Telomeres are nucleoprotein structures that protect the ends of linear chromosomes. They are composed of long tracts of TTAGGG repeats, telomere specific proteins that form the shelterin complex and several telomere accessory proteins that co-operate to telomere metabolism. Proper telomere maintenance is a crucial process to protect the genome against instability and telomere dysfunction has been linked to tumorigenesis and premature aging. AKTIP gene is the human homologue of Drosophila peo, a gene that was recently linked to telomere metabolism.
The aim of this study was to understand if AKTIP could have a role in human telomere metabolism, in analogy with the telomeric function of its homologous in fly. For this purpose we have analyzed the phenotype of human cells in which AKTIP expression was downregulated by RNA interference. In human primary cells AKTIP downregulation triggered the reduction of the mitotic index, proliferation impairment and premature senescence. AKTIP reduction induced a strong DNA damage response proved by the accumulation of the phosphorylated form of proteins involved in DNA damage sensing and signaling such as ATM, p53 and Chk1, by the accumulation of p21 mRNA and by the formation of foci containing DNA damage response proteins. About half of these foci were located at telomeres (TIFs) indicating the presence of dysfunctional telomeres in AKTIP knocked down cells. These data were consistent with the accumulation of aberrant telomeres in MEFs p53-/- observed following the downregulation of murine homologue of AKTIP (named Ft1). AKTIP involvement in telomere metabolism was further suggested by its interaction with telomeric repeats observed by ChIP analysis. Altogether, these findings indicate that AKTIP takes part in telomere maintenance. Interestingly, immunostaining assays showed that AKTIP is not a stable component of telomeres but was found located in the nucleus, mainly at nuclear rim. This particular localization, in addition with the telomeric role outlined for AKTIP, suggest that AKTIP is a telomeric nonshelterin protein. Consistent with this hypothesis, we observed that Ft1 downregulation caused the formation of chromosomal aberrations in addition to telomeric abnormalities, indicating that AKTIP/Ft1 plays a role not only in telomere maintenance but also in the overall genomic stability, possibly contributing to DNA replication. Indeed, the most prominent telomeric aberration observed in Ft1 downregulated MEFs was the formation of multiple telomeric signals at the ends of chromosomes, also known as fragile telomeres, indicative of replication impairment. In addition, AKTIP downregulation was found to induce an S-phase block of cell cycle progression and a strong reduction of PCNA positive cells in primary fibroblasts, along with an increased sensitivity to drugs that impair DNA replication, as aphidicolin. Collectively, these data demonstrate that AKTIP is a protein needed for proper DNA maintenance in mammalian cells. In the telomeric context AKTIP likely is a telomeric accessory protein, rather than a shelterin-like protein, because it’s conserved in fly, differently from shelterin proteins, has a role in telomere maintenance but is not stably located at telomeres. AKTIP, in addition to its telomeric function, seems to have a more general role in cellular metabolism, as all the other telomeric nonshelterin proteins. In particular our data indicate that AKTIP could be involved in DNA replication.
Considering all the collected data together, our current hypothesis is that AKTIP plays a role in replication of complex DNA structures, including telomeric repeats. Its downregulation could impair the replication fork progression through these DNA regions leading to chromosomal aberrations, DNA damage response and cell cycle alterations, the most prominent phenotypic traits of AKTIP knocked down cells
