2 Penev et al.,High telomerase activity is restricted to the blastocyst stage of embryonic development when telomere length is reset, and is characteristic of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, the pathways involved in telomerase regulation as a function of pluripotency remain unknown. To explore hTERT transcriptional control, we compare genome-wide interactions (4C-seq) and chromatin accessibility (ATAC-seq) between human ESCs and epithelial cells and identify several putative hTERT cis-regulatory elements. CRISPR/Cas9-mediated deletion of candidate elements in ESCs reduces the levels of hTERT mRNA but does not abolish telomerase expression, thus implicating post-transcriptional processes in telomerase regulation. In agreement with this hypothesis, we find an hTERT splice variant lacking exon-2 and prone to degradation, to be enriched in differentiated cells but absent from ESCs. In addition, we show that forced retention of exon-2 prevents telomerase silencing during differentiation.Lastly, we highlight a role for the splicing co-factor SON in hTERT exon-2 inclusion and identify a SON mutation in a Dyskeratosis congenita patient with short telomeres and decreased telomerase activity. Altogether, our data uncover a novel alternative splice switch that is critical for telomerase activity during development.Telomerase counteracts telomere erosion and promotes cellular immortality. This specialized ribonucleoprotein complex is minimally composed of a reverse-transcriptase protein subunit (TERT) and an integral telomerase RNA component (TR). While the expression of telomerase RNA is ubiquitous (1, 2), TERT levels are tightly regulated (3). Human TERT (hTERT) is turned on during the blastocyst stage of embryonic development when telomere length is reestablished (4), and subsequently downregulated in most somatic cells (4,5). Similarly, hTERT becomes activated during the process of nuclear reprogramming (6) and is essential for telomere maintenance in pluripotent cells (7). Upregulation of telomerase occurs in approximately 90% of cancers, allowing tumor cells to escape crisis and proliferate indefinitely (5,8). Somatic mutations in the hTERT promoter have been reported in a subset of cancers and shown to increase telomerase activity by creating a binding site for ETS family transcription factors (9-11). To date, the underlying mechanism that activates hTERT during development and its subsequent silencing in somatic tissues remains unknown.The core hTERT promoter contains numerous binding sites for pluripotency and growthrelated transcription factors, including Myc, Klf4, and Sp1, but expression of these different factors 3 Penev et al., is not sufficient for hTERT accumulation and telomerase activation in somatic cells (12). We therefore sought to identify cis-regulatory elements (REs) that could account for the difference in hTERT mRNA levels between pluripotent and somatic cells ( Figure S1A). Active REs are known to directly contact the promoters they regulate and h...

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