRF therefore constitute key components in the regulatory pathway leading from mitogenic stimulation to cellular proliferation.

Amino Acid Sequence↗
PubMed1989

Regulated expression of the overlapping ubiquitous and erythroid transcription units of the human porphobilinogen deaminase (PBG-D) gene introduced into non-erythroid and erythroid cells.

The human gene coding for porphobilinogen deaminase (PBG-D) is transcribed into two distinct transcription units giving two mRNAs. These units originate from two adjacent promoters distant of 3 kilobase pairs. The upstream promoter is active in all cell types, whereas the downstream promoter is active only in erythroid cells. We have studied the expression of this gene either after introduction of the corresponding human chromosome into murine erythroid cells using somatic hybrids or after transfection into both erythroid and non-erythroid cells. Using somatic hybrids, we showed that activation of the erythroid-specific promoter of the PBG-D gene did not reduce the rate of initiation of the ubiquitous promoter. Transfection experiments in erythroid cells showed that the PBG-D erythroid transcription unit, controlled by the PBG-D erythroid promoter, was correctly transcribed and regulated. Furthermore, we found that the PBG-D erythroid promoter alone was sufficient for correct expression and regulation of a reporter gene during erythroid differentiation. When the human PBG-D gene was transfected into non-erythroid cells, only the ubiquitous promoter was active. Deletion of the ubiquitous promoter did not lead to any activation of the erythroid promoter, suggesting that its inactivity in non-erythroid cells was not due to promoter occlusion but to a strict erythroid specificity.

Ammonia-Lyases↗
PubMed1989

Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse.

The process of segmentation, in which repeated homologous structures are generated along the anterior-posterior axis of the embryo is a widespread mechanism in animal development. In vertebrates, segmentation is most apparent in the somites and the peripheral nervous system, but the existence of repetitive bulges, termed neuromeres, in the early neural epithelium of vertebrates suggests that the CNS may also be segmented. Consistent with this, cranial ganglia and certain neurons are associated with specific hindbrain neuromeres. Here, we report that Krox-20, a zinc-finger gene, is expressed in two alternate neuromeres in the mouse early hindbrain. This pattern subsequently decays and Krox-20 is transiently expressed in specific hindbrain nuclei. In addition, Krox-20 is expressed in early neural crest cells, and then in the neural crest-derived boundary caps, glial components of the cranial and spinal ganglia. The demonstration that neuromeres are domains of gene expression provides molecular evidence for the segmentation of the CNS.

Animals↗
PubMed1989

Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.

Amino Acid Sequence↗
PubMed1988

Expression of human G gamma globin genes carrying a T or a C at position -158 in COS and MEL cells.

Increasing data strongly suggest that, in adults, the G gamma/A gamma globin chains ratio is genetically controlled by determinants linked in cis to the beta-globin gene cluster. The presence of the C----T substitution at position -158 5' to the G gamma globin gene in subjects displaying high G gamma/A gamma globin chains ratio led to the hypothesis that this substitution is involved in the determination of high G gamma globin gene expression. In order to test this hypothesis, we have compared the expression of two G gamma globin genes carrying a C or a T at position -158, upon transfection into a COS cell line or stable integration into a MEL cell line. Our results showed no significant effect of the nucleotide at position -158 on the level of human G gamma mRNA produced in these cells.

Cell Line↗
PubMed1988

A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells.

Zinc fingers are DNA-binding domains present in several eukaryotic regulatory proteins. We have identified a mouse gene, Krox-20, encoding a protein with three zinc fingers and whose expression is activated during G0/G1 transition in cultured cells. Serum stimulation of quiescent cells leads to rapid and transient accumulation of Krox-20 mRNA, with kinetics similar to those of the c-fos proto-oncogene. The induction does not require de-novo protein synthesis. In the mouse, Krox-20 is expressed at low levels in tissues which contain rapidly dividing cells. These properties suggest that Krox-20 encodes a transcription control factor, possibly involved in the modulation of cell proliferation.

Amino Acid Sequence↗
PubMed1988

Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells.

We previously reported the identification of a mouse gene, Krox-20, encoding a protein with three "zinc fingers" (DNA-binding domains with coordinated zinc ions) whose expression is regulated during G0/G1 transition (cell-cycle reentry). We now have isolated cDNAs corresponding to a related gene, Krox-24. Krox-24 encodes a protein with zinc fingers nearly identical to those encoded by Krox-20 and similar to those of transcription factor Sp1. Similarity between Krox-20 and Krox-24 proteins also extends to several blocks of amino acid sequence located upstream of the finger region. Like Krox-20, Krox-24 is transiently activated in quiescent cells after treatment with fetal bovine serum or purified growth factors. The kinetics of activation are similar to those of the protooncogene c-fos. The induction does not require de novo protein synthesis, and cycloheximide treatment of the cells leads to superinduction due, at least in part, to mRNA stabilization. In the mouse, the two genes are expressed in a tissue-specific manner, with slightly different patterns. These properties suggest that Krox-20 and Krox-24 may encode transcription factors with identical DNA target sequences and that these factors may be involved in the modulation of cell proliferation and differentiation.

Amino Acid Sequence↗
PubMed1988

A DNA element responsible for the different tissue specificities of Friend and Moloney retroviral enhancers.

