FIG. 4.

Amino terminus of Rtf1 is required for physical interaction with Chd1. (A) Two-hybrid analysis of the interaction between Rtf1 mutants and Chd1. Tenfold serial dilutions, ranging from 1 × 10⁸ cells/ml to 1 × 10⁴ cells/ml, of the two-hybrid reporter strain PJ69-4A expressing GAD-Chd1 (pKA202) and GBD fusions to wild-type Rtf1 (top row) or the Rtf1 deletion mutants were spotted on SC-His-Leu-Trp medium (−His) to monitor HIS3 activation and SC-Leu-Trp medium to control for growth. Plates were incubated at 30°C for 3 days. A GBD fusion to Rtf1Δ11 was not analyzed for technical reasons. (B) Coimmunoprecipitation of HA-Chd1 in strains expressing Rtf1 region 1 mutants. Extract from strains expressing untagged wild-type Rtf1 (KY1213; lanes 1 to 4), Rtf1Δ1 (KY1214; lanes 5 to 8), or Rtf1-1 (KY1215; lanes 9 to 12) was incubated with anti-Rtf1 antibody or preimmune (Pre-I) serum. Immunoblot analysis was performed to assess the presence of Rtf1 and HA-Chd1 in the immunoprecipitated fraction. Lanes 1, 3, 5, 7, 9, and 11 contain 20 μg of unbound (U) material; lanes 2, 4, 6, 8, 10, and 12 contain the total bound (B) fraction. (C and D) ChIP analysis of Chd1 recruitment to active ORFs in rtf1 mutants. HA-Chd1 and associated DNA were immunoprecipitated (IP) from extracts of a formaldehyde-treated rtf1Δ strain (KY623) that had been transformed with an empty vector or plasmids that express untagged wild-type Rtf1 or Rtf1Δ1. An untagged Chd1 strain (KY452) expressing untagged wild-type Rtf1 (pLS20) was used as a control. Association of Chd1 at the 5′ ORF of PYK1 (C) or TEF2 (D) was assessed by PCR. The means of three independent experiments with standard errors are shown. The signal from strains expressing full-length Rtf1 is set at 1. Rel., relative.