Transcription elongation factor Spt4 mediates loss of phosphorylated RNA polymerase II transcription in response to DNA damage

Abstract

Previously, we found that Rad26, the yeast Cockayne syndrome B homolog and the transcription elongation factor Spt4 mediate transcription-coupled repair of UV-induced DNA damage. Here we studied the effect of DNA damage on transcription by directly analyzing the RNA polymerase II localization at active genes in vivo. A rad26 defect leads to loss of Ser5 phosphorylated RNA polymerase II localization to active genes, while localization is only transiently diminished in wild type cells. In contrast, loss of Ser5-P RNAP II localization is suppressed in spt4 cells. Interestingly, even when DNA damage is persistent the absence of Spt4 leads to a delayed loss of transcription suggesting that Spt4 is directly involved in mediating transcription shutdown. Comparative analysis of phosphorylated and non-phosphorylated RNA polymerase II localization revealed that Ser5-P RNAP II is preferentially lost in the presence of DNA damage. In addition, we found evidence for a transient Rad26 localization to active genes in response to DNA damage. These findings provide insight into the transcriptional response to DNA damage and the factors involved in communicating this response, which has direct implications for our understanding of transcription-repair coupling.

Figure 1

Spt4 and Rad26 affect Ser5-P RNAP II localization after UV irradiation. ChIP experiment: chromatin extracts were prepared from cells either unirradiated or from irradiated cells at indicated time points post UV (in minutes). Relevant genotype of strains analyzed is indicated on the right. RNAP II was pulled down with H14 (S5-P). DNA extracted from non- precipitated WCE was included as control for difference in PCR efficiency for different loci. (A) Amplification of PMA1 promoter and coding sequences. (B) GAL7 promoter and coding sequences. HML locus was used as a negative control locus (lower bands in each panel).

Figure 2

Absence of Spt4 suppresses loss of RNAP II localization independent of repair. ChIP experiment as in Figure 1. (A) Amplification of PMA1 promoter and coding sequences. (B) GAL7 promoter and coding sequences. Quantification of the PCR product signals are displayed in histograms below corresponding PCR series. Fold cross-linking of is Ser5-P RNAP II to the gene is indicated as determined by the ratio of PCR product signals from the genes compared with the inactive HML locus.

Figure 3

Dose-dependent differential loss of Ser5-P RNAP II or non-phosphorylated RNAP II. ChIP experiment. Chromatin extracts were prepared from rad14 cells 30 min after irradiation with indicated UV doses. RNAP II was pulled down using H14 antibody (S5-P) or 8WG16 antibody (CTD). Top bands in each gel correspond to the promoter region or coding sequence of genes indicated flanking the gel. Lower bands are amplified from inactive HML locus.

Figure 4

Rad3 promoter localization is not altered upon UV irradiation. ChIP experiment. Triple HA-tagged Rad3 was immunoprecipitated from indicated strains using 12CA5 antibodies. (A) Rad3 localizes to the ACT1 promoter. Chromatin extracts were prepared from cells either unirradiated or from cells irradiated with 70 J/m² 10 min after irradiation. DNA was amplified from precipitates using primers corresponding to the promoter and CDS of the ACT1. (B) Rad3 localization is not altered in the absence of NER and Spt4 after a high UV dose. Rad3 was immunoprecipitated from chromatin extracts prepared from indicated strain either unirradiated or irradiated with 140 J/m² after indicated time points (minutes). Promoter and coding sequences were amplified from the GAL7 gene.

Figure 5

Rad26 co-localizes with transcription upon UV irradiation. ChIP experiment. Triple HA-tagged Rad26 was immunoprecipitated from indicated strain using 12CA5 antibodies. (A) Rad26 localizes to the ACT1 promoter after UV. Chromatin extracts were prepared from cells either unirradiated or from irradiated cells at indicated time points (min) post 70 J/m² of UV. DNA was amplified from precipitates using primers corresponding to the promoter and CDS of the ACT1 gene. Quantification of the PCR product signals are displayed in histograms below corresponding PCR series as in Figure 2. (B) Rad26 cross-links to RNAP II after UV. Rad26 was immunoprecipitated from chromatin extract prepared from cross-linked cells either unirradiated or from cells irradiated with indicated doses (in J/m²) at indicated time points (minutes) post UV. Precipitates were run across a SDS–PA gel blotted to a PVDF membrane and RNAP II was detected using H14 antibody.