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P Charnay

Publications and source records attributed to P Charnay.

At least 55 records · Page 3Linked to original sources
PubMed1994

Krox-20 controls myelination in the peripheral nervous system.

The molecular mechanisms controlling the process of myelination by Schwann cells remain elusive, despite recent progress in the identification and characterization of genes encoding myelin components (reviewed in ref. 1). We have created a null allele in the mouse Krox-20 gene, which encodes a zinc-finger transcription factor, by in-frame insertion of the Escherichia coli lacZ gene, and have shown that hindbrain segmentation is affected in Krox-20-/- embryos. We demonstrate here that Krox-20 is also activated in Schwann cells before the onset of myelination and that its disruption blocks Schwann cells at an early stage in their differentiation, thus preventing myelination in the peripheral nervous system. In Krox-20-/- mice, Schwann cells wrap their cytoplasmic processes only one and a half turns around the axon, and although they express the early myelin marker, myelin-associated glycoprotein, late myelin gene products are absent, including those for protein zero and myelin basic protein. Therefore Krox-20 is likely to control a set of genes required for completion of myelination in the peripheral nervous system.

Animals↗
PubMed1994

Characterization of an Krox-24/Egr-1-responsive element in the human tumor necrosis factor promoter.

We have analyzed in various human leukemic cell lines a previously unrecognized region within the human TNF gene promoter that contains the sequence motif 5'-CCGCCCCCGCG-3'. This GC-rich sequence maps to bps -170 and -160 of the TNF gene. Electrophoretic mobility shift assays (EMSA) combined with methylation interference analysis revealed the binding of two distinct proteins with overlapping recognition sites. Supershift assays identified the constitutive transcription factor Sp1 and the immediate-early growth-response transcription factor Egr-1/Krox-24. Interestingly, this Egr-1-related factor was induced by PMA but not by TNF. The TNF gene GC-rich sequence conferred PMA responsiveness when linked to a heterologous minimal c-fos promoter. To examine the involvement of Egr-1/Krox-24 in TNF gene regulation, a Krox-24 expression vector was used, pSCTKr24. In Jurkat T cells pSCTKr24 stimulated pTNF-286CAT that contains sequences -286 to +34 of the human TNF gene fused to the chloramphenicol acetyltransferase (CAT) gene. Moreover, pSCTKr24 also stimulated the TNF gene GC-rich sequence linked to the minimal c-fos promoter. However, deletion of this site did not result in markedly reduced TNF promoter activity, suggesting that the Egr-1/Krox-24 response element may play an auxiliary role in TNF gene regulation.

Base Sequence↗
PubMed1994

Several receptor tyrosine kinase genes of the Eph family are segmentally expressed in the developing hindbrain.

Pattern formation in the hindbrain involves a segmentation process leading to the formation of metameric units, manifested as successive swellings known as rhombomeres (r). In search for genes involved in cell-cell interactions during hindbrain segmentation, we have screened for protein kinase genes with restricted expression patterns in this region of the CNS. We present the cloning of three novel mouse genes, Sek-2, Sek-3 and Sek-4 (members of the Eph subfamily of putative transmembrane receptor protein tyrosine kinases (RTKs)), the identification of their chromosomal locations, and the analysis of their expression between 7.5 and 10.5 days of development. Before morphological segmentation, Sek-2 is transcribed in a transverse stripe corresponding to prospective r4 and the adjacent mesoderm, suggesting possible roles both in hindbrain segmentation and signalling between neuroepithelium and mesoderm. Sek-3 and Sek-4 have common domains of expression, including r3, r5 and part of the midbrain, as well as specific domains in the diencephalon, telencephalon, spinal cord and in mesodermal and neural crest derivatives. Together with our previous finding that Sek (Sek-1) is expressed in r3 and r5 (Gilardi-Hebenstreit et al., 1992; Nieto et al., 1992), these data indicate that members of the Eph family of RTKs may co-operate in the segmental patterning of the hindbrain.

Amino Acid Sequence↗
PubMed1993

Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain.

