Methodology development for imaging histone modifications and for site-specific protein labeling in vitro and on the surface of living cells

摘要

A new methodology for monitoring post-translational modifications of histone H3 in living cells was developed using genetically encoded fluorescent reporters. These reporters were constructed for sensing histone phosphorylation and methylation by fluorescence resonance energy transfer (FRET). These reporters are four-part chimeric proteins with the domains from N- to C-terminus as follows: a cyan fluorescent protein (CFP), a phosphoserine recognition domain (14-3-3 tau) or a methyllysine recognition domain (HP1 or Pc chromodomain), a peptide whose sequence corresponds to the Nterminus of histone H3, and a yellow fluorescent protein. The histone phosphorylation reporter exhibited a 25 % YFP/CFP emission ratio increase upon phosphorylation in vitro by Msk-1 kinase. Site-directed mutagenesis studies suggested that Ser28 phosphorylation gave rise to the reporter FRET response. When tested in living HeLa cells, the reporter exhibited a rapid increase in the emission ratio 5-15 min prior to the nuclear membrane breakdown and the FRET peaked during cell division. Another reporter, in which the 14-3-3 tau and the H3 peptide were swapped, exhibited the FRET response to both Ser10 and Ser28 phosphorylation. Two methylation reporters, K9 and K27, were constructed for sensing H3-lysine9 and H3-lysine27 methylation.