Circular Engineered Sortase for Interrogating Histone H3 in Chromatin

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.