The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain. To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E. coli lacZ gene within Krox-20. Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5. Mice homozygous for the mutation die during the first two weeks after birth. Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5. We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.

Aging↗
PubMed1993

The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation.

The zinc finger gene Krox20 and many Hox homeobox genes are expressed in segment-restricted domains in the hindbrain. The restricted expression patterns appear before morphological segmentation, suggesting that these transcription factors may play an early role in the establishment and identity of rhombomeric segments. In this paper, we show that the HoxB2 (Hox2.8) gene is normally upregulated in rhombomeres (r) 3, 4, and 5, and we identify an enhancer region upstream of the gene that imposes r3/r5 expression in transgenic mice. This enhancer contains three Krox20-binding sites required in vitro for complex formation with Krox20 protein and in vivo for rhombomere-restricted expression. In transgenic mice, Krox20 expressed in ectopic domains can transactivate a reporter construct containing the HoxB2 r3/r5 enhancer. These data demonstrate that Krox20 is a part of the upstream transcriptional cascade that directly regulates HoxB2 expression during hindbrain segmentation.

Animals↗
PubMed1993

[Molecular analysis of the development of the rhombencephalon].

Hindbrain development involves a segmentation process which in particular governs cranial-motor nerves organization. We have initiated a molecular analysis of this morphogenetic process by searching for genes expressed in specific hindbrain segments (rhombomeres). We have identified a gene encoding a transcription factor, Krox-20, which is specific of rhombomeres 3 and 5 and several genes encoding receptor tyrosine kinases which are also segment-specific. The study of Krox-20 has allowed the identification of its nucleotide target, the localization of the functional domains of the protein and the demonstration of its involvement in the transcriptional activation of the Hoxb-2 gene and in the control of normal hindbrain development.

Animals↗
PubMed1992

Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis.

Zinc fingers of the Cys2/His2 class are conserved 28-30 amino acid motifs that constitute an important and widespread family of eukaryotic DNA-binding domains. It is therefore of great interest to understand the rules that govern specific recognition of DNA by zinc fingers. The DNA-binding domain of the transcription factor Krox-20 consists of three zinc fingers, each of them making its primary contacts with a three-base pair subsite. We have performed a data base-guided site-directed mutagenesis analysis of Krox-20: nine derivatives were generated, in which one to three amino acid changes had been introduced within finger 2, at positions which were likely to affect the specificity of DNA recognition. The affinities of the different proteins for a panel of potential DNA binding sites were estimated by gel retardation assay. Six of the derivatives bound specific targets with affinities comparable to that of wild type Krox-20 for its consensus binding site. However, the specificity of recognition was dramatically modified at the expected bases, in a manner that could be explained by examining the newly introduced amino acids within the context of the overall finger/triplet interaction. These data provide new insights into the details of zinc finger-DNA interactions and, combined with the modular nature of zinc fingers, illustrate both the potential and the difficulties of utilising these motifs for designing DNA-binding proteins with novel specificities.

Amino Acid Sequence↗
PubMed1992

Mapping functional regions of the segment-specific transcription factor Krox-20.

Krox-20, a zinc finger transcription factor with similarity to Sp1, is likely to play an important role in the development of the vertebrate central nervous system. A knowledge of its molecular properties will help to understand its physiological functions. We have therefore performed a structure-function analysis of the protein to identify the regions involved in DNA-binding and transcriptional activation. Our data suggest that only the zinc fingers are required for high affinity, specific DNA-binding. Transcriptional activation was not affected by deletion of the C-terminal tail of the protein. In contrast, deletion of the N-terminal half, upstream of the zinc fingers, completely abolished transactivation without affecting DNA-binding or nuclear localization. Two transcriptional activation domains were identified in this region. They cooperate to establish full activity. They are rich in negatively-charged amino acids and are therefore may constitute acidic activation domains. Comparative analysis of the amino acid sequences of several zinc finger proteins belonging to the Krox-20 subfamily indicates that they contain acidic regions at similar locations within their N-terminal region, suggesting that the functional organization of these proteins has been conserved during evolution.

