Rif2 Promotes a Telomere Fold-Back Structure through Rpd3L Recruitment in Budding Yeast
Figure 2
Chromatin immunoprecipitation confirms structural defects.
(A) The immunoprecipitation of Rap1 following cross-linking should be associated with subtelomeric sequences at natural telomere 6R if the fold-back structure is intact (upper diagram); however the subtelomeric ChIP will be lost upon loop opening (lower diagram). (B) Upon Rap1 ChIP from exponentially growing cells, a subtelomeric signal was detected up to 1 kb away from the base of the telomeric repeats in wild type cells, whereas the signal was largely diminished in hda1Δ, sin3Δ and sir4Δ mutants. DNA stemming from the actin locus (ACT1) was not detected following Rap1 ChIP and was used as a background control. Error bars represent SD from three independent experiments. (C) Cdc13-TAP (13) was also able to precipitate subtelomeric DNA up to 1 kb away from the start of the telomeric sequence at telomere 6R following cross-linking (n = 3, error as SD) in comparison to wild type (non-tagged controls). This ChIP signal at -1000 was reduced to that of non-tagged controls in the sin3Δ strain. The difference in ChIP signal distribution between Rap1 (B) and Cdc13-TAP (C) is likely due to the different positioning of the two proteins on the telomere (compare diagrams in A and C for explanation). For all experiments above error bars represent SD of the mean from at least 3 independent experiments and * indicates statistically significant differences as determined through unpaired student's t-tests whereby * = p<0.05, ** = p<0.01, *** = p<0.001.