Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo

Abstract

TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.

FIG. 1.

(A) Cdk7 colocalizes with phosphorylated Pol II. Non-heat-shocked polytene chromosomes from wild-type larvae were stained with monoclonal antibodies recognizing phosphoserine 5 of the polymerase CTD (green) and Cdk7 (red). At the bottom is a high magnification of chromosome 3R in which the chromosome images from the different antibody stains were split down their axes using Photoshop software and aligned. (B) Cdk7 is recruited to native and transgenic heat shock loci. Bg9 larvae were immunostained with HSF (green) and Cdk7 (red) antibodies after a 20-min heat shock. Site 61A contains an hsp70 hybrid transgene. 87A and 87C contain two and three copies of native hsp70, respectively. 67B contains a cluster of the small heat shock genes hsp22, -23, -26, and -27. (C) Cdk7 and P-TEFb colocalize at heat shock puffs. Bg9 larvae were heat shocked and immunostained for the cyclin T subunit of P-TEFb (green) and Cdk7 (red). The yellow areas in the merge (right) indicate regions of colocalization.

FIG. 2.

Heat shock gene mRNA accumulation is reduced in Cdk7^ts flies. RNAs from the isogenic control strain line 47 (WT) and Cdk7^ts flies were used for Northern blotting. Signals from each gene were normalized to the rRNA 1:1,000 standard to account for differences in total RNA between WT and Cdk7^ts flies.

FIG. 3.

RNA polymerase density is decreased on heat shock genes in Cdk7^ts flies. (A) Polymerase densities for hsp70, hsp83, hsp26, and histone 2A and -3 (H2A and H3). The decreases (n-fold) represent the means of three independent experiments. (B) Polymerase pausing is decreased in the Cdk7^ts mutant. Paused molecules were released by the addition of 0.6% Sarkosyl to the nuclear run-on reactions containing non-heat-shocked nuclei. The mean of two independent experiments is shown. N.D., quantification not determined; WT, wild type; TS, Cdk7^ts.

FIG. 4.

Induced heat shock genes in Cdk7^ts mutant contain phosphorylated serine 5 but little Cdk7 protein and display abnormal puff pattern. (A and B) Isogenic control (WT) (A) and Cdk7^ts mutant (B) showing recruitment of HSF (green) and Cdk7 (red) to hsp70 genes at 87A and 87C. The 87A puff is smaller in the Cdk7^ts mutant and displays weak recruitment of the Cdk7 protein. (C and D) HSF and phosphoserine 5 (P-ser5) at 87A and 87C in the WT and Cdk7^ts mutant, respectively. (E) Serine 5 phosphorylation persists in the Cdk7^null mutant.

FIG. 5.

α-β transcription is reduced in the Cdk7^ts mutant. (A) Arrangement of hsp70 and α-β genes. (B) α-β transcription is sensitive to the Cdk7^ts mutation. Northern blots probed αβ for RNA and standards rRNA and U6 snRNA (see Materials and Methods). WT, wild type; ts, Cdk7^ts.

FIG. 6.

ChIP analysis of the induced hsp70 gene. Results from three or four independent cross-linking experiments for the wild type (lightly shaded bars) and the Cdk7^ts mutant (darkly shaded bars) are shown. The y axis indicates cross-linking efficiency as a percentage of input, and the error bars indicate standard error. Antibodies were against the following proteins: HSF (A), P-Ser5 Pol II CTD (B), Pol II largest subunit (C), P-Ser2 Pol II CTD (D), cyclin T of P-TEFb (E), and Cdk7 (F). Background levels of cross-linking were determined by omitting the precipitating antibody and were then subtracted from the signals. The hsp70 gene fragments (base pair coordinates relative to the start site) that were analyzed are shown at the bottom of each panel.