To visualize histone acetylation in living cells, we developed a genetically encoded fluorescent resonance energy transfer (FRET)-based indicator. Response of the indicator reflects changes in the acetylation state of both K5 and K8 in histone H4. Using this acetylation indicator, we were able to monitor the dynamic fluctuation of histone H4 acetylation levels during mitosis, as well as acetylation changes in response to structurally distinct histone deacetylase inhibitors.BRDT ͉ chromatin ͉ FRET ͉ HDAC inhibitor C ovalent modification of core histones plays an important role in the modulation of chromatin structure and function. Acetylation is a well-characterized modification regulated by two families of evolutionarily conserved enzymes, histone acetyltrasferases (HATs) and histone deacetylases (HDACs) (1, 2). Acetylation mainly occurs on lysine residues of core histone N-terminal tails; in this context, the modification induces chromatin conformational change by unfolding higher order chromatin structure, and represents an epigenetic mark recognized by regulatory factors including coactivators. Core histone acetylation influences gene expression by modifying chromatin conformation and/or the recruitment of the regulatory factors. The acetylation of histone H4 is thought to occur initially at K16, and then propagates through K12, K8, and K5, progressing in an N-terminal direction (3). Thus, the simultaneous acetylation of both K5 and K8 in histone H4 is indicative of histone H4 hyperacetylation (4, 5). Acetylated histones are recognized by regulatory proteins containing bromodomains, for example, PCAF, Brd2, Brd4, and BRDT (6). Recently, it has been suggested that each distinct combination of covalent modifications of histone tails functions as an epigenetic code by regulating the interaction of histone tails with chromatin-associated proteins (7).In vivo histone acetylation is reversibly and dynamically regulated. However, in most studies on protein acetylation, conventional biochemical methods such as immunostaining have been used. These methods do not always provide enough information about the temporal and spatial dynamics of protein acetylation in living cells. In the case of other cellular dynamics, such as intracellular Ca 2ϩ and protein phosphorylation, visualization by fluorescence resonance energy transfer (FRET) in live cells has been used to successfully overcome the limitations of conventional methods (8). Here, we report a FRET-based indicator, named Histac, developed to allow visualization of protein acetylation in living cells.
Histone acetylation constitutes an epigenetic mark for transcriptional regulation. Here we developed a fluorescent probe to visualize acetylation of histone H4 Lys12 (H4K12) in living cells using fluorescence resonance energy transfer (FRET) and the binding of the BRD2 bromodomain to acetylated H4K12. Using this probe designated as Histac-K12, we demonstrated that histone H4K12 acetylation is retained in mitosis and that some histone deacetylase (HDAC) inhibitors continue to inhibit cellular HDAC activity even after their removal from the culture. In addition, a small molecule that interferes with ability of the bromodomain to bind to acetylated H4K12 could be assessed using Histac-K12 in cells. Thus, Histac-K12 will serve as a powerful tool not only to understand the dynamics of H4K12-specific acetylation but also to characterize small molecules that modulate the acetylation or interaction status of histones.
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