Amino Acid Sequence↗
PubMed1992

A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm.

Pattern formation in the hindbrain and paraxial mesoderm of vertebrates occurs by the formation of a series of repeated segments. These processes of segmentation appear different at the morphological level, since hindbrain segments, the rhombomeres, form by the subdivision of the neural epithelium into compartments, whereas the mesodermal somites form by the sequential aggregation of mesenchymal cells into epithelial balls. Previous studies have implicated genes encoding transcription factors in the development of hindbrain segments, but nothing is known of genes involved in the formation of somites. Cellular interactions and signal transduction must be an important aspect of hindbrain segmentation, so we have screened for tyrosine kinases expressed in rhombomere-restricted patterns in the developing mouse embryo. We have identified a receptor protein tyrosine kinase, Sek, that has high relative levels of expression in rhombomeres 3 and 5. This alternating pattern is established coincidentally, both spatially and temporally, with the expression of Krox-20, a zinc-finger gene expressed prior to the morphological formation of rhombomeres. In addition, Sek expression occurs in several other developing tissues, including a dynamic regulation in the developing forebrain, spinal cord, early mesoderm and anterior presomitic mesoderm (segmental plate). The latter expression occurs in two stripes that correlate with, and presage, the formation of somites. Sek expression initially occurs throughout the presumptive somite, then becomes restricted anteriorly, and finally is down-regulated as the definitive somite is formed. These data suggest that despite the morphological differences in the segmentation of the hindbrain and mesoderm, Sek is involved in the segmental patterning of both of these tissues.

Animals↗
PubMed1992

Difference in the genomic organizations of the related transcription factors Sp1 and Krox-20; possible evolutionary significance.

We report here part of the genomic sequence of the mouse homolog of the human gene encoding the transcription factor Sp1. This gene belongs to a subfamily of zinc finger proteins, which includes another transcription factor, Krox-20. The analysis of the mouse Sp1 nucleotide sequence revealed that (i) the C-terminal part of the protein is conserved between man and mouse, consistent with a possible role in transcriptional activation; (ii) while the there Krox-20 zinc fingers are encoded by a unique exon, an intron separates the regions encoding fingers 2 and 3 of Sp1. On the basis of this latter observation, we propose a model for the evolution of the Sp1/Krox-20 gene family which might shed some light on the role of introns in the evolution of modular proteins.

3T3 Cells↗
PubMed1992

An Eph-related receptor protein tyrosine kinase gene segmentally expressed in the developing mouse hindbrain.

In search of genes possibly involved in the regulation of hindbrain segmentation, we have isolated mouse cDNA clones corresponding to putative protein kinase genes by polymerase chain reaction amplification of cDNA from 9.5-day-old embryo hindbrains. In situ hybridization analysis revealed that one of these genes, Sek, was expressed in an alternating segment-restricted pattern in the developing hindbrain. Isolation and analysis of Sek cDNAs covering the entire coding sequence indicated that Sek encoded a putative receptor protein tyrosine kinase, belonging to the Eph family. These data are consistent with a role of the Sek gene product in a signal transduction process involved in pattern formation in the hindbrain.

Amino Acid Sequence↗
PubMed1991

Base sequence discrimination by zinc-finger DNA-binding domains.

Zinc fingers constitute important eukaryotic DNA-binding domains, being present in many transcription factors. The Cys2/His2 zinc-finger class has conserved motifs of 28-30 amino acids which are usually present as tandem repeats. The structure of a Cys2/His2 zinc finger has been determined by nuclear magnetic resonance, but details of its interaction with DNA were not established. Here we identify amino acids governing DNA-binding specificity using in vitro directed mutagenesis guided by similarities between the zinc fingers of transcription factors Sp1 and Krox-20. Krox-20 is a serum-inducible transcription activator which is possibly involved in the regulation of hindbrain development; it contains three zinc fingers similar to those of Sp1 and binds to a 9-base-pair target sequence which is related to that of Sp1. Our results show that each finger spans three nucleotides and indicate two positions in Krox-20 zinc fingers that are important for base-pair selectivity. Modelling with molecular graphics suggests that these residues could bind directly with the bases and that other amino acid-base contacts are also possible.

