SWI/SNF-dependent chromatin remodeling of RNR3 requires TAF(II)s and the general transcription machinery

Abstract

Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters. Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established. Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II. We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter. In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment. Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities.

Figure 1

TAF_IIs are required for MMS-induced chromatin remodeling of RNR3. (A) Nucleosome mapping in TAF_II mutants. Wild type (SW87) and strains containing temperature-sensitive mutations in yeast taf1-2 (YSW93) and taf12-9 (YJR21-9) were grown and treated with MMS at 37°C as described in the text. Cells were then harvested to prepare nuclei for chromatin mapping and RNA for Northern blotting. The nucleosome organization of the RNR3 promoter is illustrated on the left side of the panel and is based on previous detailed mapping studies (Li and Reese 2001). (B) Northern blot analysis of RNR3 mRNA levels in repressed and MMS-treated cells. scR1 is a loading control. Quantification of Northern blots was performed using the ImageQuant software, and was normalized to the signal in the −MMS sample of the wild type, which was arbitrarily defined as a unit of 1.

Figure 2

RNA polymerase II and Kin28 are required for MMS-induced chromatin remodeling of RNR3. Chromatin mapping and transcriptional analysis were performed in temperature-sensitive rpb1-1 and kin28-16 mutants using the same procedure described in the legend for Figure 1. (A) MNase mapping, as in Figure 1. The first lane (M) contains a DNA marker. (B) Northern blot analysis of RNR3 mRNA levels. (C,D). Analysis of the expression and chromatin remodeling of the SUC2 promoter in the rpb1-1 mutant. Wild-type cells and a strain containing a temperature-sensitive mutation in rpb1-1 were grown in YPD (2% dextrose) at the permissive temperature (23°C) and then mixed with an equal volume of prewarmed fresh YPD medium (50°C) to rapidly elevate the temperature of the culture to 37°C. After a 45-min preinactivation, cells were collected by filtration and washed with 37°C YP media. The cell pellets were then resuspended in 37°C low-dextrose (0.05%) YP medium and incubated at 37°C for 2 h. Cells were harvested and subjected to chromatin mapping and Northern blotting. (C). Northern blot analysis of SUC2 and scR1 mRNA levels. (D) MNase mapping. The arrows indicate the regions that are hypersensitive to MNase under the derepressing condition and correspond to the TATA box and the B and C boxes (Hirshhorn et al. 1992). The first lane (M) contains a DNA marker, and digested naked DNA (ND) is shown to the right.

Figure 3

Mediator (SRB4), but not NUT1 specifically, is required for the DNA damage-induced chromatin remodeling of RNR3. Wild type (Z579) and temperature-sensitive srb4-138 (Z628) mutant were grown and processed as in Figure 1. (A) Northern blot analysis of RNR3 mRNA. (B) MNase mapping. Analysis of expression and chromatin structure of RNR3 in the srb4-138 mutant. (C). Northern blot analysis of RNR3 mRNA levels in wild type and Δnut1 mutant. Cells were treated with (+) or without (−) MMS for 2.5 h at 30°C. Quantification of RNR3 transcripts was performed using the ImageQuant software program. The expression level in wild-type cells without MMS treatment is arbitrarily defined as a unit of 1. scR1 is a loading control. (D) MNase chromatin mapping was performed using wild type (PH499) and the Δnut1 (YJR709) mutant treated with or without MMS for 2.5 h at 30°C. ND, digested naked DNA; M, a DNA size marker.

Figure 4

SWI/SNF regulates RNR3 expression and nucleosome remodeling. (A). Northern blot analysis of RNR3 mRNA levels. (B). Chromatin immunoprecipitation assay (ChIP) for TBP and RNA polymerase II cross-linking. SNF2 and Δsnf2 strains were grown at 30°C and treated with MMS for 2 h (gray bars) or left untreated (white bars). Immunoprecipitations (IPs) were carried out using a polyclonal antibody against TBP, a monoclonal antibody against RNA polymerase II (8WG16), and preimmune serum (PI) or anti-HA (HA) as controls. Immunoprecipitated and input DNA were amplified by PCR using primers specific for the core promoter regions of RNR3. Data are expressed as the means and standard deviations of the cross-linking from three independent experiments. The amount of cross-linking in untreated wild-type cells was arbitrarily set at a value of 1. (C) MNase mapping. The lane on the right (ND), indicates digestion of naked DNA by MNase. (D) ChIP assay for Snf2-myc13 binding. Strain YJR589 (SNF2-MYC13) was treated with (2 h) and without MMS prior to cross-linking as indicated. ChIPs were carried out using monoclonal antibodies to the MYC tag and HA tag (as a negative control for background). Similar background was observed in IPs using untagged strains. Immunoprecipitated and input DNA were amplified by PCR using primers directed to the regions of RNR3 indicated in the panel. For reference, +1 is the major transcription start site; the TATA box is located at −75 and the URS between −330 and −200.

