Spt6-mediated chromatin assembly and transcriptional repression at the PHO5 promoter. (a) UASp1 and UASp2 are binding sites for the Pho2 and Pho4 transactivators. During activation, chromatin disassembly of the four yellow nucleosomes is promoted by Asf1 to allow access of the general transcription machinery to the promoter. During repression, chromatin is reassembled over the PHO5 promoter by the histone H3/H4 chaperone Spt6 to compete with the general transcription machinery for DNA binding. (b) Strain JKT0010 (WT), JMY0002 (spt6-140), and MAY0067 (spt6-1004) were grown in phosphate-depleted media to activate PHO5 transcription. Following a 4 hour shift to 39°C to inactivate Spt6, phosphate was added as a signal for PHO5 repression. Samples were assayed for phosphatase activity at the indicated times after addition of phosphate. (c) Strain JKT0010 (WT) and MAY0067 (spt6-1004) were initially grown in phosphate-rich media (+Pi) where PHO5 is repressed, then shifted to phosphate-depleted media to activate PHO5 transcription (-Pi). Following a 2 hour shift to 39°C, phosphate was added. Samples were taken at the indicated times, and analyzed for histone occupancy at PHO5 UASp2 by chromatin immunoprecipitation (ChIP) analysis. The amount of immunoprecipitated DNA was determined by quantitative PCR. As a control, primer sets were used for TELVIR. Quantitation of H3 levels over the UASp2 region is a ratio of immunoprecipitated UASp2 product relative to the immunoprecipitated TELVIR product divided by the ratio of input UASp2 product relative to the ratio of input TELVIR product. Averages of three independent experiments are shown; error bars indicate the 95% confidence interval.