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Biomedical subjects

M Schorpp

Publications and source records attributed to M Schorpp.

At least 19 recordsLinked to original sources
PubMed2000

Formation of regulator/target gene relationships during evolution.

The Foxn1-like forkhead/winged-helix transcription factor genes have been maintained in single copy throughout chordate evolution. Among other functions, Foxn1 (formerly known as Whn) regulates the expression of hair keratin genes in the hair follicle, which represents an evolutionarily novel organ characteristic of mammals. We show here that fish and mouse Foxn1-like genes are functionally equivalent in hair keratin gene activation, suggesting the absence of functionally relevant changes over the course of several hundred million years of vertebrate evolution. In contrast, the Foxn1-like gene from the cephalochordate Branchiostoma lanceolatum is inactive in this assay because of changes in the region located N-terminal to DNA binding and transcriptional activation domains of the protein. Our results indicate that functionally relevant changes in cis-regulatory regions are not necessarily accompanied by corresponding changes in transcription factor proteins in the formation of evolutionarily novel regulator/target gene relationships.

Amino Acid Sequence↗
PubMed2000

Evolutionary conservation of gene structures of the Pax1/9 gene family.

Based on amino acid sequence comparisons, Pax1 and Pax9 genes are considered to form a subgroup of vertebrate Pax genes. We show here that the gene structures of mouse Pax1, human PAX9 genes are similar to that of a single Pax1/9 related gene in Branchiostoma lanceolatum, AmphiPax1. This supports the hypothesis that Pax1 and Pax9 genes were derived from a single ancestral gene. A refined protein alignment of AmphiPax1, mouse Pax1 and human PAX9 proteins based on the determined exon boundaries indicates that sequence divergence at the C-termini may be related to the unique functions of the Pax1 and Pax9 genes in vertebrates. AmphiPax1 is expressed in adult amphioxus in the pharyngeal endoderm.

Amino Acid Sequence↗
PubMed2000

Forkhead/winged-helix transcription factor Whn regulates hair keratin gene expression: molecular analysis of the nude skin phenotype.

The molecular characteristics of the nude phenotype (alopecia and thymic aplasia) in humans and rodents are unknown. The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Expression of Whn in HeLa cells induced expression of human hair keratin genes Ha3-II and Hb5. Correspondingly, in nude mice, which are homozygous for a loss-of-function mutation of Whn, expression of mouse mHa3 and mHb5 hair keratin genes is severely reduced. Characterization of a previously identified nude allele, nu(Y), revealed a mis-sense mutation (R320C) in the DNA binding domain of Whn. This mutant protein is unable to activate hair keratin gene expression in HeLa cells. When the Whn transcription factor was expressed in two parts, one containing the N-terminal DNA binding domain and the other the C-terminal activation domain, no activation of hair keratin genes in HeLa cells was observed. However, when these two proteins were noncovalently linked by means of synthetic dimerizers, hair keratin gene expression was induced. This finding suggests that target gene activation by Whn depends on the structural integrity and physical proximity of DNA binding and activation domains, providing a molecular framework to explain the loss-of-function phenotypes of all previously characterized nude mutations. Our results implicate Whn as a transcriptional regulator of hair keratin genes and reveal the nude phenotype as the first example of an inherited skin disorder that is caused by loss of expression rather than mutation of keratin genes.

Animals↗
PubMed2000

Genetically separable determinants of hair keratin gene expression.

The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Mutations in Whn cause failure of differentiation of thymic epithelium with a corresponding lack of intrathymic T-cell development; in the skin, differentiation of follicular keratinocytes is disturbed resulting, in the formation of fragile hair shafts. Here, we describe the identification and characterization of a novel nude allele, nu(StL). nu(StL) encodes a truncated Whn transcription factor protein, designated Whn(StL), lacking the activation domain but retaining the characteristic DNA binding domain. In contrast, the previously described Whn(nu) mutant protein lacks both domains. nu(StL)/nu(StL) mice show an alymphoid thymic rudiment and lack of peripheral T cells, similar to nu/nu mice. In the skin, impaired expression of hair keratin genes mHa1, mHa2, mHa3 and mHa4, mHb3, mHb4, mHb5, and mHb6 is observed in a pattern that parallels that of nu/nu mice: both mutant alleles behave as hypomorphs with respect to the expression of these hair keratin genes. However, a significant difference between these two alleles exists for mHa5 expression, which is reduced in nu(StL)/nu(StL) but not in nu/nu mice. We show that the mutant Whn protein in nu/nu mice cannot enter the nucleus, whereas the mutant Whn protein in nu(StL)/nu(StL) mice is present in the nucleus. The antimorphic characteristic of the activation-deficient Whn(StL) protein with respect to mHa5 expression is therefore most likely caused by its non-productive interaction with other proteins at cis-regulatory regions of the mHa5 gene. Our results indicate that the molecular consequences of mutations of the Whn gene can be different and demonstrate an unexpected complexity of transcriptional control mechanisms of hair keratin genes.

