Telomeres and Cancer

Telomeres cap the ends of eukaryotic chromosomes and are indispensable chromatin structures for genome protection and replication. Telomere length maintenance has been attributed to several functional modulators, including telomerase, the shelterin complex, and the CST complex, synergizing with DNA replication, repair, and the RNA metabolism pathway components. As dysfunctional telomere maintenance and telomerase activation are associated with several human diseases, including cancer, the molecular mechanisms behind telomere length regulation and protection need particular emphasis. Cancer cells exhibit telomerase activation, enabling replicative immortality. Telomerase reverse transcriptase (TERT) activation is involved in cancer development through diverse activities other than mediating telomere elongation. This review describes the telomere functions, the role of functional modulators, the implications in cancer development, and the future therapeutic opportunities.

A Na+/K+ ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice

The genetic and developmental mechanisms involved in limb formation are relatively well documented, but how these mechanisms are modulated by changes in chondrocyte physiology to produce differences in limb bone length remains unclear. Here, we used high throughput RNA sequencing (RNAseq) to probe the developmental genetic basis of variation in limb bone length in Longshanks, a mouse model of experimental evolution. We find that increased tibia length in Longshanks is associated with altered expression of a few key endochondral ossification genes such as Npr3, Dlk1, Sox9, and Sfrp1, as well reduced expression of Fxyd2, a facultative subunit of the cell membrane-bound Na+/K+ ATPase pump (NKA). Next, using murine tibia and cell cultures, we show a dynamic role for NKA in chondrocyte differentiation and in bone length regulation. Specifically, we show that pharmacological inhibition of NKA disrupts chondrocyte differentiation, by upregulating expression of mesenchymal stem cell markers (Prrx1, Serpina3n), downregulation of chondrogenesis marker Sox9, and altered expression of extracellular matrix genes (e.g., collagens) associated with proliferative and hypertrophic chondrocytes. Together, Longshanks and in vitro data suggest a broader developmental and evolutionary role of NKA in regulating limb length diversity.

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hairs cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its 2-fold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Telomere length regulation by Rif1 protein from Hansenula polymorpha

Rif1 is a large multifaceted protein involved in various processes of DNA metabolism – from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension – a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.

Chromosome-specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing

We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long-read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome-specific telomere lengths may be explained by slow clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosome end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y’ sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared to the tel1Δ single mutant, suggesting an influence of Y’ elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be around 75 bp. In these est2Δ mutants there were apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, and suggests possible new biological mechanisms regulating telomere length.

Mechanistic Model of Telomere Length Homeostasis in Yeast

Cells maintain homeostatic telomere length, and this homeostatic disruption leads to various types of diseases. Presently, it is not clear how telomeres achieve homeostasis. One of the prevailing hypotheses is a protein-counting model with a built-in sensor mechanism that counts proteins that directly regulate the telomeric length. However, it does not explain telomere length regulation at the mechanistic level. Here, we present a mathematical model based on the underlying molecular mechanisms of length regulation needed to establish telomere length homeostasis in yeast. We perform both deterministic and stochastic simulations to validate the models with the experimental data of Teixeira et al., rate-balance plot, and phase plane analysis to understand the nature of dynamics exhibited by the models. For global analysis, we constructed bifurcation diagrams. The model explains the role of negative and positive feedback loops and a delay between telomerase and telomere-bound proteins, leading to oscillations in telomere length. We map these in-silico results to proposition by Teixeira of telomeres making a transition between extendible and non-extendible states.

Abstract P501: Comprehensive Analysis Of Alternative Polyadenylation In Human Left Ventricle Failure By Using The Novel Algorithm: Polya-miner

Background: Alternative polyadenylation (APA) is an emerging post-transcriptional mechanism for gene regulation that generates distinct isoforms of mRNA with different 3′ untranslated regions (3’UTR) lengths. APA plays an important role in different biological processes and dysregulation of APA leads to many human diseases. However, the functional consequences of APA events in the left ventricle (LV) failure in humans remain unexplored. Objective: To identify whether the 3′UTR length is modulated by APA in the LV failure in humans compared to healthy LV. Methods and Results: We used Poly(A)-ClickSeq RNA sequencing and PolyA-miner algorithm to measure the global patterns of APA in healthy and failing human LV specimens. We determined shortening versus lengthening of 3′UTRs based on the PolyA index, a metric unit that determines the length of 3′UTR. Based on these scores, we identified 129 genes with a significant shift of cleavage site usage in failing LV compared to healthy LV specimens. By examining polyadenylation events in these hearts, we identified disease-specific APA signatures in many genes. In addition, differential APA events in LV failure regulate many pathways important for the progression of LV failure. Finally, the regulator proteins of APA including cleavage and polyadenylation specificity factor (CPSF) 6 and 7, cleavage factor Im (CFIm) 25 and 59 have been regulated in LV failure compared to healthy LV specimens. Conclusions: Our results provide genome-wide, polyadenylation maps of the human heart and show that APA of mRNA is dynamic in the progression of LV failure in humans. Demonstrating that APA mediated 3’UTR length regulation provides the additional layer of gene expressions in LV failure.