TY - JOUR
T1 - Combinatorial profiling of chromatin binding modules reveals multisite discrimination
AU - Garske, Adam L.
AU - Oliver, Samuel S.
AU - Wagner, Elise K.
AU - Musselman, Catherine A.
AU - Leroy, Gary
AU - Garcia, Benjamin A.
AU - Kutateladze, Tatiana G.
AU - Denu, John M.
N1 - Funding Information:
We thank G. Barrett-Wilt and G. Sabat (University of Wisconsin Biotechnology Center) for their help with MALDI-TOF mass spectrometry.We are grateful to G. Case (University of Wisconsin Peptide Synthesis Facility) for helpful conversations and advice on peptide synthesis.We thank G. Musco (Dulbecco Telethon Institute),Y. Shi (Harvard Medical School),W.Yang (National Institute of Health) and R.-M. Xu (New York University) for expression plasmids used in this study.We thank S. Bednarek (University of Wisconsin-Madison) for the GST-specific antibody. This work was supported by the US National Institutes of Health (grant GM059785 to J.M.D. and predoctoral fellowship T32GM008505 to S.S.O.) and an American Society for Mass Spectrometry research award (B.A.G.).
PY - 2010/4
Y1 - 2010/4
N2 - Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.
AB - Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.
UR - http://www.scopus.com/inward/record.url?scp=77949874334&partnerID=8YFLogxK
U2 - 10.1038/nchembio.319
DO - 10.1038/nchembio.319
M3 - Article
AN - SCOPUS:77949874334
SN - 1552-4450
VL - 6
SP - 283
EP - 290
JO - Nature Chemical Biology
JF - Nature Chemical Biology
IS - 4
ER -