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. 2007 Sep;177(1):101–112. doi: 10.1534/genetics.106.067140

Figure 3.—

Figure 3.—

Analyses of the association of Spt16 and RNA Pol II across the PMA1 gene in the context of wild-type or mutant versions of histone H3 and Spt16. (A) H3-L61W causes Spt16 to accumulate at the 3′-UTR of PMA1, and the two Spt16 mutants isolated in this study alleviate this defect. ChIP experiments were performed on H3 WT (yAAD1052 and yAAD1053), H3-L61W (yAAD1048 and yAAD1049), H3-L61W Spt16-E857Q (yAAD1060 and yAAD1061), and H3-L61W Spt16-E790K (yAAD1127 and yAAD1128) using an Spt16-specific polyclonal antibody (see materials and methods). Nine primer sets were used to assess Spt16 binding across the PMA1 ORF (shaded area) as well as within the 5′- and 3′-UTRs in the different genetic backgrounds. The percentage of immunoprecipitation values (%IP) indicated in the bar graphs for each of the strains at each region across the PMA1 locus are relative to the corresponding input material. Each %IP value represents the average with standard error from three independent experiments. As a reference point, the average levels of Spt16 binding to an untranscribed region (see materials and methods) were as follows: H3 WT, 0.22% ± 0.006; H3-L61W, 0.45% ± 0.009; H3-L61W Spt16-E857Q, 0.47% ± 0.007; and H3-L61W Spt16-E790K, 0.38% ± 0.008. (B) H3-L61W does not appear to greatly affect the distribution of RNA Pol II across the PMA1 locus. ChIP experiments were performed on H3 WT (yAAD1052) and H3-L61W (yAAD1049) strains using a monoclonal antibody specific for the Rpb3 subunit of RNA Pol II (see materials and methods). The two regions assayed are indicated above the PMA1 locus diagram. The %IP values are relative to the input material and in each case represent the average of two independent experiments. As a reference point, the average levels of Rpb3 binding to the untranscribed region were as follows: H3 WT, 0.09% ± 0.01; and H3-L61W, 0.10% ± 0.01. For both sets of experiments, the analysis and quantitation of the signals were carried out using the method described by Martens and Winston (2002) (see materials and methods).