Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.

Gametes deficient for Pot1 telomere binding proteins alter levels of telomeric foci for multiple generations

Deficiency for telomerase results in transgenerational shortening of telomeres. However, telomeres have no known role in transgenerational epigenetic inheritance. C. elegans Protection Of Telomeres 1 (Pot1) proteins form foci at the telomeres of germ cells that disappear at fertilization and gradually accumulate during development. We find that gametes from mutants deficient for Pot1 proteins alter levels of telomeric foci for multiple generations. Gametes from pot-2 mutants give rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci throughout development, which persists for six generations. In contrast, gametes from pot-1 mutants or pot-1; pot-2 double mutants induce diminished Pot1 foci for several generations. Deficiency for MET-2, SET-25, or SET-32 methyltransferases, which promote heterochromatin formation, results in gametes that induce diminished Pot1 foci for several generations. We propose that C. elegans POT-1 may interact with H3K9 methyltransferases during pot-2 mutant gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that causes constitutively high levels of heterochromatic Pot1 foci.

A Germline Mutation in the POT1 Gene Is a Candidate for Familial Non-Medullary Thyroid Cancer

Non-medullary thyroid cancer (NMTC) is a common endocrine malignancy with a genetic basis that has yet to be unequivocally established. In a recent whole-genome sequencing study of five families with occurrence of NMTCs, we shortlisted promising variants with the help of bioinformatics tools. Here, we report in silico analyses and in vitro experiments on a novel germline variant (p.V29L) in the highly conserved oligonucleotide/oligosaccharide binding domain of the Protection of Telomeres 1 (POT1) gene in one of the families. The results showed a reduction in telomere-bound POT1 levels in the mutant protein as compared to its wild-type counterpart. HEK293T cells carrying POT1 p.V29L showed increased telomere length in comparison to wild-type cells, suggesting that the mutation causes telomere dysfunction and may play a role in predisposition to NMTC in this family. While one germline mutation in POT1 has already been reported in a melanoma-prone family with prevalence of thyroid cancers, we report the first of such mutations in a family affected solely by NMTCs, thus expanding current knowledge on shelterin complex-associated cancers.

A novel germline mutation in the POT1 gene predisposes to familial non-medullary thyroid cancer

Non-medullary thyroid cancer (NMTC) is a common endocrine malignancy with a genetic basis that has yet to be unequivocally established. In a recent whole genome sequencing study of five families with recurrence of NMTCs, we shortlisted promising variants with the help of bioinformatics tools. Here, we report in silico analyses and in vitro experiments on a novel germline variant (p.V29L) in the highly conserved oligonucleotide/oligosaccharide binding domain of the Protection of Telomeres 1 (POT1) gene in one of the families. The results showed that the variant demonstrates a reduction in telomere-bound POT1 levels in the mutant protein as compared to its wild-type counterpart. HEK293Tcells carrying POT1V29L showed increased telomere length in comparison to wild type cells, strongly suggesting that the mutation causes telomere dysfunction and may play a role in predisposition to NMTC in this family. This study reports the first germline POT1 mutation in a family with a predominance of thyroid cancer, thereby expanding the spectrum of cancers associated with mutations in the shelterin complex.

Protection of telomeres 1 (POT1) of Pinus tabuliformis bound the telomere ssDNA

Protection of telomeres 1 (POT1) is a telomeric protein that binds to the telomere single-stranded (ss) region. It plays an essential role in maintaining genomic stability in both plants and animals. In this study, we investigated the properties of POT1 in Pinus tabuliformis Carr. (PtPOT1) through electrophoretic mobility shift assay. PtPOT1 harbored affinity for telomeric ssDNA and could bind plant- and mammalian-type ssDNA sequences. Notably, there were two oligonucleotide/oligosaccharide binding (OB) folds, and OB1 or OB2 alone, or both together, could bind ssDNA, which is significantly different from human POT1. Based on our data, we hypothesized that the two OB folds of PtPOT1 bound the same ssDNA. This model not only provides new insight into the ssDNA binding of PtPOT1 but also sheds light on the functional divergence of POT1 proteins in gymnosperms and humans.

RARE-48. GROWING KNOWLEDGE AND EMERGING QUESTIONS RELATED TO POT-1 GERMLINE MUTATIONS AND ASSOCIATED MALIGNANCIES: A CASE REPORT

Protection of telomeres 1 (POT1) is a member of a family of genes responsible for maintenance of telomere integrity. Germline mutations of the POT1 gene have been associated with tumorigenesis of multiple tissue types, conferring an increased risk of development of malignancies such as melanoma, chronic lymphocytic leukemia, gliomas, and cardiac sarcomas. We present the case of a 28 year old male who first presented at the age of 15 with personality changes. Several years later, he was found to have a left frontal, IDH mutated, WHO grade III anaplastic astrocytoma. Following successful treatment with gross total resection, radiation therapy, and adjuvant chemotherapy, the patient represented with an MRI showing enhancement concerning for osteomyelitis and possible tumor recurrence. Pathology of the dura was consistent with myxofibrosarcoma. Staging PET scan at that time revealed multiple hypermetabolic lung lesions found to be Lanagerhans cell histiocytosis. Given the patient’s complex oncological history, genetic testing was performed. He was found to carry a heterozygous mutation of the POT1 gene. This case raises the suspicion for a broader POT1-associated cancer predisposition than previously described in the literature. Moreover, this prompts further questioning of an association between POT1 mutations and IDH status in related gliomas. Patients with POT1 mutations might also be at increased risk of secondary malignancies after radiation exposure, which may require closer observation after treatment.