2. Academic Validation
  3. A circular engineered sortase for interrogating histone H3 in chromatin

A circular engineered sortase for interrogating histone H3 in chromatin

  • bioRxiv. 2024 Sep 10:2024.09.10.612318. doi: 10.1101/2024.09.10.612318.
Samuel D Whedon 1 2,  Kwangwoon Lee 1 2,  Zhipeng A Wang 1 2,  Emily Zahn 3,  Congcong Lu 4,  Maheeshi Yapa-Abeywardana 1 2,  Louise Fairall 5,  Eunju Nam 1 2,  Sarah Dubois-Coyne 1 2,  Pablo De Ioannes 6,  Xinlei Sheng 7,  Adelina Andrei 1 2,  Emily Lundberg 1 2,  Jennifer Jiang 1 2,  Karim-Jean Armache 6,  Yingming Zhao 7,  John W R Schwabe 5,  Mingxuan Wu 8,  Benjamin A Garcia 3,  Philip A Cole 1 2
Affiliations

Affiliations

  • 1 Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, United States.
  • 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States.
  • 3 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
  • 4 Frontiers Science Center for Cell Responses, College of Life Sciences, Nankai University, Tianjin, 300071, China.
  • 5 Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.
  • 6 Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, United States.
  • 7 The Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, United States.
  • 8 Department of Chemistry, School of Science, Westlake University, Hangzhou 310030, China.
PMID: 39372790 DOI: 10.1101/2024.09.10.612318
Abstract

Reversible modification of the histone H3 N-terminal tail is critical in regulating 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 Epigenetics discovery and therapeutic development.

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