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ALLELE-SPECIFIC ENGINEERING OF METHYLYSINE WRITERS AND READERS FOR CONTROLLING CHROMATIN-DEPENDENT PROCESSES

Arora, Simran (2020) ALLELE-SPECIFIC ENGINEERING OF METHYLYSINE WRITERS AND READERS FOR CONTROLLING CHROMATIN-DEPENDENT PROCESSES. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Arora, Simransimaqua91@gmail.comsia29
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairIslam, Kabirulkai27
Committee MemberWeber, Stephensweber
Committee MemberHorne, Sethhorne
Committee MemberBerman, Andreaajb190
Date: 16 January 2020
Date Type: Publication
Defense Date: 20 August 2019
Approval Date: 16 January 2020
Submission Date: 7 January 2020
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 196
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: NA
Date Deposited: 16 Jan 2020 16:32
Last Modified: 16 Jan 2020 16:32
URI: http://d-scholarship.pitt.edu/id/eprint/38103

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