We utilized a protein microarray approach to screen for protein domains that interact with histones and non-histone proteins at are specific methylated at lysine or arginnine residues. Our protein microarrays harbor over 250 protein domains that are arrayed on a microscopy slide (FAST slide), at high-density. The protein domains that are immobilized on these nitrocellulose coated slides generally retain their native structure and are capable of recognizing a specific methylated motifs.

Here we show the use of a protein domain micoarray for four different approaches: the screening of synthetically generated histone tail peptides that harbor different degrees of lysine methylation, enzymatic modified nucleosomes which more closely resemble the in vivo setting, peptides containing lysine methylation from non-histone proteins as p53, and finally, methylated peptides containing glycine- and arginine-rich patches (GAR motifs) to search for methyl specific protein-protein interactions.

CADOR chips probed with Cy3 labeled peptides that were mono-, di-, or tri-methylated at lysines 4 and 9 of histone H3 and lysine 20 of histone H4. Chromo domain interactions are blocked with a white square. Tudor domains are highlighted with an oval: PHF20 (red) and JMJD2A (turquoise). MBT domain interactions are marked with rectangles: PHF20L (orange) and L(3)MBTL (yellow).

Kim J, Daniel J, Espejo A, Lake A, Krishna M, Xia L, Zhang Y, Bedford MT. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep 7:397-403, 4/2006.

Cy3 labeled biotinylated recombinant nucleosomes were incubated with the CADOR protein array. To generate methylated nucleosomes, H2B was biotin-tagged and assembled in nucleosomes which were then modified in vitro using the Suv4-20h2 and G9a HMTase.

Schotta G, Sengupta R, Kubicek S, Malin S, Kauer M, Callen E, Celeste A, Pagani M, Opravil S, De La Rosa-Velazquez IA, Espejo A, Bedford MT, Nussenzweig A, Busslinger M, Jenuwein T. A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse. Genes & Dev 22:2048-2061, 2008.

Top panel shows a CADOR array probed with an anti-GST primary antibody and detected with a AlexaFluor555-conjugated secondary antibody, the section blocked with a white square is shown below along with the peptides used to probe the array; Cy3 labeled peptides p53K370me0, p53K370me1, p53K370me2 and p53K370me3. The K370me2-dependent interaction with double Tudor domains of 53BP1 is circled.

Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, and Berger SL. p53 is regulated by the lysine demethylase LSD1. Nature 449:105-109, 2007.

The array was probed with Cy3 labeled symmetrically arginine methylated peptides from the splicing factor SmD3 (SmD3-Rme2s), which has previously been demonstrated to bind to the Tudor domain of SMN. Fluorescently labeled GAR glycine/arginine motif peptides harboring either aDMA or sDMA residues are probed on a focus microarray that has a selection of Tudor-domain-containing proteins: Pombe, SMN, TDRD3, SPF30 and 53BP1.

Bedford MT. Arginine methylation at a glance. J Cell Sci 120:4243-4246, 2007.

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