Defining the Role of DNA Secondary Structures and Transcriptional Factors in the Control of c-myc and bcl-2 Expression

Persistent Link:
http://hdl.handle.net/10150/195655
Title:
Defining the Role of DNA Secondary Structures and Transcriptional Factors in the Control of c-myc and bcl-2 Expression
Author:
Dexheimer, Thomas Steven
Issue Date:
2006
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
In this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.
Type:
text; Electronic Dissertation
Keywords:
G-quadruplex; transcription; c-myc; bcl-2; DNA secondary structure
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Pharmaceutical Sciences; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Hurley, Laurence H.
Committee Chair:
Hurley, Laurence H.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleDefining the Role of DNA Secondary Structures and Transcriptional Factors in the Control of c-myc and bcl-2 Expressionen_US
dc.creatorDexheimer, Thomas Stevenen_US
dc.contributor.authorDexheimer, Thomas Stevenen_US
dc.date.issued2006en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractIn this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectG-quadruplexen_US
dc.subjecttranscriptionen_US
dc.subjectc-mycen_US
dc.subjectbcl-2en_US
dc.subjectDNA secondary structureen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHurley, Laurence H.en_US
dc.contributor.chairHurley, Laurence H.en_US
dc.contributor.committeememberHurley, Laurence H.en_US
dc.contributor.committeememberYang, Danzhouen_US
dc.contributor.committeememberFlynn, Garyen_US
dc.contributor.committeememberEbbinghaus, Scoten_US
dc.identifier.proquest1773en_US
dc.identifier.oclc659747535en_US
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