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M Bellard

Publications and source records attributed to M Bellard.

At least 19 recordsLinked to original sources
PubMed1992

A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically.

We have identified an estrogen-responsive enhancer element (DH3 ERE) in the estrogen-induced DNAase I-hypersensitive region III of the chicken ovalbumin gene, which is located approximately 3.3 kb upstream from the mRNA start site and does not contain palindromic ERE. Four TGACC half-palindromic motifs, separated from each other by more than 100 bp, are responsible for conferring estrogen inducibility either to the proximal ovalbumin gene promoter or to heterologous promoters. Thus, widely spaced half-palindromic ERE motifs can act synergistically. Each half-palindromic motif was shown to bind the estrogen receptor (ER) with a low efficiency in vitro. However, two widely spaced half-palindromic motifs bound the ER cooperatively, much more efficiently than expected from binding to isolated half-ERE motifs. The ovalbumin promoter half-palindromic ERE motif located close to the TATA box was required for the activity of the distal DH3 ERE, but could be replaced by the binding sites of other transactivators.

Animals↗
PubMed1992

GEBF-I in Drosophila species and hybrids: the co-evolution of an enhancer and its cognate factor.

The activation of the Drosophila melanogaster salivary gland secretion protein gene Sgs-3 is marked by important changes in chromatin structure in the distal regulatory region at -600 bp from the Sgs-3 start site. A stage- and tissue-specific glue enhancer binding factor, GEBF-I, binds in vitro to sequences from this region. Previous studies have revealed considerable variation in the DNA sequences of comparable regions in the related Drosophila species, D. simulans, D. erecta and D. yakuba. We detected GEBF-I-like proteins in these species, which appear to evolve as rapidly as the corresponding DNA sequences, and studied in detail the binding characteristics of the GEBF-I proteins of the two most closely related species, D. melanogaster and D. simulans. In crosses between these species, certain strains produce hybrid larvae which, unexpectedly, synthesised a single intermediate form of the protein. This suggests that the factor is subject to species-specific post-transcriptional modifications. In these hybrid larvae, which carry one D. melanogaster and one D. simulans Sgs-3 gene, the hybrid GEBF-I protein appears equally effective in the induction of both target genes.

Animals↗
PubMed1991

Control of transcription of the chicken progesterone receptor gene. In vitro and in vivo studies.

To study the promoter of the chicken progesterone receptor (cPR) gene and the relevance of several progestin-responsive elements therein, chimeric genes were constructed which contained the 5'-flanking region of the cPR gene linked to promoterless globin or chloramphenicol acetyltransferase sequences. Cell-specific initiation of transcription was observed in transiently transfected chicken embryo fibroblasts when using 876 base pairs of the cPR gene upstream region. Transcription from these reporter genes could be induced by progestins in the presence of cPR form A but not of form B. In keeping with these data, three in vitro progesterone receptor (PR)-binding sites were identified in the cPR promoter region by DNase I protection assays. However, in vivo, nuclear run-on transcription demonstrated that neither primary stimulation with progestins, nor treatment of secondarily estrogen-stimulated chicks with progestins, glucocorticoids, or androgens resulted in any significant change of cPR gene transcription in the oviduct, thus suggesting a cell- and/or development-specific role for these progestin-responsive elements. Although estrogen is known to increase PR levels in the chick oviduct, this effect does not involve stimulation of PR gene transcription, as demonstrated here by nuclear run-on experiments, the analysis of DNase I hypersensitive sites, and transient cotransfection studies. Since acute withdrawal from estrogen-stimulation markedly decreased the level of cPR mRNAs in chick oviduct when analyzed by Northern blotting, we conclude that estrogen-dependent stimulation of PR levels in the oviduct is a post-transcriptional process.

Animals↗
PubMed1991

Sgs-3 chromatin structure and trans-activators: developmental and ecdysone induction of a glue enhancer-binding factor, GEBF-I, in Drosophila larvae.

The transcription of the Drosophila melanogaster 68C salivary gland glue gene Sgs-3 involves the interaction of a distal and a proximal regulatory region. These are marked in vivo by a specific chromatin structure which is established sequentially during development, starting early in embryogenesis. The distal region is characterized by a stage- and tissue-specific DNase I hypersensitive site. A stage- and tissue-specific factor, GEBF-I, binds in this region and is missing in 2B5 mutant larvae which lack Sgs-3 transcripts. This binding involves the simultaneous interaction with two distinct DNA sequences which induces conformational changes in the protein. Salivary glands acquire competence to respond to ecdysone in the mid-third larval instar, whereafter the hormone rapidly induces both the GEBF-I protein and Sgs-3 transcription.

