Functional analysis of Rad14p, a DNA damage recognition factor in nucleotide excision repair, in regulation of transcription in vivo

Abstract

Rad14p is a DNA damage recognition factor in nucleotide excision repair. Intriguingly, we show here that Rad14p associates with the promoter of a galactose-inducible GAL1 gene after transcriptional induction in the absence of DNA lesion. Such an association of Rad14p facilitates the recruitment of TBP, TFIIH, and RNA polymerase II to the GAL1 promoter. Furthermore, the association of RNA polymerase II with the GAL1 promoter is significantly decreased in the absence of Rad14p, when the coding sequence was deleted. These results support the role of Rad14p in transcriptional initiation. Consistently, the level of GAL1 mRNA is significantly decreased in the absence of Rad14p. Similar results are also obtained at other galactose-inducible GAL genes such as GAL7 and GAL10. Likewise, Rad14p promotes transcription of other non-GAL genes such as CUP1, CTT1, and STL1 after transcriptional induction. However, the effect of Rad14p on the steady-state levels of transcription of GAL genes or constitutively active genes such as ADH1, PGK1, PYK1, and RPS5 is not observed. Thus, Rad14p promotes initial transcription but does not appear to regulate the steady-state level. Collectively, our results unveil a new role of Rad14p in stimulating transcription in addition to its well-known function in nucleotide excision repair.

FIGURE 1.

Rad14p is associated with the promoter and coding sequence of the GAL1 gene in a transcription-dependent manner. A, a schematic diagram shows the PCR primer pairs located at the promoter and coding sequence (or ORF) of the GAL1 gene in the ChIP assay. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). B, Rad14p is associated with the promoter and coding sequence of the active GAL1 gene. The yeast strain expressing Myc epitope-tagged Rad14p (ZDY3) was grown in raffinose (YPR)- or galactose (YPG)-containing growth medium up to an A₆₀₀ of 1.0 before cross-linking. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology). Primer pairs targeted to the promoter and coding sequence of the GAL1 gene and an inactive region of the chromosome-V (Chr-V) were used for PCR analysis of the immunoprecipitated DNA samples. The maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signals (represented as normalized occupancy) were plotted in the form of a histogram. C, the autoradiograms for the data presented in the panel B are shown. IP, immunoprecipitate. D, RNA polymerase II is associated with the promoter and coding sequence of the active GAL1 gene. The yeast strain was grown in YPR up to an A₆₀₀ of 0.9 and then switched to YPG for 90 min before cross-linking. Immunoprecipitation was performed using a mouse monoclonal antibody 8WG16 (Covance) against the C-terminal domain of the largest subunit (Rpb1p) of RNA polymerase II. The maximum ChIP signal was set to 100, and another ChIP signal was normalized with respect to 100. The normalized ChIP signal was plotted in the form of a histogram. E, the autoradiograms for the data presented in the panel D. F, RT-PCR analysis. Both the wild type and Δrad14 strains were grown as in panel D. The Δrad14 strain (PCY25) was generated in the W303a wild type background. GAL1 mRNA level in the wild type strain was set to 100, and the mRNA level in the Δrad14 strain was normalized with respect to 100. The normalized mRNA level was plotted in the form of a histogram.

FIGURE 2.

Rad14p promotes the association of RNA polymerase II with GAL1. A, analysis of Rpb1p association with the GAL1 promoter and coding sequence in the Δrad14 strain (PCY25) and its isogenic wild type equivalent is shown. The wild type and Δrad14 strains were grown, cross-linked, and immunoprecipitated as in Fig. 1D. The maximum ChIP signal for the wild type strain was set to 100, and the ChIP signal of the Δrad14 strain was normalized with respect to 100. The normalized ChIP signal was plotted in the form of a histogram. B, Western blot analysis. C and D, analysis of Rpb1p association with GAL1 in the Δrad14 strains (PCY35 and YMR201C) derived from the FM391 and BY4741 wild type backgrounds is shown. Yeast strains were grown, cross-linked, and immunoprecipitated as in panel A. E, analysis of GAL1 transcription in the Δrad14 strains (PCY35 and YMR201C) is shown. Yeast strains were grown as in panel A.

FIGURE 3.

