The role of Rif1p preferential elongation of short telomeres

Univ Of Med/Dent Nj-R W Johnson Med Sch, Piscataway NJ

Investigators

Abstract

The principal goal of this research is to understand telomerase regulation. Specifically, it will focus on elucidating the mechanism of how telomerase preferentiallyelongates short telomeres. A telomere is a complex of DNAand proteins that marks the end of a chromosome. Telomerase is an enzymethat elongates telomeres, thereby enabling cells to continuouslydivide without losingtheir genetic material. In both lower and higher eukaryotes, telomerase preferentiallyelongates short telomeres. Previous findings from our lab show that in yeast the protein content of short telomeres is different from that of WT-length telomeres. We speculate that this difference marks short telomeres for telomerase recruitment. Rifip and Rif2p, known negative regulators of telomerase, were initially predicted to be lost from short telomeres, thereby enhancing telomerase activity. Surprisingly,in these previous studies, Rifip bound equallywell to short and long telomeres while Rif2p binding was decreased. In contrast, Telip showed preferentialbindingfor short telomeres. Telip is an ATM/ATR-like kinase, shown to be necessary for recruiting telomerase subunits to short telomeres. A hypothesis consistent with these findings is that the reduced Rif2p binding at short telomeres recruits Telip, to that telomere, Telip phosphorylates Rifip, and this phosphorylation relieves its ability to inhibit telomerase in cis. Accordingto this model, the inactivation, not the displacement, of Rifip, allows telomerase recruitment and activation. This proposal will test these ideas with the following aims: (i) confirm the reduced Rif2p and WT-level Rifip binding to short telomeres using telomeres shortened from their ends as occurs in vivo, (2) determine if Rifip is regulated by Telip-dependent phosphorylation, and (3) assess if Rifip phosphorylation affects telomerase action. For aim i, a yeast strain, deleted in the telomerase catalytic subunit, will be used to shorten telomeres. Using this strain, chromatin IP and quantitativePCR will be performed to assess the binding levels of Rifip and Rif2p on short telomeres. For aim 2, a synthetic dosage lethality screen will be employed to evaluate whether or not Rifip is a phosphorylation target of Telip. For aim 3, site-directed mutagenesis of Telip phosphorylation consensus sites will be performed to identify sites of Telip action on Rifip. Subsequently, telomere lengths will be assessed to seeif phosphorylation of these residues affects the ability of telomerase to lengthen telomeres. As a large portion ofthe U.S. population reaches their retirement age, age-related diseases arebecoming more prevalent. Therefore, it is important to understand how cells ageby studying telomerase regulation. Once the clear mechanism is understood, it can be used to develop a new therapy to slow cell aging, the most fundamental problem in all age-related diseases, thereby serving the needs of public health at large. PHS 416-1 (Rev. 10/05) Page 2 Number pages consecutively at the bottom throughout Form Page 2 the application. Do not use suffixes such as 2a, 2b. NAME OFAPPLICANT (Last, first, middle initial) Kirschstein-NRSA Individual Fellowship Application McGee, Jean, S. (To becompleted byapplicant - follow PHS 416-1 instructions) 20. GOALS FOR KIRSCHSTEIN-NRSA FELLOWSHIP TRAINING AND CAREER During the period of this predoctoral fellowship, I will pursue research on the basic mechanisms of cell aging in order to prepare myself for a career as a physician-scientist. Ultimately,I plan to hold a faculty position at a medical school with my own laboratory and incorporate clinical medicine into my research. After completion of the MD/PhD program, I will receivepostgraduate trainingin one of the internal medicine subspecialties of geriatrics, oncology, or preventive medicine.As the elderly population in the U.S.increases, I find essential the fields of medicine that satisfy the healthcare needs of the agingpublic. Duringthis fellowship period, I will learn to work with a model organism,S. cerevisiae, to study the regulationof telomerase. Asits regulation is fundamental to the pathology of cancer and age-related diseases, I find this training extremely pertinentto myfuture career goals. Duringmy master'sdegree program, I worked extensively with human breast and prostate cancer cell lines to conduct more clinically-oriented research.As such, my current training in basic science will provide me with a more complete background to ultimately translate research into clinical practice. Asa predoctoral fellow, I willbroaden myknowledgebase in molecular biology, cancer biology,cell biology, genetics, and how each of these fields informs the study of age-related pathologies. I believe that my trainingat Princetonwill prepare me to become a physician- scientist who is always awareof implications of research at the bedside. 