  Citation: Dan, J.; Zhou, Z.; Wang, F.; Wang, H.; Guo, R.; Keefe, D.L.; Liu, L. Zscan4 Contributes to Telomere Maintenance in Telomerase-Deficient Late Generation Mouse ESCs and Human ALT Cancer Cells. Cells 2022, 11, 456. https://doi.org/ 10.3390/cells11030456 Academic Editor: Mehdi Najar Received: 3 January 2022 Accepted: 25 January 2022 Published: 28 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Zscan4 Contributes to Telomere Maintenance in Telomerase-Deficient Late Generation Mouse ESCs and Human ALT Cancer Cells Jiameng Dan 1,2, *, Zhongcheng Zhou 1 , Fang Wang 3 , Hua Wang 1 , Renpeng Guo 1 , David L. Keefe 3 and Lin Liu 1, * 1 State Key Laboratory of Medicinal Chemical Biology, Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China; loyal.zhongcheng@163.com (Z.Z.); wangh2@mskcc.org (H.W.); guorp@njau.edu.cn (R.G.) 2 State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China; Yunnan Key Laboratory of Primate Biomedical Research, Kunming, Yunnan 650500, China 3 Department of Obstetrics and Gynecology, New York University Langone Medical Center, New York, NY 10016, USA; fang.wang@nyulangone.org (F.W.); david.keefe@nyulangone.org (D.L.K.) * Correspondence: danjm@lpbr.cn (J.D.); liulin@nankai.edu.cn (L.L.) Abstract: Proper telomere length is essential for indefinite self-renewal of embryonic stem (ES) cells and cancer cells. Telomerase-deficient late generation mouse ES cells and human ALT cancer cells are able to propagate for numerous passages, suggesting telomerase-independent mechanisms responding for telomere maintenance. However, the underlying mechanisms ensuring the telomere length maintenance are unclear. Here, using late generation telomerase KO (G4 Terc -/- ) ESCs as a model, we show that Zscan4, highly upregulated in G4 Terc -/- ESCs, is responsible for the prolonged culture of these cells with stably short telomeres. Mechanistically, G4 Terc -/- ESCs showed reduced levels of DNA methylation and H3K9me3 at Zscan4 promoter and subtelomeres, which relieved the expression of Zscan4. Similarly, human ZSCAN4 was also derepressed by reduced H3K9me3 at its promoter in ALT U2 OS cells, and depletion of ZSCAN4 significantly shortened telomeres. Our results define a similar conserved pathway contributing to the telomere maintenance in telomerase-deficient late generation mESCs and human ALT U2OS cancer cells. Keywords: Zscan4; telomere; ES cells; ALT; DNA methylation; H3K9me3 1. Introduction Mammalian telomeres consist of repetitive G-rich sequences and associated proteins at the ends of linear chromosomes and function in the maintenance of chromosomal stability and integrity [1,2]. Telomeres are primarily maintained by active telomerase which is composed of telomerase reverse transcriptase (TERT), telomerase RNA (TERC), and dyskerin [2]. Telomerase is expressed highly in a subset of stem cells, as well as in most immortal and cancer cells, presumably to support their indefinite proliferation and self- renewal [3,4]. Most mammalian somatic cell types do not express telomerase activity, such that telomeres shorten progressively with each cell cycle and the end replication problem ensues [5,6]. Previously, we have generated ES cell lines with high efficiency from wild type (WT, Terc +/+ ), heterozygous (Terc +/- ), and early to late generation of Terc -/- mouse blastocyst (G1, G3 and G4 telomerase RNA null blastocyst) [7]. Interestingly, late generation telomerase- deficient mTerc -/- mouse ES cells (G4 Terc -/- ) show telomere shortening and dysfunction compared with WT ES cells, yet can sustain their proliferation for numerous passages without telomerase activity [7], suggesting the involvement of telomerase-independent alternative lengthening of telomeres (ALT) mechanisms responding for telomere mainte- nance. Similarly, about 10–15% of human cancer cells lack detectable telomerase activity, Cells 2022, 11, 456. https://doi.org/10.3390/cells11030456 https://www.mdpi.com/journal/cells