Rad14p promotes the formation of the PIC assembly. A, regulation of Rpb1p occupancy at the GAL1 promoter by Rad14p in the absence of the coding sequence. Both the wild type and Δrad14 strains without GAL1 ORF (PCY31 and PCY32) were grown, cross-linked, and immunoprecipitated as in Fig. 1D. B, the autoradiograms for the data presented in panel A. C, Rad3p is predominantly associated with the GAL1 promoter. The yeast strain expressing Myc epitope-tagged Rad3p (PCY2) was grown, cross-linked, and immunoprecipitated as in Fig. 1B. The maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signals were plotted in the form of a histogram. D, the autoradiograms of the data presented in the panel C. E, Rad14p promotes the recruitment of Rad3p. Both the wild type and Δrad14 strains expressing myc-tagged Rad3p (PCY2 and PCY35) were grown and cross-linked as in Fig. 1D. F, the autoradiograms for the data presented in the panel E. G, Western blot analysis is shown.

FIGURE 4.

Rad14p regulates the recruitment of TBP, but not Gal4p and SAGA, to GAL1. A, Rad14p facilitates the recruitment of TBP. Both the wild type and Δrad14 strains were grown and cross-linked as in Fig. 3E. Immunoprecipitation was performed using an anti-TBP antibody against TBP. B, the autoradiograms for the data presented in the panel A. C, Western blot analysis. D, Rad14p does not regulate the recruitment of Gal4p and SAGA (TAF12p) to the GAL1 UAS. Both the wild type and Δrad14 strains were grown and cross-linked as in Fig. 3E. Immunoprecipitations were performed using anti-Gal4p (Santa Cruz Biotechnology) and anti-TAF12p (Michael R. Green, University of Massachusetts Medical School) antibodies against Gal4p and TAF12p, respectively. Immunoprecipitated DNAs were analyzed by PCR using the primer pair targeted to the GAL1 UAS.

FIGURE 5.

Rad14p associates with the promoters and coding sequences of the GAL7 and GAL10 genes in a transcription-dependent manner. A, the schematic diagrams show the PCR primer pairs located at the promoters and ORFs of the GAL7 and GAL10 genes in the ChIP assay. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). B and C, Rad14p associates with the promoters and coding sequences of the active GAL7 and GAL10 genes. The yeast strain expressing Myc epitope-tagged Rad14p (ZDY3) was grown, cross-linked, and immunoprecipitated as in Fig. 1B. D, RT-PCR analysis is shown. Both the wild type and Δrad14 strains (ZDY2 and PCY25) were grown for analysis of GAL7 and GAL10 transcriptions as in Fig. 1F. E and F, analysis of Rpb1p association with the promoters and coding sequences of the GAL7 and GAL10 genes in the Δrad14 strain (PCY25) and its isogenic wild type equivalent. The wild type and Δrad14 strains were grown, cross-linked, and immunoprecipitated as in Fig. 2A.

FIGURE 6.

Effect of Rad14p on the association of RNA polymerase II with GAL genes under steady-state conditions. A and B, kinetic analysis for the association of Rpb1p with the GAL1 promoter and coding sequence following transcriptional induction. Yeast cells were initially grown in YPR at 30 °C and then switched to YPG for different induction time periods before cross-linking. C, RT-PCR analysis is shown. D and E, analysis of Rpb1p association with the GAL7 and GAL10 promoters and coding sequences in the Δrad14 strain (PCY25) and its isogenic wild type equivalent after continuous growth in YPG up to an A₆₀₀ of 1.0 before cross-linking. F, RT-PCR analysis is shown.

FIGURE 7.

Effect of Rad14p on transcription of constitutively active genes. A and B, analysis of Rpb1p association with the ADH1 promoter and coding sequence in the Δrad14 strain (PCY25) and its isogenic wild type equivalent following continuous growth in YPD up to an A₆₀₀ of 1.0 before cross-linking. C, RT-PCR analysis of ADH1 is shown. D, RT-PCR analysis of PGK1 and PYK1. E and F, analysis of Rpb1p association with the promoters and coding sequences of PGK1 and PYK1 in the wild type and Δrad14 strains. G, RT-PCR analysis of RPS5. H, analysis of Rpb1p association with RPS5 in the wild type and Δrad14 strains.

FIGURE 8.

Rad14p promotes transcriptional induction of CUP1, STL1, and CTT1. A, Rad14p promotes the association of RNA polymerase II with CUP1. B, RT-PCR analysis of CUP1. C, RT-PCR analysis of STL1 and CTT1 in the wild type and Δrad14 strains following transcriptional inductions by NaCl is shown. D, analysis of Rpb1p association with STL1 and CTT1 in the wild type and Δrad14 strains.