Amino Acid Sequence↗
PubMed1991

Krox-20: a candidate gene for the regulation of pattern formation in the hindbrain.

The mouse gene Krox-20 was isolated on the basis of cross-hybridization with Krüppel, a Drosophila segmentation gene. During recent years, an accumulation of structural, biochemical and expression data has indicated that Krox-20 encodes a transcription factor which may play a key role within a regulatory network involved in pattern formation in the developing hindbrain. The DNA-binding domain of Krox-20 consists of 3 zinc fingers and the protein recognizes a specific GC-rich nucleotide sequence. It can activate the transcription of genes located in the vicinity of this sequence. Krox-20 transcription itself is modulated by growth factors. During formation of the vertebrate central nervous system (CNS), the hindbrain is organized into segmental units, called rhombomeres. Prior to the appearance of morphological segmentation, Krox-20 is expressed in 2 stripes within the hindbrain. Later on, the Krox-20 expression domains match with 2 alternate rhombomeres. These data suggest that Krox-20 may regulate aspects of the segmentation process. The observation of segment-specific expression boundaries for homeobox containing genes in the hindbrain suggests that these genes may also be part of the regulatory network governing pattern formation in this region of the CNS. The possible interactions between Krox-20 and homeobox containing genes as well as the search for other members of the network will be discussed.

Animals↗
PubMed1991

Transactivation of Krox-20 and Krox-24 promoters by the HTLV-1 Tax protein through common regulatory elements.

The HTLV-1 Tax protein has been shown to induce the expression of host cellular genes, some of which play crucial roles in cell proliferation and differentiation. We have examined the effect of Tax on the expression of two immediate-early genes, Krox-20 and Krox-24, which encode transcription factors. Several HTLV-1-infected T-cell lines and a HeLa cell line that constitutively expresses the Tax protein have a high level of expression of the Krox-20 and Krox-24 genes. In addition, Tax transactivates the promoters of both Krox-20 and Knox-24 in a co-transfection assay. Tax-responsive elements in Krox-20 and Krox-24 include the serum response elements (SREs) and the putative cAMP-responsive element (CRE). A correlation exists between the ability of these elements to mediate Tax transactivation and their affinity for their cognate factors, the serum response factor (SRF) or the CRE-binding protein (CREB) respectively. Since Tax is also able to transactivate the human c-fos promoter through the SRE and the CRE-60, our findings support the idea that the HTLV-1 Tax protein uses common mechanisms for transactivation of these three immediate-early genes. Deregulation of their expression may contribute to malignant transformation associated with HTLV-1 infection.

Base Sequence↗
PubMed1990

The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1.4 gene.

Krox-20 is a mouse zinc finger gene expressed in a segment-specific manner in the early central nervous system, which makes it a potential developmental control gene. In this report, we show that the Krox-20 protein binds in vitro to two specific DNA sites located upstream from the homeobox containing gene Hox-1.4. The nucleotide sequence recognized by Krox-20 is closely related to the Sp1 target sequence, which is consistent with the similarity existing between the zinc fingers of the two proteins. In co-transfection experiments in cultured cells, Krox-20 dramatically activates transcription from the herpes simplex virus thymidine kinase promoter when an oligomer of its binding site is present in cis close to the promoter. Analysis of mutated binding sites demonstrates that the level of activation by Krox-20 correlates with the affinity of the protein for the mutant sequence. These data indicate that Krox-20 constitutes a sequence-specific DNA-binding transcription factor. Parallel analysis of the expression of Krox-20 and Hox-1.4 in the neural tube by in situ hybridization revealed no overlap, arguing against direct interactions between these two genes. The possible involvement of Krox-20 in the regulation of the transcription of other homeobox genes is discussed in view of their respective patterns of expression.

Animals↗