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Transcriptional regulation and mechanistic insights on the telomeric lncRNA TERRA

Periodic Reporting for period 1 - EnterTerra (Transcriptional regulation and mechanistic insights on the telomeric lncRNA TERRA)

Reporting period: 2016-10-01 to 2018-09-30

Telomeres are transcribed into a novel class of long noncoding (lnc) RNAs, called telomeric repeat containing RNAs (TERRA) whose transcriptional regulation and functions are largely unknown. Telomeres are dynamic nucleoprotein complexes that protect the physical extremities of eukaryotic chromosomes from degradation and inappropriate recombination events. Following each round of cell division, telomeres progressively shorten due to limitations of the semiconservative DNA replication, ultimately leading to rampant genomic instability, cellular senescence, and apoptosis. Telomere erosion is counteracted by the telomerase, a ribonucleoprotein enzyme that maintains the length of the telomeres in highly proliferative cell lines, such as the human germ lines and stem cell lineages. However, in most somatic cells, telomerase is inactive. Telomere shortening in the absence of telomerase limits the replicative potential of cells providing a powerful tumor suppressive mechanism. Up to 90% of human cancers reactivate telomerase to bypass the senescence barrier and ensure chromosomal stability and cellular immortalization. Thus, the ability to regulate the length and function of the telomeres is an attractive research target for cancer therapy. In previous studies we found that, in spite of the heterochromatic state of the chromatin, telomeres are transcriptionally active producing a novel class of lncRNAs. The telomeric repeat-containing RNAs (TERRA) remain in the nucleus and partially co-localize with the telomeres. The identification of TERRA revealed a surprising unknown telomeric mechanism and opened up new research direction on telomere functions. The exact roles of TERRA in telomere length regulation remain to be elucidated. TERRA has been implicated in recruiting chromatin modulators to dysfunctional telomeres during senescence but the postulated roles of TERRA as protein recruiter remain to be tested. Overall, the importance of TERRA binding to telomerase and other telomere modifying enzymes is not well understood representing a fundamentally important question in telomere research that will be tackled in this study.

Our objective is to investigate the biogenesis of TERRA and it role in the cell. We plan to systematically identify the transcriptional factors that bind TERRA promoters and characterize their role in TERRA production.Although TERRA transcripts stem from multiple chromosomes ends, it is unclear whether they act on the telomere they originated from or if they can associate with multiple chromosome ends in. In addition, TERRA has been found to interact with some telomeric proteins and heterochromatin modulators; however it remains to be tested if they are recruited by TERRA. In this study, I will explore the dynamics of TERRA localization and determine if the transcripts remain attached to the sequence of origin or they can act at other chromosomal ends or loci. I will elucidate the role of TERRA in stabilizing or recruiting telomeric or heterochromatic proteins at both telomeric and non-telomeric loci. I will assess the function of TERRA-recruited proteins on the chromatin and explore if TERRRA acts as a scaffold to assemble a wide variety of enzymatic activities.
TERRA has been implicated to control crucial telomeric roles including telomere length regulation, cellular senescence and telomere end fusions during cell crisis leading to severe genome instability. Furthermore, abnormally elevated TERRA levels are associated with the ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome, a telomere-associated disorder caused by mutations in the DNA methyltransferase DNMT3b gene. ICF patients have extremely short telomeres and high TERRA transcription. Although TERRA levels are regulated by DNA methylation in humans, they are not severely affected by changes in telomere length, which suggests that the extreme high levels of TERRA in ICF patients are not a consequence of short telomeres but rather the cause. On the other hand, TERRA transcription is downregulated in telomerase positive cancer cell-lines in order to overcome replication-induced telomeric erosion and promote uncontrollable proliferation and immortalization. Given that telomerase is inactive in healthy somatic cells, the combination of telomerase inhibitors and exogenously-induced TERRA transcription might preferentially induce senescence and death in cancer cells; a highly attractive characteristic for any aspiring successful cancer treatment. Thus, it is imperative to develop systems that will allow genetic manipulation of TERRA transcription to elucidate TERRA transcriptional network and ultimately provide a glimpse on the endogenous TERRA functions. I anticipate that the choice of integrated strategies and methodologies will be decisive to understand the tight regulation of TERRA transcription, which will provide us with the tools to effectively regulate this lncRNAs and expand our knowledge on telomere maintenance. I expect that our innovative approaches will decipher TERRA’s localization preference on the chromatin, explore the multilayered ribonucleoprotein TERRA cluster, and it will provide new insights on plethora of telomere functions. This study will have a profound impact on our current understanding of the structural and functional complexity of telomeres.