• The Effect of ZSCAN4 on Telomere Chromatin Remodeling

      Lin, Phyo Nay; Zalzman, Michal (2020)
      Telomeres are repetitive sequences at the ends of chromosomes that protect the coding regions of DNA. Telomeres shorten with every cell division and therefore operate as a biological clock. Thus, factors regulating telomeric chromatin impact cell replicative lifespan, tumor formation and growth. The murine Zinc Finger and SCAN Domain Containing 4 (mZscan4) promotes telomere homeostasis and genomic stability in mouse embryonic stem cells (mESCs). A transient expression of mZscan4 was shown to correlate with chromatin de-repression in mESCs. However, the function of human ZSCAN4 in its contribution to the epigenetic landscape changes at telomeric chromatin remains to be determined. In this study, we defined the effect of ZSCAN4 on histone 3 and 4 hyperacetylation at the telomere region which is associated with telomere extension. Understanding the mechanism by which ZSCAN4 affects the telomeric chromatin is important for designing new therapeutic approaches to target cancer cell replicative lifespan.
    • The Role of ZSCAN4 in Cancer Stemness

      Portney, Benjamin Andrew; Zalzman, Michal (2018)
      Cancer stem cells (CSCs) represent a subset of cells within tumors that maintain the ability to self-renew, drive tumor heterogeneity, and contribute to therapeutic resistance and cancer recurrence. Over the past decade, embryonic factors have emerged as key regulators of cancer stemness and as therapeutic targets. Zscan4 is an early embryonic gene expressed in mouse embryonic and induced pluripotent stem cells where it plays critical roles in genomic stability, telomere maintenance, and pluripotency. Like other embryonic factors, ZSCAN4 is reactivated in cancer. In these studies, we define for the first time the role of ZSCAN4 in human CSCs of head and neck squamous cell carcinoma (HNSCC). We find that ZSCAN4 is enriched for in and marks the HNSCC CSC population. Induction of ZSCAN4 promotes the CSC phenotype, increases CSC factors and alters the epigenetic profile. Importantly, extreme limiting dilution analyses both in vitro and in vivo indicate that ZSCAN4 induction significantly increases the frequency of CSC. Consistently, ZSCAN4 depletion leads to loss of the CSC phenotype including CSC marker expression, the ability to form spheroids in non-adherent culture conditions, and hypersensitivity to genotoxic drugs. Furthermore, loss of ZSCAN4 severely impairs tumor growth in vivo. As our findings indicated that ZSCAN4 promotes the CSC phenotype, we next chose to study its regulation and turnover in cancer cells. Expression of Zscan4 is transient, and characterized by infrequent high expression peaks that are quickly down-regulated, suggesting its expression is tightly controlled. However, little is known about the protein degradation pathway responsible for regulating the human ZSCAN4 protein levels. We first determined the protein half-life of ZSCAN4 and elucidated the role of the ubiquitin proteasome system in ZSCAN4 degradation. Importantly, our data indicate an interaction between ZSCAN4 and the E3 ubiquitin ligase RNF20. RNF20 depletion stabilizes the ZSCAN4 protein half-life, suggesting that RNF20 negatively regulates ZSCAN4 stability. Due to the crucial cellular functions of ZSCAN4, these results have important implications in telomere regulation, stem cell biology, and cancer. Overall, our study suggests that ZSCAN4 plays a critical role in maintaining the undifferentiated state and survival of CSCs, indicating that ZSCAN4 is a potential therapeutic target in HNSCC.
    • ZSCAN4: A Novel Regulator of Telomere Length in Cancer

      Meltzer, W. Alex; Zalzman, Michal; 0000-0003-1175-8055 (2017)
      Replicative immortality is facilitated by telomere maintenance through activating telomerase or by mechanisms collectively called alternative lengthening of telomeres (ALT). Zscan4c regulates telomere length of mouse embryonic stem cells, however, the activity of the human ZSCAN4 and its involvement in cancer is currently unknown. In this work, we describe for the first time the function and mechanism of ZSCAN4 in human cancer. We demonstrate that ZSCAN4 is dysregulated in a variety of cancers, and that ZSCAN4 is required to maintain both culture lifespan in vitro as well as cancer tumor growth in vivo. We further show that ZSCAN4 preferably binds to short telomeres and in conjunction with the telomeric recombination machinery MRE11A and RAD50 forms a complex on the telomere DNA-RNA hybrids. Loss of ZSCAN4 results in gradual telomere shortening which triggers an induction of growth arrest and replicative senescence. Consistently, ZSCAN4 induction increases DNA-RNA hybrids, facilitates telomere recombination events and consequently, telomere extension. Remarkably, we show that ZSCAN4 activates telomere extension irrespective of both telomerase as well as the canonical ALT pathway in cancer cells. Collectively, our work demonstrates a pivotal role for ZSCAN4 in the preservation of cancer replicative lifespan. This work provides the foundation for new approaches to cancer therapies.