Telomere Biology in Arabidopsis - TEXAS A&M UNIVERSITY

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	7010	1000	100%

Progress 09/22/11 to 09/21/16

Outputs
Target Audience:My target audience is other scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral fellows, graduate students and undergraduate students received outstanding training in cutting edge biochemical and genetic methodology. How have the results been disseminated to communities of interest?Results have been disseminated through peer-reviewed research publications, conference proceedings and through departmental seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Identification and characterization of telomere capping proteins in Arabidopsis. A major effort is directed at elucidating the function and interactions of the CST (CTC1, STN1 and TEN1) telomere capping complex in Arabidopsis. We found that CST is required to protect plants from catastrophic loss of telomeric DNA, end-to-end chromosome fusions and developmental arrest (Surovtseva et al. 2009; Song et al. 2009). We are currently studying how CST proteins interface with components of the telomerase RNP complex and the DNA replication machinery to promote telomeric DNA replication (Price et al. 2010; Beilstein et al. 2012; Renfrew et al., 2014). We are also studying how the CST complex suppresses DNA damage response machinery to maintain genome integrity (Boltz et al. 2012). This research is supported by NIH (R01-GM065383). Objective 2: Characterization of telomerase RNP evolution, function and regulation in plants. Research is ongoing to explore many facets of the telomerase reverse transcriptase in Arabidopsis and other plants. Unexpectedly, we discovered that Arabidopsis encodes two distinct telomerase enzymes, with different RNA and protein subunits and different roles in telomere biology (Cifuentes-Rojas et al. 2011; Cifuentes-Rojas et al. 2012). One telomerase complex is termed TER1 RNP (named for its RNA subunit, TER1). TER1 RNP is the canonical telomerase enzyme needed to maintain telomere repeats on chromosome ends. The other enzyme, TER2 RNP, is a novel negative regulator of telomerase. We recently discovered that Arabidopsis has evolved a highly complex network for regulating telomerase activity. This network includes competitive inhibition of the catalytic subunit, sequestration of the catalytic subunit and developmentally regulated splicing of one of the TER2 RNA subunit (Cifuentes-Rojas et al. 2012; Xu et al., 2014). Current research is directed at uncovering the role of telomerase inhibition in promoting genome stability for Arabidopsis, and on a comparative analysis to determine when a second telomerase enzyme evolved in the plant kingdom. This research is supported by two grants from NSF (MCB-0843399 and MCB-1052018).

Publications

Progress 10/01/15 to 09/21/16

Outputs
Target Audience:Scientists and Academics Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the past year, the project provided an opportunity for 3 postdoctoral fellows, 5 PhD students, 5 undergraduate students and 3 visiting scientists to acquire new technical skills and to pursue independent research in the area of telomeres and telomerase. How have the results been disseminated to communities of interest?Two primary research papers were published. Dr. Shippen presented two platform talks at scientific symposia, and gave two departmental seminars. What do you plan to do during the next reporting period to accomplish the goals?We will continue our analysis of telomere proteins, telomerase and telomerase regulatory RNAs in Arabidopsis. The research will employ genomic, biochemical, genetic and cell biological approaches.

Impacts
What was accomplished under these goals? In the past year, we discovered 1) a new gene important for telomere biology; 2) novel non-telomeric functions for several telomere genes; 3) biochemical basis for different behaviors of telomere binding proteins and telomere-related RNAs.

Publications

Type: Journal Articles Status: Published Year Published: 2016 Citation: Arora, A., Beilstein, M.A. and Shippen, D.E. (2016) Evolution of Arabidopsis Protection of Telomeres 1 alters nucleic acid recognition and telomerase regulation. Nucleic Acids Research, 44:9821-9830.
Type: Journal Articles Status: Published Year Published: 2016 Citation: Lee, J.R., Zhang, J., Yang, K., Lee, S.Y., Xie, X. and Shippen, D.E. (2016) Dynamic interactions of Arabidopsis TEN1: stabilizing telomeres in response to heat stress. Plant Cell, Sept 8 pii: tpc.00408.2016. [Epub ahead of print].

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the past year, the project provided an opportunity for 4 postdoctoral fellows, 6 PhD students, 6 graduate rotation students, 4 undergraduate students and three visiting scientists to acquire new technical skills and to pursue independent research in the area of telomeres and telomerase. How have the results been disseminated to communities of interest?Three primary research papers were published as well as one review. A graduate student gave a platform talk and five other students and postdocs gave poster presentations at the Cold Spring Harbor Telomeres and Telomerase meeting. What do you plan to do during the next reporting period to accomplish the goals?We will continue our analysis of telomere proteins and telomerase in Arabidopsis and in the model plant, Physcomitrella. The research will employ genomic, biochemical, genetic and cell biological approaches.

