[Nucleosome remodeling on the regulatory region of the rat tryptophan dioxygenase (tdo) gene during transcription in vivo].

Two DNase I-hypersensitive regions are identified on the regulatory region of the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically under control ofglucocorticoid hormones. DNase I-hypersensitive regions are identified in position -470 and in the vicinity of the first upstream gene. Micrococcal nuclease digestion pattern of this region shows disturbances in the regular cleavage and appearance of shorter DNA molecules than nucleosomal DNA. However the control experiments demonstrate that the same DNA region could be involved in the regular nucleosome core particles under in vitro reconstitution. Taken together, these data show that the nucleosome array in the regulatory region of the actively transcribed tdo gene in vivo is disturbed.

[DNAase I hypersensitivity of the 1,5'-flanking region of the tryptophan oxygenase gene in recombinant DNA and chromatin reconstituted with it]. / Giperchuvstvitel'nost' k DNKaze 1,5'-flankiruiushchei oblasti gena triptofanoksigenazy v rekombinantnoi DNK i rekonstruirovannom na nei khromatine.

We have examined DNase I hypersensitive sites in the 5'-flanking region of the rat tryptophan oxygenase gene in recombinant plasmids. We have found two DNase I-hypersensitive sites in regions that map between -350 to -210 and -180 to -90 bp from the cap site. Both hypersensitive sites are found in supercoiled plasmids but not in linear or relaxed DNA molecules. The position of the hypersensitive sites of the 5'-flanking sequence of the rat tryptophan oxygenase gene in recombinant plasmids correlate with the chromatin sites first determined by Becker et al. The hypersensitive regions in the recombinant plasmids include the DNase I-hypersensitive sites in chromatin but extend somewhat upstream and downstream from these. Computer analysis of the 5'-flanking DNA region of the gene suggests that the DNA fragment spanning nucleotides -231 to -207 forms a potential hairpin loop with 15 unpaired bases. Chromatin reconstitution with histones on supercoiled plasmids carrying the 5'-flanking region of the rat tryptophan oxygenase gene suppresses both in vitro DNase I-hypersensitive sites. We have also shown that the DNA region containing the supercoil-dependent DNase I-hypersensitive site in the position between -350 and -210 bp from the cap site may form nucleosomes after reconstitution with histones.

[Interaction of the regulatory region of the tryptophan oxygenase gene with transcription factors of the nuclear factor 1 (NF1) family]. / Vzaimodeistvie reguliatornoi oblasti gena triptofanoksigenazy s transkriptsionnymi faktorami semeistva iadernykh faktorov 1 (NF1).

Inducible hormone-dependent tryptophan oxygenase gene is expressed mostly in the liver under the control of glucocorticoid hormones. In the regulatory region of this gene there are three constitutive sites independent of hormone presence and gene expression. While investigating transcription factors responsible for the formation of specific chromatin structure in the regulatory region of inducible genes it is necessary to identify proteins which can bind to DNA specifically in these sites. The present paper reports investigation of transcription factors which bind to DNA from the -292nd to the -178th nucleotide of gene to in vitro. This DNA region contains a site corresponding to the constitutive DNase I hypersensitive site in vivo. Using electrophoretic mobility shift assay, we have analysed binding of rat liver proteins to this DNA region. NF1-rich nuclear protein fraction was purified from the rat liver nuclear extract by DEAE-cellulose and heparin-sepharose chromatography. Studies of the competition with a consensus sequence site for NF1 recognition sites have shown that it is the NF1 family transcription factors that are responsible for the formation of these specific complexes.

[Nuclear proteins, specifically binding the regulatory region of rat tryptophan oxygenase]. / Iadernye belki, spetsificheski sviazyvaiushchiesia s reguliatornoi oblast'iu gena triptofanoksigenazy krysy.

Using electrophoretic mobility shift assay we have analysed the binding of rat liver nuclear proteins to the fragments from -466 to -292 and from -292 to -178 relative to the transcriptional start site of the rat tryptophan oxygenase gene. Studies of the competition with a synthetic consensus sequence for the NF 1 recognition site have shown that the liver nuclear proteins, responsible for the formation of specific complexes with these fragments belong to the family of the nuclear factor 1 (NF 1). We have also found that the trans-acting factors of the NF 1 family form several complexes with each of the two tested fragments of the tryptophan oxygenase gene.