-Updates on current progress-focusing on the original aims of the project Aim 1, Progress: Determined the interacting domains between SAP1 and shelterin component TPP1 We have created multiple retroviral expression constructs carrying different deletion mutants of SAP1, and generated MEF cells stably expressing those mutants. We then determined that the region located from 800 to 830 amino acids in SAP1 is required for its interaction with TPP1, an integral component of shelterin. To study if this interaction domain of SAP1 is needed for its association with shelterin in vivo, we established proximity ligase assay (PLA), a powerful tool for in situ detection of the protein-protein interaction in a cell. We have observed the association between TPP1 and flag-tagged SAP1 wildtype and SAP1 mutant 852 in cells, but not with SAP1 mutants 696 and 770. These data are consistent with our IP-Western blotting results (Fig. B). Using similar approaches, we determined the region of TPP1 required for the interaction of SAP1 with TPP1. The identification of the interacting domains in both SAP1 and TPP1 provides a basis for studying the mechanism of how SAP1-SAP2 complex acts in telomere protection described in Aim 2 and Aim 3. Aim 2, Progress: SAP1-SAP2 complex controls telomere length homeostasis. We have employed several protocols to examine any telomere defects in SAP1 or SAP2 knockout MEF cells. Thus far, we have reproducibly identified that the major phenotypic abnormalities of those knockout cells are shortened telomeres. Notably, we have also detected telomere shortening of SAP1-ablated mouse primary B cells, providing additional evidence to conclude that SAP1-SAP2 complex is required for the maintenance of telomere length. To explore the underlying mechanism, we have generated multiple SAP1 knockout MEF cells stably expressing various deletion mutants of SAP1 identified from Aim 1. We are currently determining the importance of shelterin-interacting domain of SAP1 for telomere lengthening. Aim 3, Progress: SAP1-SAP2 complex stimulates telomerase activity to elongate telomere length. We have previously described in our proposal that both SAP1 and SAP2 knockout MEF cells exhibit shortened telomere length. To explore the underlying mechanism, we have utilized telomeric repeat amplification protocol (TRAP) assay to detect the telomerase activity in SAP1 or SAP2 knockout MEF cells, versus the wildtype cells, or KO cells complemented by re-expression of SAP1 or SAP2. The results showed that telomerase activity were decreased in both SAP1 and SAP2 knockout cells, when compared to those in wildtype cells; and this decrease can be largely recused by reintroduction of SAP1 or SAP2, indicating that SAP1-SAP2 complex regulates the telomerase activity in MEF cells. Notably, overexpression of either SAP1 mutant 830 or mutant 852 resulted in higher levels of telomerase activity than SAP1 mutant 696. This correlates with the findings that both SAP1 mutant 830 and 852 interact with TPP1, whereas mutant 696 does not, suggesting that SAP1 interaction with TPP1 may promote telomerase function. TPP1 is known to be the only component of shelterin that directly interacts with and recruits telomerase to shelterin to facilitate the elongation of telomere length (Nandakumar et al, Nature 2012; Schmidt et al, Nature 2014; Zhong et al, Cell 2012). Individuals carrying a heterozygous single amino acid deletion (del K170) exhibit shortened telomeres (Kocak et al., Genes Dev 2014; Guo et al, Blood 2014), which is the similar phenotype of both SAP1 and SAP2 knockout cells. Taken together, our data suggest a model that SAP1-SAP2 complex may interact with and stimulate the activity of TTP1 in recruiting telomerase to telomeres to maintain its normal length.