Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin.

Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin

TSPYL5 depletion induces specific death of ALT cells through USP7-dependent proteasomal degradation of POT1

A significant fraction (~10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). Currently, there are no known molecular, ALT-specific, therapeutic target. We have identified TSPYL5 (Testis Specific Y-encoded-Like Protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1 – a shelterin component – and protects POT1 from proteasomal degradation, exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies, and identify TSPYL5 as a ALT+ cancer-specific therapeutic target

Telomeric chromatin regulates human telomere transcription

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Characterization of Physical and Functional Anchor Site Interactions

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation. Telomeres are protective DNA-protein structures that defin

Proliferation of melanoma metastases in the absence of telomere maintenance mechanism

Cutaneous melanoma is a very aggressive skin cancer with high propensity to metastasize. It is generally admitted that metastasis formation requires the acquisition of a telomere maintenance mechanism. As melanomas derive from melanocytes in which the expression of hTERT catalytic subunit of telomerase is repressed, reactivation of telomerase to acquire immortality often requires acquisition of one of the two recurrent and -believed to be- mutually exclusive C>T somatic mutations in hTERT promoter. C228T or C250T mutations, detected in about 80% of melanoma cell lines, create binding sites for GABP transcription factor. Here, we analyzed hTERT promoter mutations and telomere maintenance in a first series of lymph node metastases from ten melanoma patients and in cell lines freshly derived from these metastases. C228T and/or C250T mutations in hTERT promoter were found in eight cell lines that all expressed hTERT mRNA and were also detected in paired melanoma metastases. One cell line lacked expression of hTERT while another one failed to express hTR. Both telomerase-deficient cell lines were also negative for ALT features and displayed continuous telomere shortening in culture. Huge genomic instability was observed at late passages, characterized by sister chromatid/chromosome fusions, before massive cell death arose. Ectopic hTR overexpression in hTRneg cell line allowed continuous proliferation of the cells, showing that telomere shortening was the cause of cellular crisis. Intriguingly, one of these two telomeraseneg tumors was the most aggressive one from the series as, within less than one year, multiple visceral metastases appeared and patient died. Xenograft experiments with both telomerasepos and telomeraseneg cell lines in immuno-compromised mice are ongoing and preliminary results will be presented. We analyzed telomere maintenance mechanisms in additional metastases and primary melanoma cell lines and will discuss our first results. Our data provide evidence for the unexpected ability of melanoma tumors to form aggressive metastases in the absence of a telomere maintenance mechanism. These observations support the emerging notion brought up by R. Reddel’s group that telomere maintenance is not an absolute pre-requisite for metastasis formation and lethality in cancer patients and suggest that telomere-driven genomic instability may contribute to tumor aggressivity