Impacts
What was accomplished under these goals? In the past year, we defined novel dynamic interactions between telomerase and the telomere. We also discovered a link between a telomerase-associated RNA and the DNA damage response.

Publications

Type: Journal Articles Status: Published Year Published: 2015 Citation: Gonz�lez-Garc�a, M.P., Pavelescu, I., Canela, A., Sevillano, J., Leehy, K.A., Nelson, A.D.L., Shippen, D.E., Blasco, M. and Ca�o-Delgado, A. (2015) Telomere length gradients preserve the meristematic potential and stem cell function in the root of Arabidopsis thaliana. Cell Reports, 11: 977-989.
Type: Journal Articles Status: Published Year Published: 2015 Citation: Beilstein, M.A., Renfrew, K.B., Song, X., Shakirov, E.V. and Shippen, D.E. (2015) Evolution of the telomerase-associated protein POT1a is characterized by positive selection to reinforce protein-protein interaction. Molecular Biology and Evolution. 32:1329-1341
Type: Journal Articles Status: Published Year Published: 2015 Citation: Xu, H., Nelson, A.D.L. and Shippen, D.E. (2015) A transposable element within Arabidopsis TER2 modulates telomerase enzyme activity in response to DNA damage. PLoS Genetics, 11: e1005281.
Type: Journal Articles Status: Published Year Published: 2015 Citation: Nelson, A.D.L and Shippen, D.E. (2015) Evolution of TERT-interacting lncRNAs: expanding the regulatory landscape of telomerase. Frontiers in Genetics 6: 277.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? In the past year, the project provided an opportunity for 3 postdoctoral fellows, 5 PhD students, 2 graduate rotation students, 4 undergraduate students and two visiting scientists to acquire new technical skills and to pursue independent research in the area of telomeres and telomerase. How have the results been disseminated to communities of interest? Two primary research papers were published as well as an online review. A graduate student presented findings at the International Plant Genome Stability and Change Conference in Pacific Grove, California. The PI gave a departmental seminar at the University of Indiana and the Toxicology Program at TAMU. What do you plan to do during the next reporting period to accomplish the goals? We will continue our analysis of telomere proteins and telomerase in Arabidopsis and in the model plant, Physcomitrella. The research will employ genomic, biochemical, genetic and cell biological approaches.

Impacts
What was accomplished under these goals? In the past year, we defined novel dynamic interactions between telomerase and the telomere. We also discovered a link between a telomerase-associated RNA and the DNA damage response.

Publications

Type: Journal Articles Status: Accepted Year Published: 2014 Citation: The role of Poly(ADP-ribose) polymerases in Arabidopsis telomere biology, PLoS One, 9:e88872.
Type: Journal Articles Status: Accepted Year Published: 2014 Citation: POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis. PLoS Genetics 10:e1004738.
Type: Book Chapters Status: Accepted Year Published: 2014 Citation: Plant telomeres and Telomerase: evolution in the field. in M. Tester and R.A. Jorgensen (eds.) The Plant Sciences Molecular Biology, Springer Press, New York.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The results have been published in peer-reviewed journals and discussed at interntional scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? We will continue to pursue the goals of our research in the same manner we have in the past.

Impacts
What was accomplished under these goals? Our research has led to a greater understanding of the fundamental properties of chromosomes, and the response of cells to DNA damage. These studies have made an impact on the research of plant scientists as well as scientists studying genome stability and cell proliferation. Although we work with plants, some of the research we have conducted has implications for cancer research. We are now collaborating with scientists in the Texas A&M Health Science Center to determine whether phenomenon we observe in plants are also relevant to human breast cancer cells.