The transcriptional enhancers of the Moloney murine sarcoma virus (MuSV) and Moloney murine leukemia virus (MuLV) have different cell type specificities from that of the Friend MuLV. While the three enhancers are approximately equally active in erythroid cells, the Moloney MuSV and Moloney MuLV enhancers are 20- to 40-fold more active than the Friend MuLV enhancer in T-lymphoid cells. Using mutant enhancers, we have shown that specific differences between the nucleotide sequences of the Moloney MuSV and Friend MuLV enhancers are responsible for their different activities in T cells. Our data allow the localization of a DNA element, repeated several times within the enhancer, which modulates the activity of the enhancer in T cells without affecting it in erythroid cells. This element therefore appears to be one of the determinants of the tissue specificity of the enhancer.

Base Sequence↗
PubMed1988

Characterization of a mouse multigene family that encodes zinc finger structures.

The Drosophila segmentation gene Krüppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers. We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Krüppel finger region. Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Krüppel and potentially encoded zinc finger domains. Within these regions, the level of similarity to Krüppel was particularly high between successive fingers. Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development. Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions. In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors. These genes are likely to play a regulatory role at the transcriptional level.

Amino Acid Sequence↗
PubMed1986

Regulated expression of cloned human fetal A gamma-globin genes introduced into murine erythroleukemia cells.

We have analysed the expression of cloned human fetal gamma-globin genes introduced into murine erythroleukemia cells by a protoplast fusion procedure. Both the wild-type a gamma-globin gene and a mutant derivative related to a phenotype of hereditary persistence of fetal hemoglobin were studied. In both cases the level of gamma-globin mRNA increased by a factor of approximately sevenfold when erythroid cell differentiation was induced by treatment with hexamethylenebisacetamide. Thus, the regulation of the expression of the cloned fetal A gamma-globin gene in murine erythroleukemia cells resembled that of cloned adult beta-globin genes.

Animals↗
PubMed1986

A transcriptional enhancer with specificity for erythroid cells is located in the long terminal repeat of the Friend murine leukemia virus.

We investigated the ability of the U3 region of the long terminal repeats (LTR) of the Friend murine leukemia virus (Fr-MuLV) and Moloney murine leukemia virus (Mo-MuLV) to promote transcription in a variety of human cell lines. Our analysis reveals the presence of a transcriptional enhancer with specificity for erythroid cells in the U3 region of the Fr-MuLV. This constitutes the first example of an enhancer with such a property. Analysis of the Mo-MuLV enhancer suggests that it is active at least in erythroid and lymphoid cells and has thus a less restricted specificity than the Fr-MuLV enhancer. The different tissue specificities of the two enhancers correlate with the different tissue selectivities and pathogenic properties of the two viruses.

Acetyltransferases↗
PubMed1985

Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

Animals↗
PubMed1984

Human alpha- and beta-globin gene transcription in mouse erythroleukaemia cells.

Human beta-globin genes introduced into mouse erythroleukaemia (MEL) cells by DNA co-transformation are correctly regulated when erythroid cell differentiation is induced by dimethylsulphoxide (DMSO). In contrast, cloned human alpha-globin genes are efficiently transcribed in MEL cells before induction, and no increase in the level alpha-globin mRNA is observed when the cells differentiate. These observations suggest that the mechanisms by which alpha- and beta-globin genes are activated during erythroid cell differentiation are fundamentally different. Analysis of the transcription of hybrid human alpha-beta-globin genes in MEL cells revealed that the sequences responsible for differences in transcription of the intact alpha- and beta-globin genes are located on the 3' side of the mRNA capping site of the two genes, suggesting that cis-acting regulatory sequences are located within the structural genes.

Animals↗
PubMed1984

Differences in human alpha- and beta-globin gene expression in mouse erythroleukemia cells: the role of intragenic sequences.

Human beta-globin genes introduced into mouse erythroleukemia (MEL) cells by DNA cotransformation are correctly regulated when erythroid cell differentiation is induced by dimethylsulfoxide (DMSO). In contrast, cloned human alpha-globin genes are efficiently transcribed in MEL cells prior to induction, and no increase in the level of alpha-globin mRNA is observed when the cells differentiate. These observations suggest that the mechanisms by which alpha- and beta-globin genes are activated during erythroid cell differentiation are fundamentally different. Analysis of the transcription of hybrid human alpha/beta-globin genes in MEL cells revealed that the sequences responsible for differences in transcription of the intact alpha- and beta-globin genes are located on the 3' side of the mRNA capping site of the two genes, suggesting that cis-acting regulatory sequences are located within the structural genes.

Animals↗
PubMed1983

Transcriptional regulation of globin gene expression in the human erythroid cell line K562.

The effect of hemin on the rate of synthesis and the level of globin messenger RNA's in the human erythroid cell line K562 was examined by means of cloned hybridization probes specific for each of the human embryonic, fetal, and adult globin genes. Hemin increases both the rate of transcription and the level of accumulation of zeta-, epsilon-, gamma-, and alpha-globin messenger RNA's by a factor of 3 to 5. Thus, hemin induction of globin gene expression in K562 cells is at the level of transcription.

Cell Line↗
PubMed1983

The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

Animals↗