Endogenous expression of inactive lysine deacetylases reveals deacetylation-dependent cellular mechanisms.

Abstract

Acetylation of lysine residues is an important and common post-translational regulatory mechanism occurring on thousands of non-histone proteins. Lysine deacetylases (KDACs or HDACs) are a family of enzymes responsible for removing acetylation. To identify the biological mechanisms regulated by individual KDACs, we created HT1080 cell lines containing chromosomal point mutations, which endogenously express either KDAC6 or KDAC8 having single inactivated catalytic domain. Engineered HT1080 cells expressing inactive KDA6 or KDAC8 domains remained viable and exhibited enhanced acetylation on known substrate proteins. RNA-seq analysis revealed that many changes in gene expression were observed when KDACs were inactivated, and that these gene sets differed significantly from knockdown and knockout cell lines. Using GO ontology, we identified several critical biological processes associated specifically with catalytic activity and others attributable to non-catalytic interactions. Treatment of wild-type cells with KDAC-specific inhibitors Tubastatin A and PCI-34051 resulted in gene expression changes distinct from those of the engineered cell lines, validating this approach as a tool for evaluating in-cell inhibitor specificity and identifying off-target effects of KDAC inhibitors. Probing the functions of specific KDAC domains using these cell lines is not equivalent to doing so using previously existing methods and provides novel insight into the catalytic functions of individual KDACs by investigating the molecular and cellular changes upon genetic inactivation.