Publications

Type: Journal Articles Status: Submitted Year Published: 2014 Citation: The role of Poly(ADP-ribose) polymerases in Arabidopsis telomere biology. *These authors contributed equally.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Meristem Disorganization 1 encodes TEN1, a telomere capping protein that modulates telomerase processivity in Arabidopsis.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Plant telomeres and Telomerase: evolution in the field. in The Plant Sciences Molecular Biology. Edited by Mark Tester and Richard A. Jorgensen, Springer Press, New York

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We are studying the physical ends of eukaryotic chromosomes called telomeres. Telomeres play a crucial role in the regulating cellular proliferation in multicellular organisms, and in humans their status is linked to cancer and stem cell disease. The major goal of this work is to exploit the model plant Arabidopsis to provide new insight into fundamental aspects of telomere structure, function and regulation, and ultimately a better understanding of how the ends of chromosomes impart stability to the genome. The basic research we perform is conducted by undergraduate and graduate students as well as postdoctoral fellows. The results of our studies are published in peer reviewed journals and discussed at national and international meetings. PARTICIPANTS: PI: (TAMU Biochemistry Department) Dr. Dorothy Shippen Postdoctoral Fellows: (TAMU Biochemistry Department) Dr. Jung Ro Lee, Dr. Amit Amora Ph.D. Students: (TAMU Biochemistry Department) Katherine Leehy, Xianoyuan Xie, Kyle Renfrew, Hengyi Xu, Xintao She, Callie Koybashi (TAMU Biology Department) Kara Boltz, Undergraduate Students: (TAMU Biochemistry Department) Aditya Panta, Elena Douglass, Jennifer Townley Collaborators from outside TAMU: Dr. Carolyn Price, Univ. Cincinnati, Dr. Sarah Mathews, Harvard Univ., Dr. Robin Fuchs-Young, TAMU Health Science Center, Dr. Mark Beilstein, Arizona Univ. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research has led to a greater understanding of the fundamental properties of chromosomes, and the response of cells to DNA damage. These studies have made an impact on the research of plant scientists as well as scientists studying genome stability and cell proliferation. Although we work with plants, some of the research we have conducted has implications for cancer research. We are now collaborating with scientists in the Texas A&M Health Science Center to determine whether phenomenon we observe in plants are also relevant to human breast cancer cells.

Publications

Boltz, K.A. et al. (2012). ATR and the CST telomere complex cooperate to maintain telomeres and genome integrity in Arabidopsis. Mol Biol Cell 23:1559-68.
Shakirov, E.V. and Shippen, D.E. (2012). Selaginella telomeres: conserved and unique features in an ancient land plant lineage. Front. Plant Sci. 3:161. doi: 10.3389/fpls.2012.00161
Beilstein, M.A., Brinegar, A.E. and Shippen, D.E. (2012). Evolution of the Arabidopsis telomerase RNA. Front. Genetics 3:188. Epub 2-12 Sep 24.
Cifuentes-Rojas, C., Nelson, A.D., Kannan, K., Boltz, K.A., She, X. and Shippen, D.E. (2012). An alternative telomerase RNA represses enzyme activity in response to DNA damage. Genes Dev 26:2512-2513.
Cifuentes-Rojas, C. and Shippen, D.E. (2012). Telomerase Regulation Mutation Res 730:20-27.
Nelson, A.D.L. and Shippen, D.E. (2012). Blunt-ended telomeres: an alternative ending to the replication and end protection stories. Genes Dev. 26:1648-1652.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Objective 1 is identification and characterization of telomere capping proteins in Arabidopsis. In the past few months we have identified a new protein that is important for telomere biology in Arabidopsis. This protein links the telomere to the circadian clock. The second Objective is characterization of telomerase RNP evolution, function and regulation in plants. We recently discovered that telomerase regulation changes in response to DNA damage. PARTICIPANTS: Dr. Jung Ro Lee, postdoctoral fellow Dr. Amit Amora, postdoctoral fellow Andrew Nelson, graduate student Kara Boltz, graduate student Katherine Leehy, graduate student Kyle Renfrew, graduate student Hengyi Xu, graduate student Xintao She, graduate student Xianoyuan Xie, graduate student TARGET AUDIENCES: Scientific community through research publication. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The results we have obtained so far reveal new linkages between the chromosome terminus and other physiological responses in the cell. These findings are unexpected, and may be highly impacting.

Publications

Cifuentes-Rojas, C. and Shippen, D.E. (2012) Telomerase Regulation. Mutation Research. 730:20-27.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We have made progress in understanding the evolution of telomerase proteins and telomeres in plants. In addition, we have discovered several new genes that influence chromosome end protection in Arabidopsis. We have also defined novel components of the Arabidopsis telomerase enzyme. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our work on plant telomores is helping to provide fundamental insight into the factors that promote integrity of the genome. Since telomeres play a crucial role in human cancer and cellular aging, our work may have implications for human medicine. The intellectual merit of this work offers a unique opportunith to explore the long-term effects of genome instability that arises as a consequence of pertubing telomeres the ends of DNA molecules. Our analysis of plant telomeres will not only contribute to understanding how plant genomes are maintained, but also will be relevant to a broad range of higher eukaryotes. We employ a combination of molecular genetic cytological and biochemical approaches to examine the structure and function of telomeres and their maintenance by the enzyme telomerase. The model system for our work is Arabidopsis, a genetically tractble higher eukaryote that has many important advantages over the mammalian model systems that have previously been exploited for telemore studies.

