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Allele specific approach to study histone demethylation using engineered KDM4A- (2)-Ketoglutarate pairs

Breski, Megan (2015) Allele specific approach to study histone demethylation using engineered KDM4A- (2)-Ketoglutarate pairs. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

KDM4 proteins are epigenetic modifiers that demethylate histone lysine residues to regulate chromatin structure and gene expression. The KDM4 subfamily contributes to the larger JmjC domain containing demethylase family, which all require 2-ketoglutarate as a cofactor for catalytic activity. KDM4 members have been shown to be relevant in biological processes, such as cellular differentiation and DNA damage repair. The amplification of KDM4 proteins has been observed in various cancers and disrupts the cell cycle, normal cellular proliferation and apoptosis. Being able to monitor the members of this protein family independently will elucidate their specific functions and the genes they regulate. Because this family of proteins is involved in various mechanisms related to pathogenesis it is of significant public health importance. The Bump-Hole approach was used to develop an engineered KDM4A-2-Ketoglutarate system that would be independent of the native demethylase system. In total, 24 KDM4A mutants and 12 2-ketoglutarate analogs were generated, providing 288 engineered systems to be screened for activity. Half of the KDM4A mutants showed activity with 2-KG compounds and three of them provided a bioorthologous enzyme-cofactor system. The most significant engineered KDM4A-2-KG pairs were tested in KDM4 isoforms B and D and gave similar results. Engineering the enzyme did not disrupt substrate specificity and the engineered systems were active following methylation of histone peptides. In conclusion, half of the KDM4A mutants rendered the enzyme inactive when paired with the 2-KG compounds obtained (including the native compound). Half of the KDM4A mutants showed activity with at least one of the 2-KG compounds, where three of them resulted in a bioorthologous enzyme-cofactor system. The observations were alike in the KDM4 isoforms and substrate specificity was maintained. These results allow for the KDM4A protein to be monitored independently of the KDM4 isoforms and other JmjC domain containing proteins.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Breski, Meganmmb123@pit.edu
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKabirul, IslamKAI2@pitt.eduKAI2
Committee MemberUrban, Zsolturbanz@pitt.eduURBANZ
Committee MemberKammerer, Candace Mcmk3@pitt.eduCMK3
Date: 29 June 2015
Date Type: Publication
Defense Date: 1 April 2015
Approval Date: 29 June 2015
Submission Date: 8 June 2015
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 77
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Human Genetics
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Epigenetics, Histones, Demethylation, Demethylase, KDM4A, JMJD2A, Genetics, cancer, Histone Demethylases, KDM4, lysine demethylases, 2-ketoglutarate
Date Deposited: 29 Jun 2015 13:52
Last Modified: 01 May 2017 05:15
URI: http://d-scholarship.pitt.edu/id/eprint/25363

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