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. 2013 Sep 19;9(9):e1003776. doi: 10.1371/journal.pgen.1003776

Figure 8. Histone dynamics during transcription elongation is impaired in pfd1Δ.

Figure 8

A. The absence of Pfd1 impairs the characteristic difference in histone occupancy along the transcribed region that occurs between active and inactive genes. Binding of H3 and H4 to transcribed (YPGAL) and untranscribed (YPD) GAL1p-YLR454w was measured by ChIP in wild-type (left) and pfd1Δ (right) exponentially growing cells. Values correspond to the mean and the standard deviation of three biological replicates. B. Sensitivity of GAL1p-YLR454w chromatin to micrococcal nuclease, under activating (YPGAL) and non activating (YPD) conditions is not affected by the absence of Pfd1. Note that the characteristic up and down pattern caused by the positioned nucleosomes present in the 5′ and 3′ ends of the gene under repressed conditions is equally absent in the wild type and in pfd1Δ. Cells exponentially growing in the indicated media were permeabilized and treated with MNase, as described in the Materials and methods section. Mononucleosomal DNA was then extracted and analyzed by quantitative PCR. Values were normalized to naked DNA digested with MNase and processed in parallel to the chromatin samples. Values correspond to the mean and the standard deviation of two biological replicates. C. The kinetics of the increase in histone occupancy that occurs after transcriptional repression is not influence by the absence of Pfd1. Histone occupancy in the 4 kb region of GAL1p-YLR454w was analyzed during the last wave of transcription after switching the GAL1 promoter off. Glucose was added at time 0 to wild-type and pfd1Δ cells growing exponentially in galactose. At the indicated times samples were taken to measure H3 binding to the 4 kb region by ChIP. Values were normalized to the levels of H3 occupancy measured 60 min after the addition of glucose to the cultures. The mean and the standard deviation of three biological replicates are shown. D and E. The difference in H3 occupancy produced by pfd1Δ and dst1Δ across GAL1p-YLR454w does not correlate with their effect on RNA polymerase II occupancy. Ratios of H3 occupancy between YPGAL and YPD in the indicated mutants across GAL1p-YLR454w (D), and levels of RNA polymerase II bound to GAL1p-YLR454w (E). H3 and Rpb3 occupancy was measured by ChIP, as described in the Materials and methods section. Values correspond to the mean and the standard deviation of three biological replicates.