TERF2

The TERF Homology Domain (TRFH; InterPro: IPR013867) is an area that helps to promote homodimerization of TERF2 with itself. This results in the formation of a quaternary structure that is characteristic of this protein. This TRFH domain also allows TERF2 to bind to and act as a dock for many other types of proteins. The Apollo nuclease, a shelterin accessory factor, uses the TRFH domain as a dock. The recruitment of Apollo by TERF2 allows for telomeric ends formed by DNA synthesis to be processed. By doing so, the telomere ends are able to avoid ATM kinase activation through the creation of a terminal structure.[8] SLX4, which is important in DNA repair by acting as a scaffold for structure-specific DNA repair nucleases, also binds to the TRFH domain of TERF2.[9] The TRFH domain is responsible for other binding events, including RTEL1, and proteins that contain a TBD site.

The Myb domain (InterPro: IPR017930) acts by binding to double-stranded telomeric DNA. This region gets its name from a viral protein called Myb derived from the avian myeloblastosis virus. Specifically, the sequence that this Myb domain targets on the DNA is (GGTTAG/CCAATC)n.

Two other domains also work to bind and influence the activity of proteins associated with the TERF2 protein. Both are unique to TERF2. The basic domain sits at the N-terminal, and has two main functions: the prevention of t-loop excision by XRCC3, and the inhibition of SLX4. The final domain of TERF2 is called the hinge domain (InterPro: IPR031902). This domain contains a motif for binding the shelterin protein TIN2, which acts as a stabilizing protein, connecting units that are attached to double stranded and single stranded DNA.[10] This domain also is responsible for binding to RAP1, and helps to inhibit RNF168 recruitment at telomeres.

Specific domains on the TERF2 protein sequence and their functionalities.[11]

Telomeric ends are structurally similar to double-stranded breaks on the chromosome. To prevent the cellular DNA repair machinery from mistakenly identifying telomeres as chromosome breaks, t-loops are formed in which the 3’ TTAGGG overhang of the telomere loops back into the DNA duplex. TERF2 promotes t-loop formation by preferentially binding to a telomeric double-stranded DNA duplex containing a 3’ TTAGGG single-stranded overhang. If the 3’ TTAGGG overhang is not present, TERF2 will not bind. Once bound, it migrates to the t-loop junction where the single-stranded overhang invades the double-stranded region upstream. No other shelterin protein has been shown to promote this process but interaction of TERF2 with TERF2IP is shown to promote higher t-loop formation in vitro.[12] Studies have demonstrated that deletion of TERF2 prevents t-loop formation, leading to excessive loss of telomeric DNA and early cell death.[13]

TERF2 plays a central role in preventing ATM kinase DNA damage response. It binds telomeric dsDNA and prevents telomeres from activating ATM kinase. This interaction of TERF2 with ATM is believed to be relevant to the mechanism by which TERF2 blocks ATM signaling. Because of its oligomeric nature, TERF2 could potentially cross-link ATM monomers and hold the kinase in its inactive dimeric state, thereby blocking amplification of the ATM signal at an early step in its activation. However, because mutations in the TERF2 dimerization domain destabilize the protein, it has not been possible to test the contribution of TERF2 oligomerization on ATM repression directly. Removal of TERF2 induces ATM-dependent apoptosis by localizing the active, phosphorylated form of ATM to unprotected chromosome ends. Since TERF2 specifically binds at telomeres and remains there when DNA damage is induced, it is unlikely to interfere with activation of the ATM kinase at different sites of DNA damage. Therefore, TERF2 could act as a telomere-specific inhibitor of ATM kinase.[14]

Conditional deletion of TERF2 in mice cells effectively removes the shelterin nucleoprotein complex. As a result of removing this complex, several unwanted DNA damage response pathways are activated, including ATM kinase signaling, ATR kinase signaling, non-homologous end-joining (NHEJ), alt-NHEJ, C-NHEJ, 5' resection, and homology directed repair (HDR).[15] These repair pathways (in the presence of P53 knockout and Cre) often contribute to the phenotype where chromosome ends are connected to each other in a very long chain, which can be visualized by a combination of a DAPI stain and fluorescence in situ hybridization (FISH) technique.

Telomerase is an enzyme that works to create telomeric ends for DNA, and it is thought to play important roles in the development of cancer. Specifically, telomeric stability is known to be a common occurrence in cancer cells.[23] Along with the telomerase, the shelterin complex, and TERF2 and TERF1 specifically, also have been noted to control the lengths of telomeres formed by these telomerases. Shelterin works to protect telomeres against unsuitable activation of the DNA damage response pathway, as noted in the function section above. TERF2 as part of the shelterin complex, has been known to block the ATM signaling pathways and prevent chromosome end fusion. In cancer cells, TERF2 phosphorylation by extracellular signal-regulated kinase (ERK1/2) is a controlling factor in the major pro-oncogenic signaling pathways (RAS/RAF/MEK/ERK) that affect telomeric stability.[23] Additionally, when TERF2 was non-phosphorylated in melanoma cells, there was a cell induced DNA damage response, arresting growth and causing tumor reversion.[23] Studies have found that in tumor cells, TERF2 levels are observed to be high, and this raised level of TERF2 contributes to oncogenesis in a variety of ways.[24][25][26] This high level of TERF2 decreases the ability to recruit and activate natural killer cells in human tumor cells.[24] One study used a dominant negative form of TERF2^ΔBΔC, to inhibit TERF2, and found that it could induce a reversion malignant phenotype in human melanoma cells.[25] Therefore, over-expression of TERF2^ΔBΔC, causing blocking of TERF2, induced apoptosis and reduced tumourigenicity in certain cell lines.[25] Additionally, upregulation of TERF2 may be the cause of the establishment and maintenance of short telomeres.[26] These short telomeres increase chromosomal instability, and increase the chances of certain cancers progressing in the body, such as with leukemia.[26] In gastric mucosa tissues, the expression of TERF2 proteins was significantly higher than normal, and this over-expression of TERF2, along with over-expression of TERF1, TIN2, TERT, and BRCA1 protein transposition, may cause a reduction in telomere length, further contributing to multistage carcinogenesis of gastric cancer.[27]