Publications

Shakirov EV, Perroud P-F, Nelson AD, Cannell ME, Quatrano RS and Shippen DE (2010) Protection of telomeres is required for telomere integrity in the moss Physcomitrella patens. Plant Cell 22:1838-1848.
Cifuentes-Rojas C., Kannan K., Tseng, L. and Shippen, D.E. (2010) Two RNA subunits and POT1a are components of Arabidopsis telomerase. Proceedings of the National Academy of Sciences USA [Epub ahead of print]. Dec 16.
Price, C.M., Boltz, K., Chaiken, MX, Stewart, J.A. and Beilstein, M.A. and Shippen, D.E. (2010) Evolution of CST function in telomere maintenance. Cell Cycle. 9:3157-3165

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: OUTPUTS: In the past year we have made considerable progress in understanding the evolution of telomere proteins in plants. In addition, we have discovered several new genes that influence chromosome end protection in Arabidopsis. We have also defined novel components of the Arabidopsis telomerase enzyme. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our work on plant telemores is helping to provide fundamental insight into the factors that promote integrity of the genome. Since telomeres play a crucial role in human cancer and cellular aging, our work may have implications for human medicine. The intellectual merit of this work offers a unique opportunity to explore the long-term effects of genome instability that arises as a consequence of pertubing telomeres, the ends of DNA molecules. Our analysis of plant telomeres will not only contribute to understanding how plant genomes are maintained, but also will be relevant to a broad range of higher eukaryotes. We employ a combination of molecular, genetic, cytological and biochemical approaches to examine the structure and function of telomeres and their maintenance by the enzyme telomerase. The model system for our work is Arabidopsis, a genetically tractable higher eukaryote that has many important advantages over the mammalian model systems that have previously been exploited for telemore studies.

Publications

Shakirov, E.V., McKnight, T.D. and Shippen, D.E. (2009) POT1-independent single-strand telomeric DNA-binding activities in Brassicaceae. Plant Journal 58:1004-15.
Surovtseva, Y, Churikov, D., Boltz, K.A., Song, X., Boltz, Lamb, J.C., Warrington, R., Leehy, K., Heacock, M., Price, C.M. and Shippen, D.E. (2009)* Conserved Telomere maintenance Component 1 interacts with STN1 and protects chromosome ends in plants in higher eukaryotes. Molecular Cell 36:207-218.
Shakirov, E.V., Song, X., Joseph, J.A. and Shippen, D.E. (2009) POT1 proteins in green algae and land plants: DNA binding properties and evidence of co-evolution with telomeric DNA. Nucleic Acids Research 37: 7455-7467.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: In the past year we have made considerable progress in understanding the evolution of telomere proteins in plants. In addition, we have discovered several new genes that influence chromosome end protection in Arabidopsis. We have also defined novel components of the Arabidopsis telomerase enzyme. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our work on plant telemores is helping to provide fundamental insight into the factors that promote integrity of the genome. Since telomeres play a crucial role in human cancer and cellular aging, our work may have implications for human medicine. The intellectual merit of this work offers a unique opportunity to explore the long-term effects of genome instability that arises as a consequence of pertubing telomeres, the ends of DNA molecules. Our analysis of plant telomeres will not only contribute to understanding how plant genomes are maintained, but also will be relevant to a broad range of higher eukaryotes. We employ a combination of molecular, genetic, cytological and biochemical approaches to examine the structure and function of telomeres and their maintenance by the enzyme telomerase. The model system for our work is Arabidopsis, a genetically tractable higher eukaryote that has many important advantages over the mammalian model systems that have previously been exploited for telemore studies.