Alleles↗
PubMed1997

The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system.

In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to a 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulated reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution.

Amino Acid Sequence↗
PubMed1997

Characterization of mouse and human nude genes.

The differentiation of primitive epithelial precursor cells in the thymic primordium into subcapsular, cortical, and medullary epithelial cells of the mature thymus requires the activity of the nude gene product Whn. Whn is also required for proper keratinization of the hair shaft. We determined the nucleotide sequence of a 58 kilobase region on mouse chromosome 11 that encompasses the mouse nude gene and part of the two neighboring genes, encoding a sodium/dicarboxylate co-transporter and the retinal protein 4. Using cross-hybridization, the human orthologue of the mouse nude gene was isolated. The human WHN protein also consists of 648 amino acids, 85% of which are identical to the mouse protein. Like the mouse gene, the human gene consists of eight coding exons and utilizes two alternative first exons in a tissue-specific fashion. Sequences upstream of the two alternative first exons display promoter activity in heterologous reporter assays. Whereas both promoters appear to be active in skin (albeit at different levels), only the most upstream element is active in the thymus, indicating that transcriptional activity of the whn gene is subject to complex regulation. Nucleotide sequence database comparisons reveal that among other winged-helix genes, the HTLF and HTLFL1 genes are most closely related to whn, although the exon/intron structure of the human HTLF gene in the DNA binding domain differs from that of whn.

Alternative Splicing↗
PubMed1996

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

Amino Acid Sequence↗
PubMed1995

Structural organization and chromosomal localization of the mouse collagenase type I gene.

A clone containing genomic sequences of part of the murine collagenase type 1 (MMP-1) gene was isolated. It contains exons 1-6 encoding all the domains required for collagenase function and 9 kb of 5'-flanking sequences. The gene organization and exon/intron borders are highly similar to the already described human and rabbit MMP-1 genes. However, neither the intron sequences, nor the promoter region up to position -660 exhibit significant sequence homologies with rabbit and human MMP-1, except for an AP-1-binding site and two PEA-3 consensus sequences. Binding studies in vitro revealed that the AP-1-binding site is recognized by Fos/Jun heterodimers with very high affinity. By in situ hybridization the mouse MMP-1 gene was located to the A1-A2 region of chromosome 9 in proximity to the curly whiskers (cw) locus. Based on the lack of sequence homologies of the promoter and intron regions, and since the chromosomal localization of the mouse and human MMP-1 genes may not be syntenic, these data strongly support previous suggestions that the MMP-1 genes from mouse, compared with rabbit and human, have evolved from different ancestral genes. The presence of the AP-1- and PEA-3- binding sites in all mammalian MMP-1 genes isolated so far, may, however, suggest evolutionary selection for common regulatory mechanisms of MMP-1 transcription.

3T3 Cells↗
PubMed1995

Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Adenosine Triphosphatases↗
PubMed1995

An intragenic deletion in the human PTPN6 gene affects transcriptional activity.

An intragenic deletion in the human PTPN6 gene is described. The PTPN6 gene maps to chromosome 12p12-13 and is shown to possess two alternative first exons. A 1.7-kb deletion occurring in the intron between the two alternatively used first exons is the result of an illegitimate recombination between two Alu-type repeats. The deletion increases the transcriptional activity of the distal promotor.

Base Sequence↗
PubMed1995

YAC/P1 contigs defining the location of 56 microsatellite markers and several genes across a 3.4-cM interval on mouse chromosome 11.

The characterization of three YAC/P1 contigs from adjacent segments of the central region of mouse Chromosome (Chr) 11 is described. These contigs are based upon 63 YACs and 40 P1 recombinants. From these clones, 185 end sequences were obtained, of which 147 sequences could be converted into sequence-tagged sites and mapped within the three contigs. Deletions were detected in 16 out of 63 YACs; 19 of 63 YACs were found to be chimeric. No such aberrations were found in P1 recombinants. A total of 22 public and 34 newly developed microsatellite markers were unambiguously localized to and ordered in the contigs. In the cryb1/Nf1 interval of the central contig, several new genes have been identified by exon trapping and precisely localized with respect to known STS markers.

