Tudor, MBT and chromo domains gauge the degree of lysine methylation

Abstract

The post-translational modification of histones regulates many cellular processes, including transcription, replication and DNA repair. A large number of combinations of post-translational modifications are possible. This cipher is referred to as the histone code. Many of the enzymes that lay down this code have been identified. However, so far, few code-reading proteins have been identified. Here, we describe a protein-array approach for identifying methyl-specific interacting proteins. We found that not only chromo domains but also tudor and MBT domains bind to methylated peptides from the amino-terminal tails of histones H3 and H4. Binding specificity observed on the protein-domain microarray was corroborated using peptide pull-downs, surface plasma resonance and far western blotting. Thus, our studies expose tudor and MBT domains as new classes of methyl-lysine-binding protein modules, and also demonstrates that protein-domain microarrays are powerful tools for the identification of new domain types that recognize histone modifications.

Figure 1

Methylated peptides bind to tudor domains on the CADOR chip. (A) A collection of 109 glutathione S-transferase (GST) fusion proteins were arrayed in duplicate on a nitrocellulose slide. The layout of the array is shown. The middle position (M) contains GST alone as a background indicator. (B) The arrayed GST fusion proteins are listed. The accession numbers and regions cloned can be found in the supplementary information online. (C) The array was first probed with an anti-GST antibody and detected with a fluorescein isothiocyanate-conjugated secondary antibody to establish roughly equal loading. The array was subsequently probed with Cy3-labelled peptides, including a peptide from SmD3 that harbours symmetrically dimethylated arginine residues and peptides harbouring K79 from histone H3 in dimethylated and unmethylated forms. The H3K79me2 peptide shows a high degree of methyl-independent binding. A methyl-dependent interaction is seen with the tudor domain of C20orf104 (red oval). The tudor domains of 53BP1 are marked (blue oval).

Figure 2

Probing the CADOR chip with a series of methylated peptides from the tails of histones H3 and H4. The protein-domain microarray was probed with Cy3-labelled peptides. The unmethylated H3(1–18) and H4(11–28) peptides showed no binding (data not shown). Chromo domain interactions are blocked with a white square. Tudor domain interactions are highlighted with ovals: C20orf104 (red), 53BP1 (blue) and JMJD2A (turquoise). MBT domain interactions are marked with rectangles: CGI-72 (orange) and L(3)MBTL (yellow).

Figure 3

A pull-down-based approach detects methyl-dependent interactions between domains (chromo, tudor and MBT) and methylated histone tail peptides. Biotinylated peptides were immobilized on streptavidin beads and used to pull down the indicated glutathione S-transferase fusion proteins.

Figure 4

Tudor and chromo domains bind to histones. (A) Tudor domains bind to histones H3 and H4, and CDY1 binds to histone H3. Core histones were acid purified from G9a wild-type and knockout cells and Suv39h wild-type and double-knockout cells. Core histones were subjected to far western analysis by incubation with the indicated glutathione S-transferase (GST) fusion proteins. Western analysis was performed with methyl-specific antibodies to confirm the null status of the cell lines. The arrowheads denote the possition of histones H3 (closed) and H4 (open). Further description of western analysis is given in the supplementary information online. (B) Green fluorescent protein (GFP)–CDY1 and DsRed–HP1β were co-transfected into Suv39h wild-type and double-knockout mouse embryonic fibroblasts (MEFs). GFP–CDY1 and DsRed–HP1β colocalize to heterochromatin. This colocalization is lost in Suv39h−/− MEFs.