Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling

Abstract

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high-affinity inorganic phosphate (P_i) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID-dependent manner in the absence of P_i in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of P_i in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, P_i in the growth medium switches transcriptional initiation of PHO84 from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, P_i, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.

Keywords: 19S RP; NuA4; SAGA; TFIID; transcription.

Figure 1

SAGA is required for PIC formation at the PHO84 core promoter to initiate transcription in the presence of inorganic phosphate (P_i) in the growth medium. (A) ChIP analysis of TBP and RNA polymerase II (Rpb1) at the PHO84 core promoter in the Δspt20 and WT strains in the growth medium with P_i (i.e., YPD or +Pi). The ChIP signal of the WT strain was set to 100, and the ChIP signal of the mutant strain was normalized with respect to 100. (B) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and Δspt20 strains in YPD. (C) ChIP analysis for the recruitment of the Spt20 component of SAGA to the PHO84 promoter in YPD. Upper panel: Schematic diagram showing the locations of the primer pairs at the PHO84 promoter for ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (D) ChIP analysis of TBP and Rpb1 at the PHO84 core promoter in the Δspt20 and WT strains in the growth medium without P_i (i.e., YPD-P_i or –P_i). (E) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and Δspt20 strains in growth medium without P_i. (F) ChIP analysis for recruitment of the Spt20 component of SAGA to the PHO84 promoter in the absence of Pi in the growth medium. (G) ChIP analysis of TBP and Rpb1 at the PHO84 core promoter in the presence and absence of Pi in the growth media. (H) Results of (G) are plotted in the form of a histogram. (I) Relative PHO84 mRNA levels in the presence and absence of Pi in the growth media. ChIP, chromatin immunoprecipitation; Pi, inorganic phosphate; PIC, preinitiation complex; UAS, upstream activating sequence; WT, wild-type; Chr. -V, Chromosome V; and TBP, TATA-box binding protein.

Figure 2

TFIID is required for PIC formation at the PHO84 core promoter to initiate transcription in the absence of P_i in the growth medium. (A) ChIP analysis of Rpb1 at the PHO84 core promoter in the taf13-ts mutant and WT strains in the growth medium with or without P_i. (B) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and taf13-ts mutant strains in the growth medium without P_i. (C) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and taf13-ts mutant strains in the growth medium with P_i. (D) ChIP analysis for the recruitment of TAF1 to the PHO84 UAS, core promoter, and Chr.-V in the absence of Pi in the growth medium. Mock ChIP control was performed without using an antibody. (E) ChIP analysis for the recruitment of TAF10 and TAF12 to the PHO84 core promoter and Chr.-V in the absence of P_i in the growth media. (F) ChIP analysis for the recruitment of TAF10 and TAF12 to the PHO84 core promoter, PHO84 UAS, and Chr.-V in the absence and presence of P_i in the growth. (G) ChIP analysis for the recruitment of TAF1 to the PHO84 core promoter, PHO84 UAS, and Chr.-V in the presence of Pi in the growth medium. ChIP, chromatin immunoprecipitation; Chr.-V, chromosome-V; Pi, inorganic phosphate; UAS, upstream activating sequence; WT, wild-type.

Figure 3

NuA4 HAT is required for PIC formation at the PHO84 core promoter (and hence transcription) in the absence of P_i in the growth medium. (A and B) ChIP analysis of Esa1 at the PHO84 UAS in the presence and absence of P_i in the growth media. (C) ChIP analysis of TBP and Rpb1 at the PHO84 core promoter in the esa1-ts mutant and WT strains in the presence or absence of P_i in the growth medium. (D) Esa1 is required for recruitment of a TFIID-specific TAF1 to the PHO84 core promoter in the absence of Pi in the growth medium. (E) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and esa1-ts mutant strains in the absence of P_i in the growth medium. (F) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and esa1-ts mutant strains in the presence of P_i in the growth medium. ChIP, chromatin immunoprecipitation; Chr.-V, chromosome-V; Pi, inorganic phosphate; PIC, preinitiation complex; UAS, upstream activating sequence; WT, wild-type.

Figure 4

The 19S RP is required for recruitment of RNA polymerase II and TBP to the PHO84 core promoter, and hence transcription, in the presence as well as absence of P_i in the growth media. (A and B) ChIP analysis for association of RNA polymerase II and TBP with the PHO84 core promoter in the WT and rpt4-ts mutant strains in the growth medium with or without Pi. (C and D) RT-PCR analysis of PHO84 mRNA and 18S rRNA levels in the WT and rpt4-ts mutant strains in the presence or absence of P_i in the growth medium. ChIP, chromatin immunoprecipitation; Chr.-V, chromosome-V; Pi, inorganic phosphate; RP, regulatory particle; UAS, upstream activating sequence; WT, wild-type.

Figure 5

The 19S RP is predominantly recruited to the PHO84 UAS to promote the targeting of NuA4 HAT and SAGA to the PHO84 UAS in the absence and presence of P_i in the growth media, respectively. (A and B) ChIP analysis of Rpt2 at the PHO84 promoter and an inactive region within Chr.-V in the growth medium with or without Pi. (C) ChIP analysis of NuA4 HAT at the PHO84 UAS in the WT and rpt4-ts mutant strains in the growth medium lacking Pi. (D) ChIP analysis of TAF10 and TAF12 at the PHO84 UAS in the WT and rpt4-ts mutant strains in the growth medium containing Pi. ChIP, chromatin immunoprecipitation; Chr.-V, chromosome-V; Pi, inorganic phosphate; RP, regulatory particle; UAS, upstream activating sequence; WT, wild-type.

Figure 6

ChIP analysis of Pho2 and Pho4 at the PHO84 UAS in the presence and absence of Pi in the growth media. (A and B) ChIP analysis of Pho2 and Pho4 at the PHO84 UAS in the rpt4-ts mutant and WT strains in the growth medium lacking Pi. Maximum ChIP signal was set to 100 and other ChIP signals were normalized with respect to 100. (C) ChIP analysis of Pho2 at the PHO84 UAS in the Δspt20 and esa1-ts mutants and their isogenic WT equivalents in the absence of Pi in the growth medium. Statistical analysis was performed using four sets of biologically independent experiments. (D and E) ChIP analysis of Pho2 and Pho4 at the PHO84 UAS in the rpt4-ts mutant and WT strains in the growth medium containing Pi. (F) ChIP analysis of Pho2 at the PHO84 UAS in the Δspt20 and esa1-ts mutants and their isogenic WT strains in the presence of Pi in the growth medium. (G) ChIP analysis of Pho4 at the PHO84 UAS in the Δspt20 mutant and its isogenic WT equivalent in the growth medium with Pi. ChIP, chromatin immunoprecipitation; Chr.-V, chromosome-V; Pi, inorganic phosphate; UAS, upstream activating sequence; WT, wild-type.

Figure 7

ChIP analysis of RNA polymerase II recruitment at the PHO5 and PHO12 core promoters in the esa1-ts, taf13-ts, and rpt4-ts mutants and their isogenic WT equivalents in the absence of Pi in the growth medium. (A) Esa1 is required for TAF1 recruitment to the PHO5 and PHO12 core promoters in the growth medium lacking Pi. (B–D) TAF13, Esa1, and Rpt4 are required for recruitment of Rpb1 to the PHO5 and PHO12 core promoters in the growth medium without Pi. ChIP, chromatin immunoprecipitation; Pi, inorganic phosphate; WT, wild-type.