TY - JOUR
T1 - Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation
AU - McGinty, Robert K.
AU - Kim, Jaehoon
AU - Chatterjee, Champak
AU - Roeder, Robert G.
AU - Muir, Tom W.
N1 - Funding Information:
Acknowledgements We acknowledge H. Deng and J. Fernandez at The Rockefeller University Proteomics Resource Center for mass spectrometric analysis of methylated peptides. We thank T. J. Richmond for donating the 12_177_601 plasmid. We thank C. D. Allis for contributing the Xenopus histone plasmids for recombinant histone expression. We thank Y. Zhang for donating a plasmid containing hDot1L. We thank B. R. Rosenberg for assistance with phosphorimaging. We thank C. D. Allis, J. Tanny, and K. P. Chiang for discussions. This work was funded by the US National Institutes of Health. R.K.M. was supported by National Institutes of Health MSTP grant GM07739. J.K. was supported by the LLS SCOR grant.
PY - 2008/6/5
Y1 - 2008/6/5
N2 - Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.
AB - Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.
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U2 - 10.1038/nature06906
DO - 10.1038/nature06906
M3 - Article
C2 - 18449190
AN - SCOPUS:44849100496
SN - 0028-0836
VL - 453
SP - 812
EP - 816
JO - Nature
JF - Nature
IS - 7196
ER -