relation: http://d-scholarship-dev.library.pitt.edu/38103/
title: ALLELE-SPECIFIC ENGINEERING OF METHYLYSINE WRITERS AND READERS FOR CONTROLLING CHROMATIN-DEPENDENT PROCESSES
creator: Arora, Simran
description: One of the key players in regulating the gene pattern is the post-translational modifications (PTMs)  of histone proteins. Histone modifications regulate the transcriptional potential of genes by  interacting with reader/effector protein domains. Post-translational modifications on methyllysine  are ubiquitous in biological systems and critical for mammalian development. Specific  perturbation of such interactions has remained a challenging endeavor. We hypothesized that  incorporation of an unnatural modification with the aid of an engineered writer domain and its  recognition by reader domain would regulate the downstream genes (epigenetic editing) leading  to modification of the epigenetic landscape. The engineered orthogonal pairs together with  catalytically inactive Cas9 would specifically modulate the expression of a gene of interest,  thereby providing control on transcription machinery. We employed the allele-specific strategy  towards engineering the epigenetic landscape and protein-protein interface orthogonal to the  human proteome. We generated a hole-modified methyltransferase (writer) that would install an  aryllysine moiety on histones in-cellulo. We established the orthogonality of the engineered  system, overcame the permeability issue of SAM analogues, developed an antibody and  established the applicability of the system in cells. Our data confirms successful benzylation of  histone proteins in mammalian cells at sites known to be regulated by SUV39h2 (writer protein) in cellulo. Further we engineered a chromodomain (reader) with a pocket to accommodate the  bulky modifications. We established the biochemical integrity of the engineered interface,  provided structural evidence for domain integrity, demonstrated the generality of the approach,  and validated its applicability to identity transcriptional regulators. We have shown that the  orthogonal reader domain on binding to the unnatural modification remains functionally intact.  The interactions of reader proteins with its binding partners are transient, weak and cell-cycle  dependent thereby challenging to identify. We applied the interactome-based protein-profiling  (IBPP) approach to the chromodomain in cellulo to identify its native binding partners. We  confirmed established biochemical integrity of the mutant proteins, established their crosslinking  efficiency in vitro, crosslinked them to their native binding partners in vivo and pulled them down.  On LCMS/MS data validation, we envision translating this approach to other chromodomains  containing proteins and identifying their binding partners.
date: 2020-01-16
type: University of Pittsburgh ETD
type: PeerReviewed
format: application/pdf
language: en
identifier: http://d-scholarship-dev.library.pitt.edu/38103/7/Arora%20Final%20ETD.pdf
identifier:   Arora, Simran  (2020) ALLELE-SPECIFIC ENGINEERING OF METHYLYSINE WRITERS AND READERS FOR CONTROLLING CHROMATIN-DEPENDENT PROCESSES.  Doctoral Dissertation, University of Pittsburgh.    (Unpublished)