Age Factors↗
PubMed1990

Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex.

The proximal region of the ovalbumin gene promoter contains a half-palindromic estrogen-responsive element (ERE) that mediates cell-specific trans-activation by the estrogen receptor (ER). We show that the ovalbumin ERE binds a ubiquitous nucleoprotein complex containing oncoproteins c-Fos and c-Jun. Mutations altering the estrogen inducibility of the promoter prevent the complex formation, which is, however, found in the presence and absence of ER and estradiol. Mutagenesis indicates that the sequence 5'-TGGGTCA-3', containing the half-palindromic ERE, is responsible for induction by phorbol esters of the ovalbumin promoter and is a target for c-fos and c-jun trans-activation. Transfection experiments reveal that c-fos, c-jun, and ER coactivate the ovalbumin promoter. Direct ER interaction with the target sequence is not required, since an ER deleted for its DNA binding domain is functional in the coactivation with c-fos and c-jun. Our data indicate a convergence of hormonal induction and activation of signal transduction pathways at the transcriptional level.

Animals↗
PubMed1988

Cell-specific activity of a GGTCA half-palindromic oestrogen-responsive element in the chicken ovalbumin gene promoter.

Using a transient co-transfection system we have identified a functional oestrogen responsive element (ERE) in the near vicinity of the TATA box of the chicken ovalbumin gene promoter. Oestrogen induced activation of transcription was observed only in chicken embryo fibroblast cells, and not in human HeLa cells, suggesting the existence of a cell-specific transcription factor(s) that is necessary for the hormonal response. By 5'-deletion mapping and site-directed mutagenesis, a sequence encompassing the more proximal of two GGTCA half-palindromic motifs was identified as a functional ovalbumin gene ERE. Both motifs correspond to oestrogen receptor binding sites in vitro as revealed by DNase I footprinting. Optimal response to the hormonal stimulus apparently requires stereoalignment of the ERE and the TATA box.

Animals↗
PubMed1988

Analysis of a DNase I-hypersensitive site in transgenic Drosophila reveals a key regulatory element of Sgs3.

We have undertaken chromatin studies on transformed Drosophila strains carrying DNA sequences modified in the region of the DNase I (EC 3.1.4.5)-hypersensitive sites -750 and -600 base pairs upstream from the Sgs3 start site. Although both sites are developmentally specific, modifications in the -750 site have little or no effect on Sgs3-encoded transcript levels, whereas either deletion or replacement of sequences at the -600 site causes an important reduction in transcript levels. The element associated with the -600 site enhances Sgs3 transcription when displaced with respect to the start site. This combined approach has defined sequence elements necessary both for normal transcript levels as well as the chromatin structure characteristic of Sgs3 activity in vivo.

Animals↗
PubMed1986

Changes in the chromatin structure of Drosophila glue genes accompany developmental cessation of transcription in wild type and transformed strains.

Three Drosophila salivary gland glue genes show a dramatic transition in their DNAse I hypersensitive sites during the short period between the late third instar and the white prepupa, which correlates with the cessation of their transcription. In culture cells, where the genes are inactive, there is a chromatin configuration similar to that of prepupal salivary glands. In two transformed fly strains where the sgs3 gene is active at new chromosomal sites, including one in which 2.6 kb of sgs3 upstream sequences have been inverted, the same DNAase I hypersensitive sites and developmental transitions are seen over the same DNA regions. These results, together with the analysis of transformants carrying rearranged sgs3 genes, suggest that there is at least one distal DNAase I hypersensitive site associated with an element of regulation which may be exchanged between sgs genes.

Animals↗
PubMed1986

Steroid hormone dependence of four DNase I-hypersensitive regions located within the 7000-bp 5'-flanking segment of the ovalbumin gene.

