Agnel Sfeir, PhD
Assistant Professor of Cell Biology
Ph.D., 2006, The University of Texas Southwestern Medical Center
Skirball Institute of Biomolecular Medicine
Developmental Genetics Program
New York University School of Medicine
540 First Ave. 4th floor
New York, NY 10016
Research Theme(s): Chromosome biology, Stem cell biology, Telomere length regulation, DNA damage signaling and repair.
Keywords: Telomeres, Telomerase, DNA damage and repair, embryonic stem cells. iPS.
Telomeres, the natural ends of linear chromosomes are essential to ensure genomic stability and promote cellular survival. In mammalian cells telomeres consist of long tracts of repetitive DNA sequences (TTAGGG), bound by a specialized six-subunit protein complex termed shelterin.
In normal human somatic cells, telomeres progressive shorten with ongoing cellular division due to the inability of the replication machinery to copy chromosomes till the very end. When a subset of telomeres within a cell becomes short, a DNA damage signal is triggered to activate the p53 and Rb pathways that ultimately induce cellular senescence. This is known as the Hayflick limit and is the basis of the tumor suppressive function of telomere shortening. Cells that accumulate mutations in the checkpoint pathways are capable of by-passing this stage and proliferate further, resulting in additional telomere shortening. This is followed by telomere erosion, which engages the DNA repair machineries, leading to a stage of extensive chromosome instability, known as crisis. Very few cells can bypass both senescense and crisis and they do so by upregulating telomere maintenance pathways, which either involve activating telomerase or inducing telomere recombination. These cells are then able to progress and become the increasingly aggressive tumors.
This scenario highlights the importance of fully understanding telomere maintenance and homeostasis in mammalian cells, which is the focus of the Sfeir laboratory. We are interested in how telomere dynamics impact stem cell function and leads to tumorigenesis.
Aim 1: Telomere length resetting
Telomere length is reset in the germ line and during early embryonic development. In recent years, accumulating evidence suggested that telomere length resetting is recapitulated during the process of nuclear reprogramming, yet the underlying mechanism is completely unknown. To elucidate the pathways that control telomere re-lengthening, our lab utilizes iPSc (induced pluripotent stem cell) induction with four transcription factors (Oct4, Sox2, c-Myc and Klf4) as a cell-reprogramming platform.
Our aim is to assess the contribution of telomerase mediated-extension as well as alternative recombination pathways to telomere elongation, using standard molecular tools of telomere length analysis, telomere recombination and telomerase activity. We also plan to pursue a non-biased approach and perform shRNA-based screens to identify candidate genes that control the extent of telomere elongation during the reprogramming process.
Aim 2: Telomerase activation and regulation
Telomerase is a reverse transcriptase that adds TTAGGG repeats to the end of chromosome and in doing so, counteracts telomere shortening. While normal human somatic cells lack the activity of the enzyme, telomerase is active in stem cells, progenitor cells of self-renewing tissues, reproductive cells and the majority of cancer cells. Being an obligate means for the indefinite growth of cancer cells, telomerase constitutes a very promising target for cancer therapy and understanding the details of its action is key to allow it to be further explored in cancer therapy. Despite recent progress in our understanding of telomerase biogenesis and enzymology, it is still unknown how cancer cells turn on telomerase expression and what factors control this step. Furthermore, it is not fully understood how this enzyme, which is present in very low abundance (<100 /cell) gets recruited to 96 chromosome ends in a timely and controlled fashion. Lastly, the similarities and/or differences in the telomerase pathway between stem cells and cancer cells require further investigation. To answer all of these questions, we plan to develop novel approaches and identify genes that regulate telomerase expression, activity, and recruitment. One such assay involves using genetically modified mouse cells expressing an altered form of telomerase that, when active is toxic to cells. In this setting, toxicity can only be reverted by inhibiting telomerase activity. These cells will be used to screen a genome wide shRNA library and identify novel factors in the telomerase pathway. Once idenitfied, the functionality of the genes will be studied in the context of cancer cells and stem cells.
Aim3: Telomere dysfunction in stem cells
Mammalian cells are constantly faced with DNA lesions and breaks that can lead to genomic instability if not fixed. Telomere dysfunction in senescent cells is a major source of genomic instability that is thought to fuel cancer progression. Telomere function is lost when the repetitive sequence becomes too short or when the shelterin complex that binds to the DNA is lacking. The outcome of telomere dysfunction has been studied in mouse fibroblasts in which shelterin was genetically targeted using conditional knockout strategy. The specific DNA damage signaling (ATM and ATR) and repair (NHEJ and HR) pathways that block cell cycle progression in these cells have been identified. A major study in our lab is to use mouse genetics as a tool to study the consequence of telomere dysfunction in embryonic and adult stem cells. We will probe for the mechanisms that are operative in stem cells to sense the DNA damage and stop cell cycle progression and to make molecular decisions to repair the damage.
- Rap1-independent telomere attachment and bouquet formation in mammalian meiosis. Scherthan H, Sfeir A, de Lange T. Chromosoma. 2011 Apr;120(2):151-7. Epub 2010 Oct 7. PMID: 20927532
- Taking apart Rap1; An adaptor protein with telomeric and non-telomeric functions. Kabir S, Sfeir A and de Lange T (2010). Cell Cycle. Oct 15;9(20):4061-7. PMID: 20948311
- Loss of Rap1 induces telomere recombination in absence of NHEJ or a DNA damage signal. Sfeir A, Kabir S, van Overbeek M, Celli G, and de Lange T (2010). Science. Mar 26;327(5973):1657-61. PMID: 20339076
- Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells. Zhao Y, Sfeir AJ, Zou Y, Buseman CM, Chow TT, Shay JW, Wright WE (2009). Cell. Aug 7;138(3):463-75. PMID: 19665970
- Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Sfeir A, Settapong K, Hockemeyer D, McRae SL, Karlseder J, Schildkraut K, and de Lange T (2009). Cell. Jul 10;138(1):90-103 (Cover) PMID: 19596237
- The MRN complex is required for the generation of proper G-overhangs at human telomeres. Chai W, Sfeir AJ, Shay JW and Wright WE. (2006) EMBO Reports. Feb;7(2):225-30. PMID: 16374507
- POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end. Hockemeyer D, Sfeir AJ, Shay JW, Wright WE and de Lange T (2005). EMBO J. Jul 20;24(14):2667-78. PMID: 15973431
- Fine-tuning the chromosome ends: the alst base of human telomeres. Sfeir AJ, Shay JW and Wright WE. (2005). Cell Cycle Nov;4(11):1467-70. PMID: 16258279
- Telomere-end processing; the terminal nucleotide of mammalian chromosomes. Sfeir AJ, Chai W, Shay JW and Wright WE. (2005). Mol Cell. Apr 1;18(1):131-8. PMID: 15808515
- Does a sentinel or a subset of short telomeres determine replicative senescence? Zhou Y, Sfeir A, Gryaznov SM, Shay JW and Wright WE. (2004). Mol Biol Cell. Aug; 15(8):3709-18. PMID: 15181152