Publications

Vespa, L., Warrington, R., Mokros, P., Siroky, J., and Shippen, D.E. (2007) ATM regulates the length of individual telomere tracts in Arabidopsis. Proceedings of the National Academy of Sciences U.S.A. 104: 18145-18150
Kannan, K., Nelson, A. and Shippen, D.E. (2008) Dyskerin is a component of the Arabidopsis telomerase RNP required for telomere maintenance. Molecular and Cellular Biology 28:2332-2341.
Riehs, N., Akimcheva, S., Puizina, J., Bulankova, P., Idol, R., Siroky, J., Schleiffer, A., Schweizer, D., Shippen, D.E., Riha, K. (2008) The Arabidopsis EST1/SMG7-like protein is required for exit from meiosis. Journal of Cell Science 121:2208-2216.
Ming R, Hou S, Feng Y, Yu Q, Dionne-Laporte A, Saw JH, Senin P, Wang W, Ly BV, Lewis KL, Salzberg SL, Feng L, Jones MR, Skelton RL, Murray JE, Chen C, Qian W, Shen J, Du P, Eustice M, Tong E, Tang H, Lyons E, Paull RE, Michael TP, Wall K, Rice DW, Albert H, Wang ML, Zhu YJ, Schatz M, Nagarajan N, Acob RA, Guan P, Blas A, Wai CM, Ackerman CM, Ren Y, Liu C, Wang J, Wang J, Na JK, Shakirov EV, Haas B, Thimmapuram J, Nelson D, Wang X, Bowers JE, Gschwend AR, Delcher AL, Singh R, Suzuki JY, Tripathi S, Neupane K, Wei H, Irikura B, Paidi M, Jiang N, Zhang W, Presting G, Windsor A, Navajas-Perez R, Torres MJ, Feltus FA, Porter B, Li Y, Burroughs AM, Luo MC, Liu L, Christopher DA, Mount SM, Moore PH, Sugimura T, Jiang J, Schuler MA, Friedman V., Mitchell-Olds T, Shippen DE, dePamphilis CW, Palmer JD, Freeling M, Paterson AH, Gonsalves D., Wang L., Alam M. (2008) Genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus) Nature 452:991-996.
Song, X., Leehy, K., Warrington, R.T., Lamb, J.C., Surovtseva, Y. and Shippen, D.E. (2008) STN1 protects chromosome ends in Arabidopsis. Proceedings of the National Academy of Sciences, USA 105:19815-19820.

Progress 01/01/07 to 12/31/07

Outputs
In the past year we have made considerable progress in understanding the evolution of telomere proteins in plants. In addition, we have discovered several new genes that influence chromosome end protection in Arabidopsis. We have also defined novel components of the Arabidopsis telomerase enzyme.

Impacts
Our work on plant telemores is helping to provide fundamental insight into the factors that promote integrity of the genome. Since telomeres play a crucial role in human cancer and cellular aging, our work may have implications for human medicine. The intellectual merit of this work offers a unique opportunity to explore the long-term effects of genome instability that arises as a consequence of pertubing telomeres, the ends of DNA molecules. Our analysis of plant telomeres will not only contribute to understanding how plant genomes are maintained, but also will be relevant to a broad range of higher eukaryotes. We employ a combination of molecular, genetic, cytological and biochemical approaches to examine the structure and function of telomeres and their maintenance by the enzyme telomerase. The model system for our work is Arabidopsis, a genetically tractable higher eukaryote that has many important advantages over the mammalian model systems that have previously been exploited for telemore studies.

Publications

Shakirov, E.V., Surovtseva, Y., Vespa, L., Osbun, N. Song, X. and Shippen, D.E. (2007) Arabidopsis POT1 associates with the telomerase RNP and is required for telemere maintenance. EMBO Journal, 26:3653-3661.
Heacock, M., Idol, R., Friesner, J., Britt, A. and Shippen, D.E.(2007) Telomere dynamics and fusion of critically-shortened telomeres in plants lacking DNA ligase IV. Nucleic Acids Research, 35:6490-6500.

Progress 01/01/06 to 12/31/06

Outputs
In the past year we have made considerable progress in understanding the evolution of telomere proteins in plants. In addition we have discovered several new genes that influence recombination at telomeres. Finally, we have identified a novel pathway for chromosome fusion in Arabidopsis.

Impacts
Our work on plant telomeres is helping to provide fundamental insight into the factors that promote integrity of the genome.

Publications

Riha, K. Heacock, M. and Shippen, D.E. (2006) Telomeres and the non-homologous end-joining DNA repair pathway. Annual Review of Genetics 40:237-277.
Watson, J.M. and Shippen, D.E. (2006) Telomere rapid deletion regulates telomere length in Arabidopsis. Molecular and Cellular Biology, Dec 22; [Epub ahead of print].

