The ends of linear chromosomes are formed by special heterochromatic structure, known as the telomere, which protects them from degradation and repair activities and, it is essential to ensure chromosomal stability (de Lange, 2005; Blasco, 2007; Palm & de Lange, 2008). Mammalian telomeric chromatin is composed of tandem repeats of the TTAGGG sequence bound by a specialized protein complex known as shelterin (de Lange, 2005; Palm & de Lange, 2008). The shelterin complex is composed of six core proteins, the telomeric repeat binding factor 1 and 2, (TRF1 and TRF2), the TRF1-interacting protein 2 (TIN2), protection of telomeres protein 1 (POT1), TPP1, (also known as ACD, TINT1, PTOP, or PIP1), and RAP1 (de Lange, 2005). TRF1, TRF2, and POT1 bind directly to telomeric DNA repeats, with TRF1 and TRF2 binding to telomeric double-stranded DNA and POT1 to the 3′-single-stranded G-overhang (Broccoli et al., 1997; Bianchi et al., 1999; Court et al., 2005; de Lange, 2005; Palm & de Lange, 2008). (Baumann & Cech, 2001; Loayza & De Lange, 2003; Lei et al., 2004; He et al., 2006; Hockemeyer et al., 2006; Wu et al., 2006; Palm & de Lange, 2008). TIN2 is able to bind TRF1 and TRF2 through independent domains and to recruit the TPP1-POT1 complex, constituting the bridge between the different shelterin components (Kim et al., 2004; Liu et al., 2004; Ye et al., 2004; Chen et al., 2008). TPP1 recruits POT1 to telomeres (Chen et al., 2007; Kibe et al., 2010). In addition, TPP1 has a crucial role in the recruitment of telomerase to chromosome ends (Xin et al., 2007; Tejera et al., 2010; Nandakumar et al., 2012; Sexton et al., 2012; Zhong et al., 2012; Zhang et al., 2013). Finally, RAP1 binds telomeric repeats through its interaction with TRF2 although it is dispensable for telomere capping (Martinez et al., 2010; Sfeir et al., 2010). Rap1 has also been shown to bind throughout chromosome arms where it regulates gene expression. (Martinez et al., 2010). Indeed, Rap1 has been recently demonstrated to protect from obesity through its role in regulating key metabolic pathways (Martinez et al., 2013; Yeung et al., 2013).
Abrogation of TRF1, TRF2, POT1b, TPP1, and TIN2 in mice results in early embryonic lethality (Karlseder et al., 2003; Chiang et al., 2004; Celli & de Lange, 2005; Hockemeyer et al., 2006; Lazzerini Denchi et al., 2006; Wu et al., 2006; Kibe et al., 2010). Conditional deletion of TRF1 and TPP1 in mouse stratified epithelia leads to perinatal lethality coincidental with skin hyperpigmentation and severe skin morphogenesis defects, including absence of mature hair follicles and sebaceous glands, which are concomitant with induction of telomere-originated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo (Martinez et al., 2009; Tejera et al., 2010). Importantly, p53 deficiency rescues both the stem cell defects and skin hyperpigmentation, as well as mouse survival, in both mouse models, indicating that the severe skin defects associated with TRF1 and TPP1 abrogation are mediated by p53. Interestingly, long-lived TRF1/p53 double null mice spontaneously develop invasive and genomically unstable squamous cell carcinomas (Martinez et al., 2009). These results suggest that TRF1 normally acts as a tumor suppressor in the context of the organism by preventing telomere-induced genetic instability in proliferating cells. In marked contrast to TRF1 and TPP1, targeted Rap1 deletion in stratified epithelia in Rap1Δ/Δ K5-cre mice does not impact on mouse viability in accordance with a dispensable role for Rap1 in telomere capping (Martinez et al., 2010).
In contrast to conditional deletion of TPP1 and TRF1 in stratified epithelia, TRF2 conditional deletion in the liver (Mx1TRF2 mice) did not impact on liver regeneration or mouse viability, indicating that TRF2 is dispensable for hepatocyte regeneration (Lazzerini Denchi et al., 2006). In particular, TRF2 deletion in hepatocytes leads to telomere damage and telomere fusions; however, these phenotypes were not accompanied by loss of liver function upon partial hepatectomy. Indeed, liver regeneration in these mice occurred by endoreduplication and cell growth, but not cell division, thereby overcoming the chromosome segregation problems associated with telomere fusions (Lazzerini Denchi et al., 2006).
To further understand the role of TRF2 in the adult organism, as well as to do a comparative analysis with conditional knock-out models for other shelterins, here we deleted TRF2 in stratified epithelia. TRF2Δ∕ΔK5-Cre mice were born at submendelian ratios indicating partial embryonic lethality. Newborn mice die immediately after birth and show complete lack of epidermis. We show that TRF2-deleted epidermis undergoes tissular necrosis accompanied by the presence of dermal infiltrates and increased IL6 levels already at E13.5 of embryonic development that becomes apparent at E16.5 by the complete absence of the epidermal layer of the skin. Comparative studies of the phenotypic effects originated by conditional deletion of either TRF1, TPP1, RAP1, or TRF2 in stratified epithelia, a highly proliferative tissue, revealed that TRF2 deficiency causes the most severe proliferative and developmental defects of all these shelterin components.