Amino Acid Sequence↗
PubMed1994

New member of the winged-helix protein family disrupted in mouse and rat nude mutations.

Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 (refs 3, 4) within a region of < 1 megabase. Here we show that one of the genes from this critical region, designated whn, encodes a new member of the winged-helix domain family of transcription factors, and that it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates.

Alleles↗
PubMed1994

A yeast artificial chromosome contig on mouse chromosome 11 encompassing the nu locus.

Mutations at the nude locus disrupt the homing process of T cell progenitor cells to the thymic rudiment, a key aspect of T cell differentiation. Here, we map the nude locus to a set of overlapping yeast artificial chromosomes (YAC) clones covering a genetic interval of about 0.5 centi Morgan on mouse chromosome 11. These results provide a suitable starting point to molecularly clone the nude gene.

Animals↗
PubMed1993

Isolation and characterization of the human melanin-concentrating hormone gene and a variant gene.

Melanin-concentrating hormone (MCH) is a cyclic peptide found expressed almost exclusively in the hypothalamus while MCH-containing fibers project throughout the brain of many vertebrates including man. In fishes, MCH induces melanin concentration within the melanophores and may inhibit ACTH secretion. In mammals, MCH modulates ACTH release in vivo and participates as a neuromediator in the control of complex behaviors such as water and food intake. Salmon, rat and human MCH cDNAs have been cloned and structures of deduced mRNAs and precursors have been elucidated. In this report we determine the nucleotide sequence of two human MCH (hMCH) genes and demonstrate that both genes are expressed in human brain. Cloning from three genomic libraries and sequencing of one class of hMCH genomic DNA reveal high similarity between coding regions and the C-terminal part of the hMCH prohormone. However no sequence identity was found in the N-terminal and 5' end non-coding regions of the gene between them even within 6.5 kilobases (kb) upstream from the truncation point. Using polymerase chain reaction (PCR) analysis we have identified RNA populations that are derived from this gene in human brain. For that reason, this gene is a variant rather than a pseudogene. The authentic hMCH gene could only be cloned by using the PCR technique. With primers specific to 5'-end and 3'-end regions of the MCH mRNA we amplified a 1400 bp fragment as well as other shorter PCR products from human genomic DNA. The longest PCR fragment contains 3 exons encompassing most of the 5' untranslated and all of the coding and 3' untranslated sequences of the hMCH mRNA, that are separated by two introns of 350 and 271 bp, respectively. Interestingly the second intron dissects the hMCH peptide sequence in both the authentic and the variant gene. A strikingly high degree of homology was found between the variant and authentic hMCH genes, including intronic sequences, suggesting that these two genomic sequences diverged very recently during evolution. A strong homology was also noted between the exons and intervening sequences of the human and rat MCH genes. Altogether, our results provide the first strong evidence for the existence of two distinct MCH genes expressing prohormones with different MCH and neuropeptide EI (NEI) sequences in human and along with in vivo and in vitro findings, suggest that these neuropeptides may influence the activity of numerous mammalian neuronal systems.

Amino Acid Sequence↗
PubMed1988

Hepatocyte-specific promoter element HP1 of the Xenopus albumin gene interacts with transcriptional factors of mammalian hepatocytes.

By transfecting various Xenopus albumin-CAT fusion genes into the mouse hepatoma cell line BW1J a 13 base-pair hepatocyte-specific promoter element (HP1) could be identified. A similar sequence element is also present in the promoter of the albumin and alpha-fetoprotein genes of other vertebrates. Introduction of single point mutations into HP1 destroys its function. Binding studies with nuclear proteins identify a factor interacting with HP1 which is specific for hepatic cells. In-vitro transcription in a rat liver nuclear extract demonstrates that HP1 leads to an increased transcriptional activity. This increased transcription is specifically inhibited by the addition of an HP1-containing oligonucleotide, establishing that the interaction of factors with HP1 is essential for increased transcription. Since HP1 derived from a Xenopus gene functions in mammalian hepatocytes, we conclude that a regulatory system involved in liver-specific gene expression has been conserved during evolution.

Albumins↗