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Harvard Medical School

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J. Am. Chem. Soc., Nov 25 2024, | https://doi.org/10.1021/jacs.4c12585 Circular Engineered Sortase for Interrogating Histone H3 in Chromatin

Samuel D. Whedon,^1⧫ Kwangwoon Lee,^1⧫ Zhipeng A. Wang,^1⧫ Emily Zahn², Congcong Lu³, Maheeshi Yapa Abeywardana¹, Louise Fairall⁴, Eunju Nam¹, Sarah DuBois-Coyne¹, Pablo De Ioannes⁵, Xinlei Sheng⁶, Adelina Andrei¹, Emily Lundberg¹, Jennifer Jiang¹, Karim-Jean Armache⁵, Yingming Zhao⁶, John W. R. Schwabe⁴, Mingxuan Wu,^1* Benjamin A. Garcia,^2* and Philip A. Cole^1*

¹Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States.
²Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, United States.
³Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
⁴Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 7RH, U.K.
⁵Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, New York 10016, United States.
⁶Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois 60637, United States.

^⧫S.D.W., K.L., and Z.A.W. contributed equally.
*Corresponding Authors : Mingxuan Wu, Benjamin A. Garcia, Philip A. Cole

Abstract

Reversible modification of the histone H3 N-terminal tail is critical in regulating the chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here, we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle-down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetic discovery and therapeutic development.

논문정보

형식Research article
게재일2024년 11월 (BRIC 등록일 2024-11-27)
연구진국외 연구진
분야 생명과학 > 생화학

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