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An Integrated Chemical Genetic Approach to Probe Biological Methylation

Scott, Valerie L (2023) An Integrated Chemical Genetic Approach to Probe Biological Methylation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Epigenetic writer, reader, and eraser proteins are responsible for the installation, recognition, and removal of methyl groups on histone tails, which instruct the cell to carry out specific biological processes, such as gene expression. S-adenosyl L-methionine (SAM) and 2-ketoglutarate (2KG) are widely used as the methyl donor and demethylase cofactor, respectively. Given that there are more than 60 lysine methyl transferases and 30 lysine demethylases in humans, determining member-specific function of these enzymes is challenging. In this work, histone demethylase proteins have been engineered with an expanded active site to first catalytically rescue an otherwise dead demethylase with a complementary bulky 2KG analog. Second, a slightly different active site modification was found to accept the canonical cofactor and activity can be modified by an engineered selective inhibitor. In each case the engineered small molecule was able to perturb enzymatic function in mammalian cells. The generality of the approaches are shown through similar active site engineering in other family members. Next, by the chemical modification of SAM to furnish a benzyl moiety instead of the methyl group, this work demonstrates a method to install a bulky abiotic modification directly on histone tails in mammalian cells by the engineering of a SAM synthetase and a methyltransferase. The benzyl mark on histone tails is recognized by a modified reader protein and removed by an engineered demethylase. This work will be able to identify specific cellular roles of these proteins and is expected to be applicable to other family members.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Scott, Valerie Lvls38@pitt.eduvls38
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorKabirul, Islamkai27@pitt.edukai27
Committee MemberWeber, Stevesweber@pitt.edusweber
Committee MemberChilders, W. Sethwschild@pitt.eduwschild
Committee MemberLee, Miler T.miler@pitt.edumiler
Date: 26 January 2023
Date Type: Publication
Defense Date: 2 September 2022
Approval Date: 26 January 2023
Submission Date: 20 November 2022
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 159
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: Bump-and-hole approach
Date Deposited: 26 Jan 2023 15:05
Last Modified: 26 Jan 2023 15:05


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