Telomeres are the physical ends of eukaryotic chromosomes. They mediate chromosome stability and function as cellular clocks and tumor suppressors. Our work should provide fundamental insight into telomere structure and function in addition to delineating novel approaches to attack telomere function in cancer cells.
Our lab’s early work uncovered the mechanisms of telomere length homeostasis revealing that telomerase recognizes and extends preferentially short telomeres in yeast. In human cancer cells, we showed in collaboration with Michael Terns’ laboratory (University of Georgia), that telomerase particles undergo maturation steps in the nuclear Cajal Bodies making telomerase competent for telomere extension. We also demonstrated that the telomeric shelterin component TPP1 recruits human telomerase to telomeres for their extension. We defined a role of the human trimeric CST complex, which binds to newly extended single stranded telomeric DNA, in terminating telomerase activity after having accomplished telomere extension in S phase. Recently, in collaboration with the laboratory of Daniela Rhodes (Nanyang Technological University, Singapore) we reported the first low-resolution structure of full-length human telomerase. Our current and future work focuses on the dynamics of telomere composition and the long noncoding RNA TERRA as described in the next paragraphs.
Telomeric states in health and disease
A fundamental aspect of telomeres is their dynamic nature. One of the biggest challenges in the telomere field is to identify the molecular changes at telomeres that occur during normal development, in cancer and in telomere syndromes. To tackle this problem, we established a Quantitative Telomeric chromatin IsolationProtocol (QTIP), to biochemically isolate nucleoprotein complexes present at telomeres. Combined with SILAC labeling and mass spectrometry (done in collaboration with the EPFL core facility http://pcf-ptp.epfl.ch/), the QTIP technique enables comparison of different physiological and pathological telomeric states. Our analysis uncovered several new telomeric factors that associate with intact or damaged telomeres, and are currently studied in our lab. Our main emphasis is to apply QTIP to a variety of cellular models in order to elucidate telomere function during normal development and telomere dysfunction in cancer and telomere syndromes.
Figure 1: QTIP: Quantitative telomeric chromatin isolation protocol. (a) Workflow of QTIP. Approach A: Cells derived from two telomeric states are cultivated separately in either light or heavy SILAC medium, mixed, crosslinked with formaldehyde, lysed and sonicated. Telomeric chromatin is immunopurified with antibodies against the two abundant telomeric proteins TRF1 and TRF2. Purified telomeric chromatin is subjected to tryptic digestion and identified and quantified by mass spectrometry. Peptides derived from the two conditions are distinguishable due to their mass differences. In Approach B, the immunoprecipitation with a nonspecific control antibody (IgG) is performed separately using a third SILAC medium. This serves as a negative control to eliminate false positives. (b) Effects of TRF2-depletion at telomeres. Scatter plot representing the log2 ratio of a QTIP experiment with cells depleted for TRF2 versus control cells (wt). Proteins on the left are reduced at telomeres upon depletion of TRF2 (c) Comparison of telomeric protein composition at long versus short telomeres. Scatter plot representing the log2 ratio of two QTIP experiments. Proteins significantly changing between short and long telomeres in both experiments are indicated by green dots. SMCHD1 and LRIF1 are enriched at long telomeres. See Grolimund, Aeby et al., Nature Communications 4: 2848 (2013) for further details.
The long noncoding RNA TERRA
Several years ago, we refuted the dogma that telomeres are transcriptionally silenced. We demonstrated that telomeres are transcribed into the long noncoding telomeric repeat containing RNA (TERRA). In the meantime, work from several labs has promoted the view that TERRA sustains several crucial telomere functions. We characterized two modes by which TERRA promotes telomere shortening. First, TERRA binds human telomerase acting as a potent natural inhibitor of enzymatic activity. However, telomerase inhibition by TERRA can be prevented by TERRA binding proteins. Second, TERRA stimulates in vivo chromosome end shortening by the telomere trimming enzyme exonuclease 1. We also found that TERRA can form RNA-DNA hybrid structures (so-called R-loops) at telomeres, which are counteracted by the THO/TREX-complex and RNaseH to prevent telomere damage. Recently, we demonstrated that TERRA also plays some roles in the processing of eroded or damaged telomeres that occur during senescence. TERRA promotes the molecular interaction and the recruitment of the LSD1 lysine demethylase to MRE11 at damaged telomeres. LSD1 stimulates the nuclease activity of MRE11 and the nucleolytic processing of uncapped telomeres. We are currently applying Q-TIP and other novel technologies to identify TERRA-protein interactions and we employ molecular genetic methods to systematically dissect TERRA function.
Figure 2: Telomeres are transcribed into telomeric repeat containing RNA (TERRA). TERRA which is detected by RNA-FISH (red signals) at chromosome ends in human fibrosarcoma (HeLa) and osteosarcoma (U2OS) cells stays associated with telomeric heterochromatin. Telomeres are detected by indirect immunofluorescence with an antibody against the telomeric Rap1 protein (green dots). Signal overlap of TERRA and Rap1 is in yellow. DAPI stained nuclei are shown in grey. See Azzalin et al., Science 318: 798-801 (2007) for further details.