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

Abstract

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.

(cont.) The reporters gave 60 % (K9) and 28 % (K27) emission ratio changes after in vitro methylation, catalyzed by the histone methyltransferase vSET. Applying the K9 reporter in cells showed different levels of reporter FRET in fibroblasts either expressing or lacking methyltransferases Suv39h1 and Suv39h2. Site-specific incorporation of biophysical probes onto cell surface proteins is critical for the study of protein trafficking. One general solution to achieve labeling specificity is the use of enzymes for the ligation of probes to a substrate peptide tagged onto a protein of interest. Transglutaminase incorporates amine probes to cell surface proteins expressing a 6- or 7-amino acid consensus sequence (Q-tag). A variety of probes such as biotin cadaverine and fluorescein cadaverine were incorporated to Q-tag-CFP and Q-tag-EGF receptor constructs expressed on the surface of living HeLa and HEK cells. The NF?B p50 transcription factor fused to a Q-tag was labeled with a benzophenone photo-affinity probe in vitro. Upon UV irradiation, elevated levels of p50 homodimerization were observed in the presence of DNA or the interacting protein myotrophin.