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J Wijnholds

Publications and source records attributed to J Wijnholds.

27 records · Page 2Linked to original sources
PubMed1994

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.

Animals↗
PubMed1994

Characterization of Pax-6 and Hoxa-1 binding to the promoter region of the neural cell adhesion molecule L1.

The neural cell adhesion molecule L1, a member of the immunoglobulin superfamily, mediates cell interactions in the developing and regenerating nervous system of mammals and is also detectable in the immune system and in the epithelia of intestine, skin, lung, and kidney. This diverse pattern of expression begs the question as to the regulatory mechanisms underlying transcription of the L1 gene. We demonstrate here that the paired domain and homeodomain containing Pax-6 protein binds to three different sites in the promoter region of the L1 gene. The promoter proximal binding site is also recognized by Hoxa-1 and lies approximately 60 bp upstream from the transcription start site only few base pairs upstream of a putative binding site for the TFII-I transcription initiation factor. On the basis of this sequence, we have characterized the binding of Pax-6 and explored two modes of its DNA binding activities.

Base Sequence↗
PubMed1993

Estrogen-inducible and liver-specific expression of the chicken Very Low Density Apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal.

Animals↗
PubMed1993

Pax: gene regulators in the developing nervous system.

In recent years, the discovery of Pax genes in mouse has played an invaluable role in furthering our understanding in mouse developmental processes and disorders. To date, eight murine paired box-containing genes have been cloned. Seven of these exhibit a distinct spatiotemporal expression pattern in the developing nervous system implying a role in the regional specification of the developing spinal cord and brain. The Pax genes encode for sequence-specific DNA binding transcription factors that play a key role in embryonic development. Three of these developmental control genes are altered in mutant mice and two are associated with human diseases. Disruption of these Pax genes leads to abnormalities in neural crest derivatives, neuroectoderm, sclerotome or myotome-derived tissues. Disruption of the Pax-3 gene causes the Splotch phenotype in mice and Waardenburg syndrome in humans. Pax-6 mutations result in Small eye mice and the human genetic disorder aniridia. The Pax-1 gene is mutated in undulated mice. Pax proteins can transform cells in culture which then form tumours following injection in nude mice. Consistent with this activity, PAX3 has been recently implicated in the generation of the tumour alveolar rhabdomyosarcoma.

Animals↗
PubMed1991

Regulatory elements and DNA-binding proteins mediating transcription from the chicken very-low-density apolipoprotein II gene.

The chicken Very-Low-Density Apolipoprotein II (apoVLDL II) gene is specifically expressed in liver in response to estrogen. In this study, we performed a functional analysis of the 300-base pair region immediately 5' to the gene by gene transfer of chloramphenicol acetyl transferase (CAT) constructs into chicken embryonic hepatocytes (CEH). Two estrogen response elements (EREs) could be distinguished which together form a potent estrogen response unit. Stimulation of transient expression by co-transfection with a plasmid expressing rat C/EBP confirmed that a similar protein in chicken liver may be involved in apoVLDL II transcription. In vitro DNaseI footprinting and band-shift analysis with liver, oviduct and spleen nuclear extract revealed the tissue distribution of the proteins binding to the promoter region. A liver-specific protein bound to multiple sites of which some resembled the recognition sequence of the CCAAT/Enhancer binding protein, C/EBP. Of the other proteins binding to the apoVLDL II promoter, one was identified as the liver-specific LF-A1 by mobility shift analysis, using purified bovine LF-A1, and another as the general COUP-transcription factor, using an antiserum against the human COUP-TF.

Animals↗
PubMed1991

Oestrogen facilitates the binding of ubiquitous and liver-enriched nuclear proteins to the apoVLDL II promoter in vivo.

Using genomic and in vitro DNasel footprinting, we have analyzed protein-DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apoVLDL II. The footprints coincide with previously detected guanosine-protein contacts in vivo. All footprints identified are present in the apoVLDL II-expressing liver exclusively and absent in hormone-naive liver, spleen and oviduct. They comprise recognition sites for the oestrogen receptor, the ubiquitous COUP-transcription factor, the liver-enriched C/EBP and/or DBP and the liver-specific LF-A1. In vitro, binding of protein to the oestrogen response element (ERE) is excluded by the prior binding of a protein, possibly C/EBP or DBP, to an adjacent element. The recognition sequence of the COUP-TF is also a target for LF-A1. The results suggests that oestrogen-dependent liver specific activation of the apoVLDL II promoter is established by the binding of the oestrogen receptor to EREs and multiple liver-enriched factors (C/EBP, DBP and LF-A1) to their nearby recognition sequences. Apparently, several DNA binding nuclear proteins cooperate to keep the promoter in a state that is accessible for the RNA polymerase complex.

Animals↗
PubMed1988

Tissue-specific and steroid-dependent interaction of transcription factors with the oestrogen-inducible apoVLDL II promoter in vivo.

Using in vivo dimethylsulphate footprinting, we have analysed protein--DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apo very low density lipoprotein II (apoVLDL II). Most of the guanosine--protein contacts found, are located within the 230-bp DNA 5' flanking the gene and can be grouped into separate protein-binding sites. Two of these sites resemble the oestrogen-responsive element (ERE) which is the target site for the oestrogen receptor. A third site has some features in common with the chicken ovalbumin upstream promoter element binding the COUP transcription factor. All protein contacts identified are present in the apoVLDL-II-expressing liver exclusively, and are not found in the hormone-naive liver, in erythrocytes or the oviduct tubular gland. Our results demonstrate the binding in vivo of a protein, presumably the oestrogen receptor, to the ERE and suggest that the hormone activates transcription by establishing a transcription complex comprising several factors at the apoVLDL II promoter.

Animals↗
PubMed1986

Plasma clearance of mitochondrial aspartate aminotransferase in the rat: competition with mitochondrial malate dehydrogenase.

In previous experiments we have shown that the rapid clearance in rats of alcohol dehydrogenase, lactate dehydrogenase M4, and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. Competition experiments indicated that the dehydrogenases as well as adenylate kinase and creatine kinase MM are endocytosed via the same receptor. We suggested that this receptor contains a group with affinity for the nucleotide-binding sites of the enzymes. We now demonstrate that competition also occurs between mitochondrial malate dehydrogenase and mitochondrial aspartate aminotransferase, which does not require a nucleotide for its activity. At low doses, mitochondrial aspartate aminotransferase was cleared following first-order kinetics (half-life: 19 min). Simultaneous injection of a high dose of mitochondrial malate dehydrogenase strongly retarded the clearance of the aminotransferase. These results make unlikely the hypothesis that a nucleotide-binding site is involved in recognition of enzymes by macrophages.

Animals↗