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Regulation of DNA Demethylation: Investigating the Effect of Catalytic Space Engineering

Sappa, Sushma (2023) Regulation of DNA Demethylation: Investigating the Effect of Catalytic Space Engineering. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Cellular differentiation utilizes the epigenetic mechanism of maintaining and regulating the methylation patterns of DNA, specifically at the carbon 5' position of the cytosine base forming 5mC, to mark genes as silenced. The erasure of this marker post-fertilization is carried out by FeII/2-KG dependent Ten-Eleven Translocation (TET) enzymes, which oxidize the methyl group of 5mC in the presence of 2-KG, producing 5hmC, 5fC and 5caC base modifications as reaction intermediates. Collectively known as 5mCox, these base modifications interact with unique chromatin-associated proteins to regulate transcription due to their distinct chemical features. However, their heterogenous mixture and fleeting nature have made challenging to assign their transcriptional role during development. The following work is aimed at developing an integrated chemical genetic approach to elucidate 5mCox specific function in mammalian gene expression. The work is divided into two modes of regulating TET's catalytic activity. Firstly, by protein engineering at the protein-cofactor interface: the catalytic space of TET2 is systematically modulated to influence the degree of 5mC oxidation towards a specific intermediate. Secondly, cellular metabolite ATP is used to fine-tune the generation of 5hmC oxidation product. Furthermore, a combination of an engineer TET enzyme with an expanded active site and a complementary bulky 2KG analog is employed to generate the desired oxidation product. This work aims to identify the specific cellular roles of 5mCox, and the generality of the approaches is shown through similar active site engineering in TET family members.

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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Sappa, Sushmasts132@pitt.edusts132
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairIslam, Kabirulkai27@pitt.edukai27
Committee MemberChaillet, John. Rchaillet@pit.educhalet
Committee MemberHorne, Sethhorne@pitt.eduhorne
Committee MemberKoide, Kazuorikoide@pitte.dukoide
Date: 6 September 2023
Date Type: Publication
Defense Date: 3 May 2023
Approval Date: 6 September 2023
Submission Date: 18 July 2023
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 165
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: Epigenetics
Related URLs:
Date Deposited: 07 Sep 2023 01:15
Last Modified: 07 Sep 2023 01:15
URI: http://d-scholarship.pitt.edu/id/eprint/45113

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  • Regulation of DNA Demethylation: Investigating the Effect of Catalytic Space Engineering. (deposited 07 Sep 2023 01:15) [Currently Displayed]

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