Progress 01/01/05 to 12/31/05

Outputs
Most of our effort in 2005 focused on the characterization of a family of genes called Pot (protection of telomeres). We discovered that Arabidopsis has three of these genes, whereas most other organisms have only one. The Arabidopsis genes appear to play different roles in telomere biology. Pot1 functions in telomere length maintenance by telomerase, Pot2 in chromosome end protection and Pot3 in both telomere length regulation and chromosome end protection. We are now examining Pot genes in related plant species to understand the evolution of this important class of telomere binding proteins. In 2005, we also worked on defining the telomere function of two major DNA damage response proteins, ATM and ATR. We found that both of these genes function at telomeres, but they play fundamentally different roles. Interestingly, ATR is absolutely required to protect chromosome ends from rapid telomere shortening when telomerase is inactivated. This is a novel function for this enzyme.

Impacts
The intellectual merit of our work is that it offers a unique opportunity to explore the long-term effects of uncapped telomeres and the accompanying genome instability relevant to a broad range of higher eukaryotes. Since telomeres play a crucial role in human cancer and cellular aging, our studies in Araidopsis may have implications for human medicine.

Publications

Shakirov, E., Surovtseva, Y., Osbun, N. and Shippen, D.E. (2005) The Arabidopsis Pot1 and Pot2 proteins function in telomere length homeostasis and chromosome end protection. Mol Cell Biol, 25:7725-7733.
Watson, J.M., Bulankova, P., Riha, K., Shippen, D.E. and Vyskot, B. (2005) Telomerase-independent cell survival in Arabidopsis. Plant J 43:662-674
Vespa, L., Couvillion, M., Spangler, E. and Shippen, D.E. (2005) ATM and ATR make distinct contributions to chromosome end protection and the maintenance of telomeric DNA in Arabidopsis. Genes and Dev 19:2111-2115.

Progress 01/01/04 to 12/31/04

Outputs
Although plants only express telomerase in a few cell types, we found that telomere length is the same in "young" tissues that have divided very few times and "old" tissues that have undergone substantially more cell divisions. One explanation for this is that cells lose relatively little DNA as a conseuqence of the end replication problem. To address this possiblity, we measured the rate of telomere shortening in "young" and "old" tissues from telomerase-deficient plants. We found that essentially all of the telomere depletion associated with telomerase inactivation occurs in a narrow window of early plant development just after meiosis. We are currently testing the possibility that a single DNA processing event, perhaps involving loss of the T-loop via recombination, occurs at this stage. If this is true, the end replication problem contributes very little to the loss of telomeric DNA in a telomerase mutant. Another approach to study telomere dynamics has been to examine the rate of telomere shortening in ku70 mutants that have been restored with a wild type copy of Ku70. In ku70 mutants, telomeres expand to more than twice the length of wild type. We expected that telomeres would drift slowly back to the wild type length due to the end-replication problem. Instead, telomeres shortened very rapidly, arguing that long telomeres are subjected to discrete DNA processing events, prehaps via recombination.

Impacts
The intellectual merit of our work is that it offers a unique opportunity to explore the long-term effects of uncapped telomeres and the accompanying genome instability relevant to a broad range of higher eukaryotes. Since telomeres play a crucial role in human cancer and cellular aging, our studies in Araidopsis may have implications for human medicine.

Publications

Karamysheva, Z.N., Surovtseva, Y.V., Vespa, L., Shakirov, E.V., and Shippen, D.E. (2004) A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis. J. Biol Chem. 47799-47807.
Shakirov, E.V. and Shippen, D.E. (2004) Length regulation and dynamics of individual telomere tracts in wild-type Arabidopsis. Plant Cell. 1959-1967.
Heacock, M., Spangler, E., Riha, K., Puizina J. and Shippen, D.E. (2004) Molecular analysis of telomere fusions in Arabidopsis:multiple pathways for chromosome end-joining. EMBO. 2304-2313.
McKnight, T.D. and Shippen, D.E. (2004) Plant telomere biology. Plant Cell. 794-803.
Ren, S., Johnston, J.S., Shippen, D.E. and McKnight, T.D.(2004) Telomerase Activator 1 induces telomerase activity and potentiates responses to auxin in Arabidopsis. Plant Cell. 2910-2922.

Progress 01/01/03 to 12/31/03

Outputs
Genomic integrity in eukaryotes is derived in part from the architecture of telomeres, the nucleoprotein complexes that form a protective cap on chromosome ends. The ability to maintain the telomere cap lies at the heart of a biological clock that defines cellular life span in humans. In my lab we are using the plant, Arabidopsis, as a model system to study aspects of telomere structure and function. In the last year, our work focused on analysis of dysfunctional telomeres and on the identification and characterization of proteins that bind to telomeric DNA.

