Functional Impact of Chromatin Remodeling Gene Mutations and Predictive Signature for Therapeutic Response in Bladder Cancer

Most missense mutations in the HAT domain of EP300 and CREBBP lead to HAT inactivation. A, Schematic showing the location of bladder cancer patient point mutations in p300 and CBP proteins relative to each other. Line is to scale. B, Each patient mutation was introduced into an EP300 construct which was then transfected into cells. Most, but not all, HAT domain point mutations lead to a loss of acetyltransferase activity as judged by the level of HistoneH3 acetylation at lysine 27. The blots are representative of three independent experiments in each cell line. Each graph shows the mean and standard error from the three independent experiments, with the data being normalized to vector alone (mean of 1). C, CREBBP HAT domain missense mutations analyzed as in B.

The computer program Mutation Assessor predicted HAT activity of this bladder cancer patient dataset with 100% accuracy. A, The chart displays the mean and standard error of the 9 experiments performed above on p300 mutants in three cell lines. The Mutation Assessor functional impact label for each mutation is listed above the chart. B, Display of the mean and SE of 9 experiments performed above on CBP mutants in 3 cell lines. C, The difference in HistoneH3-Ac27 levels in a HAT domain mutant relative to the corresponding wild-type is plotted on the y-axis. For each mutation the value is represented as a circle, color coded as in A and B above to correspond to mutation assessor functional impact scoring. D, To mathematically assess correlation between observed acetyltransferase activity and Mutation Assessor prediction we assigned the Mutation Assessor designations numerical values: high = 3; medium = 2: low = 1. There is a strong correlation (Pearson) of 0.8918 between predicted HAT inactivity and decreased levels of acetylation of K27 of HistoneH3 (P < 0.0001).

Structural analysis of the missense mutants mapped onto the p300 HAT domain. A, Ribbon representation of the HAT domain colored in gray with the bisubstrate inhibitor shown in cyan. The high impact, in activating mutations are boxed in red while the low/medium impact, wild-type like mutations are boxed in green. Panel i and iii are a view of the p300 active site, detailing the environments of the deleterious mutation sites. Panel ii and iv are close-up views, with black dotted lines representing inferred hydrogen bonds. The surface rendering of amino acids highlights the role of the hydrophobic mutants in stabilizing hydrophobic pockets. B, Structural analysis of the missense mutants mapped onto the HAT domain from the CBP homology model. Panel i is a ribbon representation of the HAT domain, colored in gray, and the bisubstrate inhibitor colored in cyan. Panel ii is a close-up view of the p300 active site detailing the environments of the deleterious mutation sites.