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A Bruhat

Publications and source records attributed to A Bruhat.

28 records · Page 2Linked to original sources
PubMed1996

Phosphorylation/dephosphorylation of the repressor MDBP-2-H1 selectively affects the level of transcription from a methylated promoter in vitro.

We have previously shown that in vivo estradiol-dependent dephosphorylation of MDBP-2-H1 (a member of the histone H1 family) correlates with the loss of in vitro preferential binding to methylated DNA. To study the effects of the phosphorylation/dephosphorylation of MDBP-2-H1 on the expression of the avian vitellogenin II gene, we optimised an in vitro transcription system using HeLa nuclear extracts. We show that in the absence of the phosphorylated form of MDBP-2-H1 from rooster, methylation of the vitellogenin II promoter does not affect the transcription. Addition of purified MDBP-2-H1 from rooster to the in vitro transcription system inhibits transcription more efficiently from a methylated than an unmethylated DNA template. Dephosphorylation of rooster MDBP-2-H1 by phosphatase treatment or estradiol treatment of rooster lead to the loss of inhibitory activity of the protein when added to the in vitro transcription assays. These findings indicate that the phosphorylation of MDBP-2-H1 is essential for the repression of the transcription. Taken together these results establish the relationship between the dephosphorylation of MDBP-2-H1 caused by estradiol, the down regulation of its binding activity to methylated DNA and the derepression of vitellogenin II transcription.

Animals↗
PubMed1995

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

Animals↗
PubMed1993

Intronic and 5' flanking sequences of the Drosophila beta 3 tubulin gene are essential to confer ecdysone responsiveness.

The expression of the beta 3 tubulin gene is regulated, at the transcriptional level, by the steroid hormone ecdysone, in Drosophila Kc cells. Using a transient expression assay, we show that 360 bp from the first intron of the beta 3 tubulin gene, associated with the 5' flanking sequences, are essential to confer ecdysone inducibility on a minimum promoter driving the chloramphenicol acetyl transferase (CAT) gene. The 5' flanking region contains ecdysone-independent cis-positive elements located in proximity to the promoter. Deletion analysis of the 360 bp intronic region reveals that a fragment of 57 bp is crucial for the ecdysone response of the beta 3 tubulin gene. This fragment contains 5'-TGA(A/C)C-3' motifs homologous to ecdysone responsive elements (EcRE) half sites. Band shift assays show that this 57-bp fragment is bound by three specific complexes. One of them appears to be involved in the level of the ecdysone response.

Animals↗
PubMed1993

Enhancer and silencer elements within the first intron mediate the transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-E) in Drosophila Kc cells. A series of hybrid genes, with different fragments of the beta 3 tubulin gene driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence and the absence of 20-E. Constructs with 0.91 kb upstream from the transcription start site and 360 bp from the first large intron allowed the hormonal regulation, i.e. a repression in the absence of 20-E and a derepression-activation in the presence of the hormone. This 360 bp fragment contains several enhancers and silencer(s) sequences. The regulation of the expression of the beta 3 tubulin gene results from the combined activity of all the positive and negative regulatory sequences of the first intron, and a dialogue with the promoter sequences. The nucleotide sequence of this intronic regulatory-fragment has been established and we have identified several EcRE (ecdysone responsive element) consensus sequences.

Animals↗
PubMed1993

In Drosophila Kc cells 20-OHE induction of the 60C beta3 tubulin gene expression is a primary transcriptional event.

In Drosophila Kc cells, the 60C beta3 tubulin transcription unit, whose expression is induced by 20-hydroxyecdysone (20-OHE), has the same structure as in Drosophila. This gene is characterized by an unusual 5' intron of regulating importance, by an alternatively spliced second intron and by a long 3' transcribed but untranslated region. This gene codes for two beta3 tubulin isoforms with one amino acid difference. We have established that beta3 tubulin gene expression is transcriptionally regulated by the steroid hormone in a time and hormonal concentration-dependent fashion, without requirement of protein synthesis. This implies that this transcriptional induction is a primary event and that this gene is probably a direct target for the 20-OHE receptor.

Animals↗
PubMed1990

Regulatory elements in the first intron contribute to transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-OH-E) in Drosophila Kc cells. A series of hybrid genes with varying tubulin gene lengths driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence or absence of 20-OH-E. We find that 0.91Kb upstream from the transcription start site contain one or several hormone independent positive cis-acting elements, responsible for the constitutive expression of the beta 3 tubulin gene. In the large (4.5 Kb) first intron of this gene, we identified additional hormone dependent negative and positive regulatory elements, which can act in both directions and in a position-independence manner. Then, the negative intron element(s), which repress the transcription in the absence of 20-OH-E has characteristics of silencer.

Animals↗