Recognition and Specificity Determinants of the Human Cbx Chromodomains

Lilia Kaustov,Alexander Lemak,Matthieu Schapira,Shili Duan,Gregory A. Wasney,Maria Amaya,Nataliya Nady,Hui Ouyang,Cheryl H. Arrowsmith,Zhihong Li,Jinrong Min,Masoud Vedadi

doi:10.1074/jbc.m110.191411

Abstract

The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1–8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1–8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.

Highlights

The heterochromatin protein HP1 and Polycomb proteins are two distinct regulators of chromatin structure in Drosophila melanogaster involved in epigenetic repression of gene expression
Binding Preferences of Human Cbx Proteins Diverge from Those of Drosophila—To elucidate the binding specificity for the chromodomains of human Cbx proteins, we measured the affinity of the recombinant chromodomains for both trimethylated Lys-9 and Lys-27 marks on histone H3 peptides using fluorescence polarization
Most human Pc homologs (Cbx2, -4, -6, -7, -8) had a wide range of affinities toward both marks without a distinct selectivity for one, similar to their mouse homologs [15, 31]

Summary

EXPERIMENTAL PROCEDURES

Expression, and Purification—Chromodomains of Cbx1-(20 –73), Cbx2-(9 – 66), Cbx3-(29 – 81), Cbx4-(8 – 65), Cbx5-(18 –75), Cbx6-(8 – 65), Cbx7-(8 – 62), and Cbx8-(8 – 61) were overexpressed as N-terminal His6-tagged proteins at 15 °C using Escherichia coli BL21 (DE3) Codon plus RIL (Stratagene) as a host organism. The bacteria were grown in M9-defined medium supplemented with [15N]ammonium chloride (0.8 g/liter) and D-[13C6]glucose (0.4 g/liter) for 13C,15N-labeled samples at room temperature These highly expressed proteins were purified by Talon (BD Biosciences) affinity chromatography under native conditions and eluted with buffer containing 500 mM Imidazole. Binding assays were performed in a 10-␮l volume at a constant labeled peptide concentration of 40 nM, and Cbx protein concentrations at saturation ranging from 800 to 1300 ␮M in buffer containing 20 mM Tris, pH 8.0, 250 mM NaCl, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, and 0.01% Tween 20. Crystallization, Data Collection, and Structure Determination—Crystals of the Cbx chromodomains were grown at 18 °C using the sitting-drop method by mixing equal volumes of the mother liquor with protein solutions. H3K27me, and Cbx8/H3K9me have been deposited with accession codes 3H91, 3FDT, 3GV6, 3I90, 2L11, 2L12, 2L1B, and 3I91, respectively

RESULTS

DISCUSSION

98 Ϯ 15 29 Ϯ 6 37 Ϯ 4 6Ϯ2 180 Ϯ 80