Figure 5

Cross-linking of transcription factors to the RNR3 promoter in the presence and absence of DNA damage. (A) YJR589 was grown at 30°C and treated with or without MMS where indicated. IPs were carried out using polyclonal antibodies against TBP, TAF_IIs, and a monoclonal antibody against RNA polymerase II (8WG16). (Upper panel) Results from one experiment. The IPs using preimmune sera shown correspond to the rabbits used to raise antiserum against TAF1p and TBP. Similar levels of background were observed using three other preimmune sera or HA monoclonal antibody. IP and input DNA were amplified by PCR using primers specific for the promoter of RNR3 or +1500 bp from the start site of transcription (ORF). Lanes 1–6 show PCR reactions using 3.3-fold dilutions of input DNA. The DNA from the RNA polymerase II IP was diluted fivefold prior to amplification to assure linearity. The ChIP results are expressed as signal above preimmune sera, which was arbitrarily set at 1.0. (Lower panel) Graphical representation of the MMS-induced increase in cross-linking from three to five experiments, using at least three independently prepared chromatin preparations. (B) Chromatin immunoprecipitation analysis of transcription factor occupancy at the RNR3 promoter in temperature-sensitive mutants. Wild-type (YJR589) and taf1-2 (YJR595), taf12-9 (YJR592), and rpb1-1 (YJR598) strains, all containing SNF2-13MYC, were grown at 25°C and then shifted to 37°C for 45 min to inactivate the transcription factors. Cells were then treated with 0.02% MMS for 2 h (gray bars) or left untreated (white bars). IPs were carried out with antibodies against TBP, TAF1p, TAF12p, Pol II (8WG16), Ac-H3 (Di-acetyl Lys 9 and Lys 14), and (9E10) c-myc. Cross-linking of Srb4-myc was performed the same as the others except strains contained SRB4-13MYC (YJR588, YJR594, YJR591, and YJR597). The data are presented as a ratio of the amount of PCR product in wild-type cells, −MMS. Data are from at least three independent chromatin preparations and experiments. The increase in TAF1p cross-linking in the taf1-2 mutant (−MMS) is the result of a single aberrant value, and is not truly indicative of increased cross-linking in all repetitions.

Figure 6

SAGA facilitates SWI/SNF remodeling at RNR3. (A) Northern blot for RNR3 in Δgcn5 and Δada2 mutants. PSY316, PSY316Δgcn5, and PSY316Δada2 were grown in YPAD at 30°C and treated with MMS for 2 h. scR1 is a loading control. (B) Analysis of histone H3 acetylation in HAT complex mutants. A single experiment is shown. Results from three independent chromatin preparations and experiments yielded similar results with errors of 10%. (C) Chromatin mapping in wild-type and Δgcn5 cells. (D) SWI/SNF and polymerase II recruitment. As in Figure 5, except strains YJR589 (SNF2-MYC13) and YJR715 (Δgcn5; SNF2-MYC13) were used. The data are presented relative to the amount of PCR product in IPs from untreated wild-type cells (−MMS), and is the result of at least three independent chromatin preparations and experiments. White bars, −MMS; gray bars, +MMS.

Figure 7

PIC formation is required to retain SWI/SNF at the promoter. (A) ChIP assay. Wild-type (YJR589), Δcrt1 (YJR732), taf1-2/Δcrt1 (YJR734), taf12-9/Δcrt1 (YJR733), and rpb1-1/Δcrt1 (YJR735) strains, all containing SNF2-13MYC, were grown at 25°C and then shifted to 37°C for 45 min to inactivate the transcription factors (white bars), treated with 0.02% MMS for 2 h at 37°C (gray bars), cross-linked with formaldehyde and processed. (B) RNA polymerase II and Kin28 are required for maintaining the fully remodeled state at the RNR3 promoter. Wild-type (SW87), Δcrt1 (YJR352), rpb1-1/Δcrt1 (YJR658), kin28-16/Δcrt1 (YJR660) and taf12-9/Δcrt1 (YJR733) strains were grown at the permissive temperature (23°C) and then rapidly shifted to 37°C by mixing with an equal volume of prewarmed fresh YPD medium (50°C). Cultures were maintained at 37°C for 2 h prior to harvesting for chromatin mapping. Note: The lane on the far left in the taf12-9/crt1 panel on the right is digested naked DNA (ND).