Reprogramming Chromosome Ends by Functional Histone Acetylation.

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between 'stemness' and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells.

ZSCAN4 facilitates chromatin remodeling and promotes the cancer stem cell phenotype.

Cancer stem cells (CSCs) are cells within tumors that maintain the ability to self-renew, drive tumor growth, and contribute to therapeutic resistance and cancer recurrence. In this study, we investigate the role of Zinc finger and SCAN domain containing 4 (ZSCAN4) in human head and neck squamous cell carcinoma (HNSCC). The murine Zscan4 is involved in telomere maintenance and genomic stability of mouse embryonic stem cells. Our data indicate that the human ZSCAN4 is enriched for, marks and is co-expressed with CSC markers in HNSCC. We show that transient ZSCAN4 induction for just 2 days increases CSC frequency both in vitro and in vivo and leads to upregulation of pluripotency and CSC factors. Importantly, we define for the first time the role of ZSCAN4 in altering the epigenetic profile and regulating the chromatin state. Our data show that ZSCAN4 leads to a functional histone 3 hyperacetylation at the promoters of OCT3/4 and NANOG, leading to an upregulation of CSC factors. Consistently, ZSCAN4 depletion leads to downregulation of CSC markers, decreased ability to form tumorspheres and severely affects tumor growth. Our study suggests that ZSCAN4 plays an important role in the maintenance of the CSC phenotype, indicating it is a potential therapeutic target in HNSCC.

The Role of Ubiquitination and SUMOylation in Telomere Biology.

Telomeres are a unique structure of DNA repeats covered by proteins at the ends of the chromosomes that protect the coding regions of the genome and function as a biological clock. They require a tight regulation of the factors covering and protecting their structure, as they are shortened with each cell division to limit the ability of cells to replicate uncontrollably. Additionally, they protect the chromosome ends from DNA damage responses and thereby, prevent genomic instability. Telomere dysfunction can lead to chromosomal abnormalities and cancer. Therefore, dysregulation of any of the factors that regulate the integrity of the telomeres will have implications to chromosomal stability, replicative lifespan and may lead to cell transformation. This review will cover the main factors participating in the normal function of the telomeres and how these are regulated by the ubiquitin and SUMO systems. Accumulating evidence indicate that the ubiquitin and SUMO pathways are significant regulators of the shelterin complex and other chromatin modifiers, which are important for telomere structure integrity. Furthermore, the crosstalk between these two pathways has been reported in telomeric DNA repair. A better understanding of the factors contributing to telomere biology, and how they are regulated, is important for the design of new strategies for cancer therapies and regenerative medicine.

ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20.

Zscan4 is an early embryonic gene cluster expressed in mouse embryonic stem and induced pluripotent stem cells where it plays critical roles in genomic stability, telomere maintenance, and pluripotency. Zscan4 expression 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. In this study we determine for the first time the ZSCAN4 protein half-life and degradation pathway, including key factors involved in the process, responsible for the regulation of ZSCAN4 stability. We demonstrate lysine 48 specific polyubiquitination and subsequent proteasome dependent degradation of ZSCAN4, which may explain how this key factor is efficiently cleared from the cells. Importantly, our data indicate an interaction between ZSCAN4 and the E3 ubiquitin ligase RNF20. Moreover, our results show that RNF20 depletion by gene knockdown does not affect ZSCAN4 transcription levels, but instead results in increased ZSCAN4 protein levels. Further, RNF20 depletion stabilizes the ZSCAN4 protein half-life, suggesting that RNF20 negatively regulates ZSCAN4 stability. Due to the significant cellular functions of ZSCAN4, our results have important implications in telomere regulation, stem cell biology, and cancer.

Harvesting multipotent progenitor cells from a small sample of tonsillar biopsy for clinical applications.

BACKGROUND: Human adult stem cells hold the potential for the cure of numerous conditions and degenerative diseases. They possess major advantages over pluripotent stem cells as they can be derived from donors at any age, and therefore pose no ethical concerns or risk of teratoma tumor formation in vivo. Furthermore, they have a natural ability to differentiate and secrete factors that promote tissue healing without genetic manipulation. However, at present, clinical applications of adult stem cells are limited by a shortage of a reliable, standardized, and easily accessible tissue source which does not rely on specimens discarded from unrelated surgical procedures. METHOD: Human tonsil-derived mesenchymal progenitor cells (MPCs) were isolated from a small sample of tonsillar tissue (average 0.88 cm3). Our novel procedure poses a minimal mechanical and enzymatic insult to the tissue, and therefore leads to high cell viability and yield. We characterized these MPCs and demonstrated robust multipotency in vitro. We further show that these cells can be propagated and maintained in xeno-free conditions. RESULTS: We have generated tonsillar biopsy-derived MPC (T-MPC) lines from multiple donors across a spectrum of age, sex, and race, and successfully expanded them in culture. We characterized them by cell surface markers, as well as in vitro expansion and differentiation potential. Our procedure provides a robust yield of tonsillar biopsy-derived T-MPCs. CONCLUSIONS: Millions of MPCs can be harvested from a sample smaller than 1 g, which can be collected from a fully awake donor in an outpatient setting without the need for general anesthesia or hospitalization. Our study identifies tonsillar biopsy as an abundant source of adult MPCs for regenerative medicine.

Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5-dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics.

MARCH5, an OMM-associated E3 ubiquitin ligase, controls mitochondrial function. Despite its importance, the mechanism and factors controlling MARCH5 activity are largely unknown. Here we report that the MARCH5 C-terminal domain plays a critical role in degradation of MARCH5 substrates, likely by facilitating release of ubiquitinated proteins from the OMM. We also found that the mitochondrial fission proteins Drp1 and Mff negatively regulate MARCH5's activity toward MiD49 and Mcl1. Knockouts of either Drp1 or Mff led to reduced expression, shorter half-lives, and increased ubiquitination of MiD49 and Mcl1. Effects of Mff and Drp1 depletion on degradation rates and ubiquitination of Mcl1 and MiD49 were eliminated in Drp1-/-/MARCH5-/- and Mff-/-/MARCH5-/- cells. Our data show that it is not mitochondrial morphology per se but rather Mff and Drp1 that directly control MARCH5. Consistently, we find that Mff is an integral component of the MARCH5/p97/Npl4 complex, which is also controlled by MARCH5's C-terminal domain. Furthermore, not only mitochondrial fission but also fusion is regulated through Mff and Drp1 protein activities. Thus, in addition to their canonical roles in mitochondrial fission, Mff and Drp1 also act as regulatory factors that control mitochondrial fission and fusion.