Impacts
The intellectual merit of our work is that it offers a unique opportunity to explore the long-term effects of uncapped telomeres and the accompanying genome instability relevant to a broad range of higher eukaryotes. Since telomeres play a crucial role in human cancer and cellular aging, our studies in Araidopsis may have implications for human medicine.

Publications

Sedivy, J.M, Shippen, D.E. and Shakirov, E. (2003) Surprise ending. Invited Commentary for Nature Genetics 33:114-116.
Riha, K. and Shippen, D.E. (2003) Telomere structure, function and maintenance in Arabidopsis. Chromosome Research 11:263-275.

Progress 01/01/02 to 12/31/02

Outputs
Genomic integrity in eukaryotes is derived in part from the architecture of telomeres, the nucleoprotein complexes that form a protective cap on chromosome ends. Telomeres consist of simple G-rich repeated DNA sequences bound by non-nucleosomal proteins. Telomeric DNA tracts are maintained by the enzyme telomerase, a specialized reverse transcriptase that compensates for the inability of the conventional DNA polymerase machinery to fully replicate the ends of linear chromosomes. The ability to maintain the telomere cap lies at the heart of a biological clock that defines cellular life span in humans. In somatic cells, telomerase is inactive, causing telomeres to inexorably shorten until they become dysfunctional. However, in cells with long-term proliferative potential (germline and stem cell populations, and more than 95 percent of human cancers) telomerase is expressed and telomeres are maintained at a stable length. The critical function of the telomere was demonstrated in plants more than 60 years ago by the pioneering work of Barbara McClintock (McClintock, 1942; McClintock, 1941). McClintock found that broken chromosomes lacking the natural terminal caps were highly unstable and subject to repeated rounds of breakage-fusion-bridge (BFB) cycles. At the molecular level the inability to maintain proper telomere architecture results in "deprotection" of chromosome ends, and signals to DNA damage checkpoint machinery that the telomere is a double-strand break that must be repaired. Indeed, recent studies demonstrate that a variety of DNA repair proteins reside at telomeres and may help to monitor telomere status. When a telomere becomes uncapped, the free ends are subject to nucleolytic degradation and end-to-end joining reactions that create chromosome fusions that undergo BFB cycles. Replicative senescence ensues, typically followed by cell cycle arrest and in metazoa, apoptosis (Lansdorp, 2000; Lundblad, 2000; Harrington, 2000). Consequently, elucidating the mechanisms that maintain and monitor the integrity of the telomere cap are widely believed to offer fundamental insight into the control of cell proliferation and genome stability.

Impacts
The intellectual merit of our work is that it offers a unique opportunity to explore the long-term effects of uncapped telomeres and the accompanying genome instability relevant to a broad range of higher eukaryotes. Since telomeres play a crucial role in human cancer and cellular aging, our studies in Arabidopsis may have implications for human medicine.

Publications

Riha, K., Watson, J., Parkey, J and Shippen, D.E. (2002) Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku. The EMBO Journal, 21:2819-2826.
Ray, S., Karamysheva, Z. and Wang, L., Shippen, D.E. and Price, C.M. (2002) Telomerase interacts with DNA replication proteins to physically associate de novo telomere formation and chromosomal replication machinery. Molecular Cellular Biology, 22: 5859-5868.
Wang, L., Dean, S. and Shippen, D.E. (2002) Oligomerization of the telomerase reverse transcriptase from Euplotes crassus. Nucleic Acids Research, 30:4032-4039.
McKnight, T.D., Riha, K. and Shippen, D.E. (2002) Telomeres, telomerase and stability of the plant genome. Plant Molecular Biology 48:331-337.

Progress 01/01/01 to 12/31/01

Outputs
My lab is studying telomeres and their maintenance by the enzyme telomerase using a biochemical model system, the ciliate, Euplotes, and a genetic model system, Arabidopsis. We made significant progress on both fronts. We found that that the catalytic subunit of telomerase from Euplotes is generated by a novel mechanism that involves two plus-one ribosomal frameshifting events. In addition we showed that this subunit can form a dimer, which may provide important insight into the structure of higher order telomerase complexes. Using the Arabidopsis model system, we have created a plant with no telomerase and have investigated the consequences of telomere dysfunction. We have also made significant progress in identifying novel proteins that bind to telomeric DNA in plants.

