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. 2007 Mar 9;128(5):1003-12.
doi: 10.1016/j.cell.2006.12.041.

The site-specific installation of methyl-lysine analogs into recombinant histones

Matthew D Simon  1 , Feixia Chu, Lisa R Racki, Cecile C de la Cruz, Alma L Burlingame, Barbara Panning, Geeta J Narlikar, Kevan M Shokat
Affiliations

The site-specific installation of methyl-lysine analogs into recombinant histones

Matthew D Simon et al. Cell. .

Abstract

Histone lysine residues can be mono-, di-, or trimethylated. These posttranslational modifications regulate the affinity of effector proteins and may also impact chromatin structure independent of their role as adaptors. In order to study histone lysine methylation, particularly in the context of chromatin, we have developed a chemical approach to install analogs of methyl lysine into recombinant proteins. This approach allows for the rapid generation of large quantities of histones in which the site and degree of methylation can be specified. We demonstrate that these methyl-lysine analogs (MLAs) are functionally similar to their natural counterparts. These methylated histones were used to examine the influence of specific lysine methylation on the binding of effecter proteins and the rates of nucleosome remodeling. This simple method of introducing site-specific and degree-specific methylation into recombinant histones provides a powerful tool to investigate the biochemical mechanisms by which lysine methylation influences chromatin structure and function.

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Figures

Figure 1
Figure 1. The Installation of Methyl-Lysine Analogs into Recombinant Proteins by Alkylating Cysteine Residues
(A) The MLA strategy is an extension of the traditional aminoethylation reaction in which a cysteine is alkylated with an electrophilic ethylamine producing aminoethylcysteine, an analog of lysine. (B) Cysteine residues can be converted into analogs of mono-, di-, and trimethylated lysine by treatment with alkylating agents 1-3, respectively. (C) Incubating 2 under basic conditions leads to aziridinium formation. The resulting aziridinium can react with a cysteine residue leading to the desired dimethyl lysine analog.
Figure 2
Figure 2. Cysteine Residues Can Be Efficiently Converted into MLAs in the Context of Peptides and Full-Length Proteins
(A) A cysteine-containing peptide (Fluroesceine-KACR-OH) is converted to the desired lysine analog under optimized reaction conditions (see Experimental Procedures) as observed by reverse phase HPLC analysis of the crude reaction products. The traces are for the starting peptide (Cys); the unmethylated lysine analog, KC; the monomethylated lysine analog, KC(me1); the dimethylated lysine analog, KC(me2); and the trimethylated lysine analog, KC(me3). (B) Mass spectra (ESI-oaTOF) demonstrate that MLAs can be cleanly installed at position 9 of full-length histone H3 protein. Starting with a K9C mutation (top left), this histone was treated under conditions to install analogs of monomethyl Lys9 H3 (top right), dimethyl Lys9 H3 (bottom left), or trimethyl Lys9 H3 (bottom right). The asterisk indicates a peak at +42 daltons corresponding to an artifact and is present in both the starting material and the methylated histone products. (C) Representative ECD spectra of full-length histones acquired from a FT-ICR mass spectrometer are consistent with the installation of desired analogs at histone H3 residue 9.
Figure 3
Figure 3. MLAs Can Be Incorporated into Histone Octamers and Are Specifically Recognized by Antibodies Raised against the Corresponding Natural Modifications
(A) Western blot analysis of octamers assembled with MLAs at position 9 of histone H3. (B) Similar analysis with MLAs incorporated at various core and tail positions. (C) The positions of modifications in (A) and (B) mapped onto a model of the nucleosome (based on Luger et al., 1997).
Figure 4
Figure 4. Methyl Lys9 Analogs Behave Similarly to Their Natural Counterparts in Binding Assays
(A) Based on the crystal structure of HP1 (green) bound to a K9me2 peptide (gray backbone, PDB:1KNA, from Jacobs and Khorasanizadeh [2002], a model of a KC9me2 peptide (yellow backbone shown as overlay) bound to HP1 was constructed and minimized using MOLOC. (B) Analysis of HP1α from 293 nuclear extracts binding to immobilized H3 tail peptides. Peptides (100 μg) with either unmodified lysine at position 9 (K9), with natural dimethylation at Lys9 (K9me2) or containing a dimethyl lysine analog, KC9me2, were used as affinity reagents and bound HP1α was monitored by western blot. In (B)–(E), input represent 5% of the starting nuclear extract. (C) HP1α from nuclear extracts is specifically enriched upon binding to immobilized nucleosomes (100 pmol) assembled with H3KC9me2 relative to unmodified nucleosomes (WT). (D) SUZ12 is enriched when using both KC9me2 modified and unmodified (WT) nucleosomes as affinity reagents. (E) DNMT1 does not pull down using either KC9me2 modified or unmodified (WT) nucleosomes as affinity reagents.
Figure 5
Figure 5. Analysis of MLAs as Substrates in Enzymatic Assays
(A) The Lys9 methyltransferase activity SUV39H1 was examined using [3H-Me]-SAM and biotinylated H3 tail peptides (residues 1–14) containing either unmodified lysine at position 9 (K), dimethyl lysine (K9me2), aminoethylcysteine (KC9), the monomethyl lysine analog (KC9me1), or the dimethyl lysine analog (KC9me2). Data are shown as white bars for peptides with natural lysine residues and as gray bars for lysine analogs. (B) Schematic depicting the assembly and hACF-mediated remodeling of positioned mononucleosomes. (C) Similar levels of hACF-mediated ATP-dependent remodeling activity are observed using nucleosomes with or without MLAs. Upon native gel electrophoresis, the migration of centrally positioned nucleomsomes is retarded relative to the migration of end-positioned nucleosomes. (D) The maximal rate of ACF-mediated nucleosome remodeling is not affected by KC20me3 or KC79me2 as demonstrated in a remodeling assay observed at several different time points. (E) Graphical analysis of the results form (D) demonstrating similar rates of remodeling of wild-type and modified nucleosomes.
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