21. ACTIVITIES PLANNED UNDER THIS AWARD: Approximate percentage of proposed award time in activities identified below. (See instructions.) Year Research Course Work Teaching Clinical First 95% 5% Second 95% 5% Third 95% 5% PREDOCTORAL FELLOWSHIPS ONLY MD/PhD FELLOWSHIPS ONLY Sixth Briefly explain activities other than research and relate them to the proposed research training. During this upcomingspring semester, I will complete two courses, which will fully satisfy the course requirements for the PhD degree at Princeton.Therefore,starting with the beginningof thisfellowship period, I will devote 95% of my time on my dissertation project. The remaining5%will be spent on shadowing a physician at hospital once a month in order to maintain continuityof clinical skills training during my PhD years. This will allow for an easier transition into medical school to complete my MD degree. 22. TRAINING SITE(S) (organization, city, state) PrincetonUniversity Princeton, NJ 23. HUMAN EMBRYONIC STEM CELLS [^ No Q Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://Stemcells.nih.qov/registrv/index.asp. Use continuation pages as needed. If a specific line cannotbe referenced at this lime, include a statement that one from the Registrywill be used. Cell Line PHS 416-1 (Rev. 10/05) Page3 Form Page 3 Kirschstein-NRSA Individual Fellowship Application NAME OF APPLICANT (Last, first, middle initial) McGee, Jean, S. Table of Contents Page Numbers (Number pages consecutively at the . bottom throughout theapplication. Section 1 - Applicant Do not use suffixes such as6a, 6b.) Face Page i Sponsor's Contact Information, Description (Form Page 2) 2 Training &Career Goals, Activities Planned Under This Award, Training Site, Human Embryonic Stem Cells (Form Page 3) 3 Table of Contents (Form Page 4) 4 Biographical Sketch - Applicant/Fellow (Not toexceed four pages) 5-7 Previous Research Experience (Form Page 5) 8-9 Research Training Plan Introduction to Revised Application (nof to exceed 1page) - A. Specific Aims [unreadable]x. f 10 B. Background/Significance !(.....(Not toexceed 10pages) ) 10-12 C. Preliminary Studies/Progress Report..?. | 13 D. Research Design andMethods J. ^ 13-16 E. Human Subjects (Required if Item 9 on the Face Page is marked "Yes") Protection of Human Subjects (Required if Item 9 on the Face Page is marked "Yes") - Data and Safety Monitoring Plan (Required if Item 9 on the Face Page is marked "Yes" and a Phase I, II, or III clinical trial is proposed - Inclusion of Women and Minorities (Required if Item 9 on the Face Page is marked "Yes" and is Clinical Research) - Targeted/Planned Enrollment Table (for new and continuing clinical research studies) - Inclusion of Children (Required if Item 9 on the Face Page is marked "Yes") - F. Vertebrate Animals (Required if Item Won theFace Page is marked "Yes") - G. Literature Cited 17-18 H. Resource Sharing " - I. Respective Contributions 18-19 J. Selection of Sponsor and Institution 19 K. Responsible Conduct of Research 19 Section 2 - Sponsor's/Co-Sponsor's Information Biographical Sketch-Sponsor 20-23 Research Support Available 24-25 Previous Trainees 25 Training Plan, Environment, Research Facilities 25-26 Number of Fellows/Trainees to be Supervised 26 Applicant's Qualifications and Potential 26-27 Checklist (Completed by Fellow/Applicant &Sponsoring Institution) 28 Section 3 - References (Minimum of 3) (See instructions for submission of references.) List full name, institution, and departmentof individuals submitting reference letters. Dr. Helen Nivison, Cornell University, Department of Molecular Biology and Genetics Dr. Noa Noy, Case Western Reserve University, Department of Pharmacology Dr. Yigong Shi, Princeton University, Department of Molecular Biology Dr. Rubina Yasmin, Case Western Reserve University, Department of Pharmacology Other Items (list): Personal Data Page for Fellowship Applicants Section 4 - Appendix (5 collated sets. Nopage numbering necessary. Not to exceed 3 publications;2 for predoctoral candidates.) K Check if Appendix is included PHS 416-1 (Rev. 10/05) Page Form Page 4