Telomere-associated Protein TIN2 Is Essential for Early Embryonic Development

Chiang YJ, Kim SH, Tessarollo L, Campisi J, and Hodes RJ. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway. Mol Cell Biol 24: 6631–4, 2004.

he ends of linear eukaryotic chromosomes consist of telomeres that contain telomeric DNA repeats, (TTAGGG)n hexanucleotide repeats in mammalian chromosomes, and a number of associated proteins. This telomeric structure is critical for distinguishing the chromosomal terminus from free ends of damaged DNA, and thus, telomeres prevent the triggering of inappropriate cell cycle arrest and/or apoptotic responses normally elicited by DNA damage. In eukaryotic cells, the mechanism of chromosomal replication during cell division results in incomplete terminal synthesis, so that in the absence of a compensatory mechanism, 50–200 bases of terminal telomeric DNA are lost with each division. Thus, successive cycles of cell proliferation can lead to progressive telomere shortening, until a critically short length is reached at which telomere function is compromised, with consequences that can include replicative senescence, apoptosis, and tumorigenic chromosomal instability. A compensatory mechanism capable of adding terminal telomeric repeats is mediated by the RNA-dependent DNA polymerase, telomerase. This enzyme consists of two essential molecular components, the telomerase RNA (TR) component, which includes a template for telomeric DNA, and the catalytic telomerase reverse transcriptase (TERT), which mediates telomere synthesis. Importantly, recent discoveries have demonstrated that maintenance of telomere function is also dependent on the influence of additional telomere-associated proteins, and elucidating the function of these proteins is, therefore, an area of considerable interest.

TIN2 (TRF1-interacting protein 2) was recently identified as a telomere-associated protein that interacts with TRF1, a molecule that binds directly to telomeric DNA and functions as a negative regulator of telomere length. TIN2 contains N-terminal basic and acidic regions, a central TRF1-binding domain, and a C-terminal region. The basic and acidic regions are required for the regulation of TRF1 activity by TIN2. The TRF1-binding domain associates with the TRF1-homodimerization domain, providing for the recruitment of TIN2 to the telomere. In vitro studies have shown that overexpression of TIN2 inhibits telomere elongation in human cell lines, whereas expression of dominant-negative mutants of TIN2 enhances telomere elongation. It has been suggested that the binding of wild-type TIN2 induces changes in TRF1 conformation that in turn favor a telomeric structure that is inaccessible to telomerase, thus preventing telomerase-mediated telomere elongation. The absence of TIN2 would conversely favor telomerase accessibility and telomere elongation.

The physiological role of TIN2 during in vivo development and in normal cell function had not previously been assessed. To better understand the in vivo function of TIN2, we have, therefore, studied the effect of TIN2 mutation on mouse development, using gene-targeting technology. No homozygous TIN2–/– mice were identified in the offspring of TIN2+/– mouse intercrosses. Furthermore, homozygous TIN2-deficient embryos were absent as early as day 7.5. This finding indicated that TIN2 is essential for mouse development and that homozygous inactivation of TIN2 is lethal before day 7.5 of embryonic development. However, day 3.5 TIN2–/– embryos were obtained in expected frequency (1/4) among offspring of TIN2+/– intercrosses. When day 3.5 TIN2–/– embryonic cells were cultured, it was striking that they were uniformly defective in their differentiation, in comparison to day 3.5 wild-type embryonic cultures. Wild-type embryonic cultures grew to form multilayered cell masses, whereas TIN2–/–embryonic cultures were flat and contained few viable cells. A growth and/or survival defect was thus apparent in TIN2–/– cells at an early stage of embryonic development.

The previously identified function of TIN2 was proposed to involve enhancing the activity of TRF1 in downregulating the telomerase elongation of telomeres. We asked whether the embryonic lethality observed in TIN2–/– mice might be telomerase dependent. To explore this possibility, TIN2+/– mice were bred to mTERT–/– mice that lacked telomerase activity. It was striking that no TIN2–/– mTERT–/– offspring were observed, whereas TIN2+/+ mTERT–/– and TIN2+/– mTERT–/– mice survived. Thus, embryonic lethality of TIN2–/– mTERT–/– mice indicated that the requirement for TIN2 in mouse development reflects a previously unappreciated telomerase-independent function of this molecule.

Recently, it was reported that inactivation of the mouse TRF1 gene results in embryonic lethality, and that TRF1 knockout blastocysts have a cell growth defect and increased apoptosis. The phenotype of TIN2 knockout mice thus appears to be similar to that of TRF1-deficient mice. These observations imply that, in addition to the telomerase-dependent functions played by TIN2/TRF1 complexes, both TIN2 and TRF1 also function in telomerase-independent roles. To understand the telomerase-independent roles of TIN2 and TRF1 in embryonic development and in adult animals, studies of inducible TIN2 or TRF1 conditional knockout mice will be informative. We have in fact generated TIN2 conditional knockout constructs using cre/loxP techniques and will use these constructs in studies of inducible and tissue-specific TIN2 inactivation. Additional telomere-associated proteins may be involved in the potentially complex functions of TIN2 and TRF1, and we are currently pursuing genetic approaches to analyze candidate components involved in these functions.

Y. Jeffrey Chiang, PhD
Staff Scientist
Experimental Immunology Branch
NCI-Bethesda, Bldg. 10/Rm. 4B10
Tel: 301-496-1376
Fax: 301-496-0887
chiangj@mail.nih.gov

Richard J. Hodes, MD
Senior Principal Investigator
Experimental Immunology Branch
NCI-Bethesda, Bldg. 10/Rm. 4B10
Tel: 301-496-3129
Fax: 301-496-0887
hodesr@31.nia.nih.gov