Impacts
(N/A)

Publications

Riha, K., McKnight, T.D., Griffing, L. and Shippen, D.E. (2001) Living with genome instability: plant responses to telomere dysfunction. Science, 291:1797-1800.
Fitzgerald, M.S., Shakirov, E., Hood, E., McKnight, T.D. and Shippen, D.E. (2001) Different modes of de novo telomere formation by plant telomerases. The Plant Journal, 26:1-12.

Progress 01/01/00 to 12/31/00

Outputs
My lab is studying telomeres and their maintenance by the enzyme telomerase using a biochemical model system, the ciliate, Euplotes, and a genetic model system, Arabidopsis. In the last year, we made significant progress on both fronts. We found that that the catalytic subunit of telomerase from Euplotes is generated by a novel mechanism that involves two plus-one ribosomal frameshifting events. In addition we showed that this subunit can form a dimer, which may provide important insight into the structure of higher order telomerase complexes. Using the Arabidopsis model system, we have created a plant with no telomerase and have investigated the consequences of telomere dysfunction. In addition, we have made significant progress in identifying novel proteins that bind to telomeric DNA in plants.

Impacts
(N/A)

Publications

Spangler, E., Rogers, K, Pustejovsky, D., Thomas, J., Boyd, S. and Shippen, D.E. 2000 Telomerase activity as a diagnostic tool to distinguish effusions of malignant and benign origin. Journal of Veterinary Internal Medicine. 14: 146-150.
Riha, K., Fajkus, J., McKnight, T.D., Vyskot, B. and Shippen, D.E. 2000 Analysis of the G-overhang structures on plant telomeres: evidence for two distinct telomere architectures. The Plant Journal, 23: 1-11.
Fitzgerald, M.S., Shakirov, E., Hood, E., McKnight, T.D. and Shippen, D.E. 2001 Different modes of de novo telomere formation by plant telomerases. The Plant Journal, in press.
Riha, K., McKnight, T.D., Griffing, L. and Shippen, D.E. 2001 Living with genome instability: plant responses to telomere dysfunction. Science, In press.
Wang, L., Karameysheva, Z., Shrode, T., and Shippen, D.E. Dimer 2000 formation by the telomerase reverse transcriptase from Euplotes crassus. In preparation.
Ray, S., Wang, L., Shippen, D.E. and Price, C.M. 2000 Telomerase associates with pola-primase during macronuclear develoment. In preparation.
Wang, L., Karmeysheva, Z., Shrode, T., Hurley, L.A. and Shippen, D.E. 2000 Expression of the telomerase catalytic subunit from Euplotes crassus requires two ribosomal frameshifts. In preparation.
McKnight, T.D., Riha, K. and Shippen, D.E. 2001 Telomere structure and function in plant growth and development. Plant Molecular Biology, In preparation.

Progress 01/01/99 to 12/31/99

Outputs
The overall objective if this project is to investigate the functions and interactions of telomerase-associated proteins. We hypothesize that the Euplotes crassus telomerase in a developing macronucleus exists as a holoenzyme, comprised of a "core" protein subunits of the E. crassus telomerase RNP and examine their functions in vitro. In addition, we will begin a biochemical characterization of higher order telomerase complexed that may correspond to a telomerase holenzyme. Finally, we have been investigating the molecular basis for a developmental switch in the ability of E. crassus telomerase to perform de novo telomere synthesis. To facilitate characterization of intrinsic and extrinsic telomerase components, recombinant telomerase proteins and antibodies will be generated. Also our specific aims for this project are to: 1)To clone and express genes encoding telomerase-associated proteins and generate specific antibodies. 2)To examine DNA,RNA and protein interactions in the "core" telomerase RNP. 3)To examine the biochemical properties and composition of higher order telomerase complexes and 4)To investigate the molecular basis for a developmental switch in DNA processing by telomerase.

Impacts
(N/A)

Publications

Hargrove, B., Bhattacharyya, A., Domitrovitch, A., Kapler, G.M, Kirk, K., Shippen, D.E. and Kunkel, G. (1999) An essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila. Nucleic Acids Research 27:4269-4275
Fitzgerald, M.S., Riha, K., Gao, F., Ren, S. McKnight, T.D. and Shippen, D.E.(1999) Disruption of the catalytic telomerase subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA. Proceedings of the National Academy of Sciences USA. 96: 14813-14818.
Spangler, E., Rogers, K, Pustejovsky, D., Thomas, J., Boyd, S. and Shippen, D.E. (2000) Telomerase activity as a diagnostic tool to distinguish effusions of malignant and benign origin. Journal of Veterinary Internal Medicine. In press.