Occupancy of the Drosophila hsp70 promoter by a subset of basal transcription factors diminishes upon transcriptional activation - PubMed (original) (raw)

Comparative Study

. 2005 Dec 13;102(50):18087-92.

doi: 10.1073/pnas.0509063102. Epub 2005 Dec 5.

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Comparative Study

Occupancy of the Drosophila hsp70 promoter by a subset of basal transcription factors diminishes upon transcriptional activation

Lyubov A Lebedeva et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The presence of general transcription factors and other coactivators at the Drosophila hsp70 gene promoter in vivo has been examined by polytene chromosome immunofluorescence and chromatin immunoprecipitation at endogenous heat-shock loci or at a hsp70 promoter-containing transgene. These studies indicate that the hsp70 promoter is already occupied by TATA-binding protein (TBP) and several TBP-associated factors (TAFs), TFIIB, TFIIF (RAP30), TFIIH (XPB), TBP-free/TAF-containg complex (GCN5 and TRRAP), and the Mediator complex subunit 13 before heat shock. After heat shock, there is a significant recruitment of the heat-shock transcription factor, RNA polymerase II, XPD, GCN5, TRRAP, or Mediator complex 13 to the hsp70 promoter. Surprisingly, upon heat shock, there is a marked diminution in the occupancy of TBP, six different TAFs, TFIIB, and TFIIF, whereas there is no change in the occupancy of these factors at ecdysone-induced loci under the same conditions. Hence, these findings reveal a distinct mechanism of transcriptional induction at the hsp70 promoters, and further indicate that the apparent promoter occupancy of the general transcriptional factors does not necessarily reflect the transcriptional state of a gene.

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Figures

Fig. 1.

Fig. 1.

Localization of HSF, TFIID, TFIIB, TFIIF, TFIIH, TFTC, and MED13, relative to RNA Pol II, in endogenous heat-shock loci on wild-type Drosophila polytene chromosomes. Shown are images focused on the native heat-shock loci at 87A and 87B that contain two and four copies of the hsp70 gene, respectively. The images in each column are collected from a nucleus in which the staining is representative of nuclei from several salivary gland squashes. Antibodies used in this study raised against the different transcription factors are rabbit polyclonal antibodies (red). Polytene chromosome staining was carried out as a double staining with a monoclonal antibody raised against the C-terminal domain of Pol II as a positive control (green). When the anti-TFIIH (XPB) monoclonal antibody was used (green), the samples were double-stained with an anti-TBP polyclonal antibody (red). White lines mark the heat-shock loci at 87A and 87B before heat shock. After heat-shock, the labeling of TBP, TAFs, TFIIB, and TFIIF (RAP30) decreases at 87A and 87B loci (B), whereas labeling of Pol II, HSF, and TFIIH (XPB), TFTC (GCN5 and TRRAP), and MED13 increases at the 87A and 87B loci (A). NHS, no heat shock; HS, 20 min of heat shock.

Fig. 2.

Fig. 2.

TBP, TAF1, TAF9, and TAF10 are present at native ecdysone-induced puffs independently of heat shock. Experiments were carried out as in Fig. 1. White lines mark ecdysone-induced loci at 74EF and 75B locations. TBP, TAF1, TAF9, and TAF10 (red) are present at the native ecdysone-induced puffs 74EF and 75B after heat shock. Pol II staining is green. NHS, no heat shock; HS, 20 min of heat shock.

Fig. 3.

Fig. 3.

Localization of HSF, TFIID, TFIIB, TFIIF, TFIIH, TFTC, and Mediator complex subunits at the hsp70 promoter-containing transgene. Kinetics of binding of the different indicated factors to the hsp70 promoter in a transgenic Drosophila line containing the _hsp70_-Penelope gene at 19E. Experiments were carried out as in Fig. 1. In each image, the 19E locus is marked by an arrowhead. The duration of the heat shock is indicated in minutes. For a better orientation of the 19E locus, the chromocenter (Ch) has also been labeled on each panel. NHS, no heat shock. DNA was stained with DAPI.

Fig. 4.

Fig. 4.

Heat-shock-induced location of GTFs and cofactors at the hsp70 promoter and coding region in Schneider cells. Crosslinked chromatin from cultured Schneider cells subjected to heat shock (black bars) or not (white bars) was immunoprecipitated with antibodies specific for the indicated factors. The results of the real-time PCR analyses of the ChIP experiments are summarized in the histograms. Percentage of target sequences in the immunoprecipitated material relative to input is shown on the y axis of each plot. Of the crosslinked chromatin, 1% was used as input. Background of immunoprecipitation (an average normalized value obtained by treatment of the chromatin with a nonspecific antibody and with beads only) was subtracted from normalized specific ChIP signals (obtained with antibodies against the transcription factors indicated) at each position. Primer sets used to amplify the hsp70 promoter or the _hsp70_-coding region (gene) are indicated on the x axis. The error bars indicate the standard deviations.

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