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B P Leblanc

Publications and source records attributed to B P Leblanc.

5 recordsLinked to original sources
PubMed2001

Remodeling of yeast CUP1 chromatin involves activator-dependent repositioning of nucleosomes over the entire gene and flanking sequences.

The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactive CUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1Delta cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

Carrier Proteins↗
PubMed2000

An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled.

Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

Carrier Proteins↗
PubMed1997

The promoter context is a decisive factor in establishing selective responsiveness to nuclear class II receptors.

The vigorous retinoic acid (RA)-dependent activation of the retinoic acid receptor beta2 (RARbeta2) gene in embryonal carcinoma (EC) cells is mediated by retinoid receptor heterodimers (RXR-RAR) binding to RAREs that are closely positioned to the TATA box and an EC cell-specific co-factor activity termed E1A-LA. Using a series of direct repeat (DR) elements, we now show that positioning RXR-RAR in close proximity to the basal transcription machinery assembled on the TATA box is decisive in RA responsiveness in EC cells. Notably, a DR1 element functions predominantly as an RAR-responsive element when placed in the context of the RARbeta2 promoter. Moreover, DR3 and DR4 elements which mediate vitamin D3 and thyroid hormone responses, respectively, in other contexts, are converted to exclusive RAR response elements when placed in the RARbeta2 promoter and EC cell context. In differentiated cells, the adenovirus E1A(13S) protein is required to achieve high level RA activation through all of the different DR elements placed in the RARbeta2 context, suggesting that the molecular bridging function of E1A-LA [E1A(13S)] is essential to redefining response element specificity. Finally, we show that the arrangement of cis-acting elements as present in the RARbeta2 promoter is not crucial, but rather the close positioning of the RAREs to the TATA. We conclude that the identity of a given cis-acting element is defined not only by its affinity for the transactivator, but also by the context in which it is placed, as well as the cell type in which the transactivator is expressed.

Adenovirus E1A Proteins↗