A chemical method for labeling lysine methyltransferase substrates

Abstract

Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

Scheme 1

A chemical biology method for labeling lysine methyltransferase substrates. A) Alkyne–SAM analogue 1. B) PKMT substrate labeling method: enzymatic transfer by a PKMT of a terminal alkyne from 1 to a protein substrate is followed by CuAAC to ligate an azido-FLAG epitope to the substrate protein.

Figure 1

Alkyne–SAM assessment in lysine methylation assays. A) SETDB1 was incubated with GST–H3_tail and 1 or controls (no cofactor, trace material from analytical HPLC prep). Following the methyltransferase reaction, the product was treated with the azide-FLAG and the samples were analyzed by SDS-PAGE and immunoblotting by using the indicated antibodies. B) Methyltransferase reactions were performed as in panel A, but by using 1 in increasing amounts.

Figure 2

Alkyne–SAM transfer onto histone H3 by SETDB1. A) Lysine methylation assays performed in the absence or presence of 1 and SETDB1. B) Lysine methylation assays conducted with SETDB2 along with SETDB1 as a control. C) Lysine methylation assays with 1 analyzed with the M5 anti-FLAG antibody to show the differential migration of the alkyne-modified substrate. D) Recombinant histone H3 was immunoprecipitated by using FLAG M2–agarose after CuAAC.