Four DNase I-hypersensitive regions (I-IV) occur in the 5'-flanking region of the ovalbumin gene in hen oviducts. One is centered close to the cap site (position +1) of transcription and the others at -0.8, -3.3 and -6.0 kb. The correlation of hypersensitivity with transcription was determined for each region in oviducts of chicks, where expression of the ovalbumin gene can be controlled by administration and withdrawal of steroid hormones. DNase I-hypersensitive regions were mapped by the indirect end-labeling technique and ovalbumin mRNA levels were determined by the dot blot assay. Diethylstilbestrol (DES) induces the appearance of hypersensitive regions I-IV whereas progesterone induces regions I, II and IV, but not region III. Upon withdrawal, regions II and III, and most of regions I and IV, disappear. A weak zone of hypersensitivity in region I near the cap site persists during withdrawal and a new zone of hypersensitivity appears between regions I and II. There is strong correlation between hypersensitivity at regions I-IV and gene transcription; ovalbumin mRNA levels are high in stimulated chicks, where hypersensitive regions are present, and drop to very low levels in withdrawn chicks, where the hypersensitivity is absent. We suggest that proteins, perhaps hormone receptors acting together with tissue-specific factors, induce DNAse I-hypersensitive regions I-IV of the ovalbumin gene.

Animals↗
PubMed1986

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Hormonally induced alterations of chromatin structure in the polyadenylation and transcription termination regions of the chicken ovalbumin gene.

We have studied the chromatin structure of a 16-kb region of the chicken genome containing the 3'-terminal 2 kb of the ovalbumin pre-mRNA coding sequence and the 14-kb segment located immediately downstream from the main mRNA polyadenylation site. Using the indirect end-labelling technique, four major and two minor DNase I-hypersensitive regions were found in the oviduct chromatin, whereas they were not present in liver, kidney or erythrocyte chromatin. The first hypersensitive region (region A) was present in chromatin of oviducts from laying hen and estrogen- or progesterone-stimulated immature chicks, in which the ovalbumin gene is expressed, but not in the chromatin of 'acute withdrawn' chicks where the gene is no longer transcribed. Region A spans 1.3 kb, from 7.2 to 8.5 kb downstream from the ovalbumin gene capsite (position +1), and encompasses the 3' moiety of the last exon including the major polyadenylation signal and polyadenylation site located at +7546 and +7564, respectively. Region A also contains a minor polyadenylation signal present at +7294 and the corresponding polyadenylation site at +7368. Two putative termination sequences at +8445 and +8483 are also found at the 3' extremity of region A in a 170-bp DNA segment within which 90% of the ovalbumin primary transcripts apparently terminate. Two minor hormone-independent DNase I-hypersensitive regions (a1 and a2) located at +8.6 and +8.8 kb are also specific to oviduct chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Nucleosome structure.

Electron microscopic and biochemical results are presented supporting the following conclusions: (1) Two molecules of each histone H2A, H2B, H3 and H4 are necessary and sufficient to form a nucleosome with a diameter of 12.5 +/- 1 nm and containing about 200 base pairs of DNA. (2) H3 plus H4 alone can compact 129 +/- 8 DNA base pairs into a sub-nucleosomal particle with a diameter of 8 +/- 1 nm. In such a particle the DNA duplex is under a constraint equivalent to negative superhelicity. (3) Chromatin should be viewed as a dynamic structure, oscillating between a compact structure (the nucleosome) and more open structures, depending on the environmental conditions.

Animals↗
PubMed1976

Subunit structure of simian-virus-40 minichromosome.

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 +/- 2 nucleosomes, each containing 190 -- 200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 15% of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190 - 200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a 'core' containing a DNA segment of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone H1 is involved in the folding of chromatin fibers.

Animals↗
PubMed1976

Biochemical evidence of variability in the DNA repeat length in the chromatin of higher eukaryotes.

Biochemical evidence is presented which confirms that the DNA repeat length in micrococcal nuclease (spleen endonuclease, nucleate 3'-oligonucleotidohydrolase, EC 3-1-4-7) digests of Chinese hamster ovary chromatin is shorter than that of rat liver chromatin [J.L. Compton, R. Hancock, P. Oudet, and P. Chambon (1976) Eur. J. Biochem., in press]. A survey of available cells has shown that the DNA repeat length of the chromatin of higher eukaryotes varies widely. A value of 196 base pairs was found for cells of all mature tissues, regardless of the source of the tissue, whereas smaller values were found for cells of actively dividing tissues and larger values were found for a genetically inactive cell. Although the DNA repeat length of the chromatin of cells in culture was usually shorter than 196 base pairs, there was no general correlation between the size of the chromatin DNA repeat length and the rate of cell division or the functional state of the cell in culture. Examination of extensive micrococcal nuclease digests suggests that the chromatin subunits of all of the higher eukaryotic cells we have studied contain a core with approximately 140 base pairs of DNA.

Animals↗