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Determination of Novel Gene Targets of TETS via Non-Canonical Protein and DNA Modifications and Selective Inhibition

Shriver, Kirsten (2017) Determination of Novel Gene Targets of TETS via Non-Canonical Protein and DNA Modifications and Selective Inhibition. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

The Ten-Eleven translocation (TET) family of enzymes mediates the oxidation of DNA methylation, but the specific gene targets and the role of these enzymes in human development and disease are not well understood. The overall goals of this work are to profile TET-specific gene targets and to develop potent and specific small molecule inhibitors for TET family members. To accomplish these goals, a photo-crosslinking system and non-canonically modified DNA substrates will be developed as chemical tools to elucidate TET member-specific gene targets. Additionally, the development of specific, small molecule inhibitors will be explored. Photo-crosslinkable TET enzymes were developed, via incorporation of a photo-activable unnatural amino acid into the catalytic domain; three mutant enzymes were found to crosslink to methyl cytosine containing DNA substrates. This crosslinking system will be generalized across family members and applied in cells to identify novel gene targets of TET enzymes. DNA substrates with non-canonical modifications were tested in the development of a DNMT-TET tandem system to functionalize oxidation products. Ethyl, allyl, and propyl substituted DNA substrates were successfully oxidized by both wild type and mutant TET proteins. Potential inhibitor compounds were screened against the TET2 wild type enzyme, and a few compounds indicated slight inhibitory activity. A fragment based approach was employed to design potential linked inhibitor compounds, which will be synthesized and tested against TET enzymes.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Shriver, Kirstenkes176@pitt.edukes176
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairIslam, Kabirulkai27@pitt.edukai27
Committee MemberHorne, Sethhorne@pitt.eduhorne
Committee MemberChilders, Sethwschilds@pitt.eduwschilds
Date: 15 June 2017
Date Type: Publication
Defense Date: 22 November 2016
Approval Date: 15 June 2017
Submission Date: 11 April 2017
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 46
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: TET, photo-crosslinking
Date Deposited: 16 Jun 2017 00:20
Last Modified: 16 Jun 2017 00:20
URI: http://d-scholarship.pitt.edu/id/eprint/31417

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