Association of the Mediator complex with enhancers of active genes

Abstract

The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro. Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo. We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Fig. 1.

Rgr1 association with GAL genes. (A) Schematic of GAL1–10 and GAL2 promoters and regulatory regions showing location of primer pairs used in ChIP analysis. For GAL1–10:A, –690/–481; B, –491/–322; C, –142/+81; and D, +252/+439 (coordinates are given relative to GAL1 ATG). For GAL2: E, –794/–524; F, –591/–538; G, –179/+70; and H, +285/+444 (coordinates are given relative to GAL2 ATG). The arrows indicate the position of transcription start sites. (B) Chromatin from cells expressing TAP-tagged (+) or untagged (–) Rgr1 and HA₃-tagged TBP grown in YP medium containing glucose (D), raffinose (R), or galactose (G) was immunoprecipitated with IgG-agarose (Rgr1), anti-HA (TBP), or anti-Gal4 (Gal4) antibodies. Immunoprecipitated (IP) and input (Total) DNA were analyzed by radioactive PCR by using primers shown in A and primers located in a transcriptionally inactive region (Control). PCR products were separated on an 8% TBE polyacrylamide gel and visualized by autoradiography. Signals shown in Total or IP derive from identical dilutions and were confirmed to be in the linear range of PCR. (C) Quantitation of the ChIP data obtained from the cells grown in galactose. Amounts of PCR products were quantified by PhosphorImager analysis, and IP/Total ratios were calculated by dividing the amount of PCR product obtained with IP DNA by the amount obtained with the total DNA, using each time at least two dilutions confirmed to be in the linear range of the PCR (standard error <15%, data not shown). The highest value obtained in each immunoprecipitation was set to 100, and other values were expressed relative to this maximum.

Fig. 2.

Mediator association with GAL genes. Equivalent amounts of crosslinked chromatin extracts prepared from cells grown in YP galactose medium and expressing Rgr1-TAP, Nut1-TAP, Med7-TAP, or Srb6-TAP and HA₃-TBP were immunoprecipitated in parallel with IgG-agarose (Mediator) or anti-HA antibodies (TBP). Immunoprecipitated (IP) and input (Total) DNA were analyzed with primer pairs described in Fig. 1.

Fig. 3.

Association of Rgr1 with GAL genes in TFIIB mutant cells. (A) TFIIB temperature-sensitive (sua7-1) and isogenic WT cells, both expressing Rgr1-TAP and HA₃-TBP, were grown in YP galactose medium at permissive temperature (24°C) and shifted for 40 min to restrictive temperature (37°C). Chromatin was immunoprecipitated with anti-pol II (Pol II), anti-HA (TBP), IgG-agarose (Rgr1), and anti-Gal4 (Gal4) antibodies. Immunoprecipitated and input (Total) DNA were analyzed with primers for GAL1 and GAL2 (TATA element region for pol II and TBP and UAS_G region for Rgr1 and Gal4) and primers for a transcriptionally inactive region (Control). (B) Quantification by PhosphorImager analysis of the ChIP data obtained in the experiment presented in A. Quantification was carried out as described in Fig. 1.

Fig. 4.

Association of Srb6 with GAL genes in TFIIB mutant cells. Same as in Fig. 3 with cells expressing Srb6-TAP instead of Rgr1-TAP.

Fig. 5.

Association of Mediator with an isolated UAS_G. (A) Schematic of GAL1–10 regulatory region and pGAL1-xylE and pGAL1ΔT-xylE chimeric genes showing location of primers used in the ChIP analysis. The arrow indicates the position of GAL1 transcription start site. The chimeric genes contain a fragment of GAL1 promoter spanning position –537 to –13 (pGAL1-xylE) and position –537 to –225 (pGAL1ΔT-xylE) fused to xylE ORF and are integrated at the URA3 locus. (B) Quantitation of GAL1 and xylE RNA levels by RT-PCR in pGAL1-xylE and pGAL1ΔT-xylE cells grown in raffinose (R) and induced for 90 min with galactose (G). GAL1 and xylE maximum RNA levels were arbitrarily set to 1,000. (C) ChIP analysis of galactose-induced pGAL1-xylE and pGAL1ΔT-xylE cells expressing either Nut1-TAP or Rgr1-TAP. Crosslinked chromatin was immunoprecipitated with anti-pol II (Pol II), anti-HA (TBP), IgG-agarose (Rgr1), and anti-Gal4 (Gal4) antibodies. Immunoprecipitated and input (Total) DNA were analyzed by radioactive PCR with primers shown in A and primers specific for a transcriptionally inactive region (Control). Pair D was used specifically for samples deriving from pGAL1-xylE cells, and pair E was specifically for samples deriving from pGAL1ΔT-xylE cells. PCR products were separated on an 8% TBE polyacrylamide gel and visualized by autoradiography (Upper). Quantification in Lower was carried out as described in Fig. 1.