Alternative splicing is a developmental switch for hTERT expression

Abstract

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...

The SON RNA splicing factor is required for intracellular trafficking structures that promote centriole assembly and ciliogenesis

“…SON was identified to be important for maintaining human embryonic stem cell (hESC) identity (Chia et al, 2010), and its depletion led to hESC differentiation into a fibroblast-like state with a consequent downregulation of pluripotency-associated genes, upregulation of differentiationassociated genes and no change to housekeeping genes (Lu et al, 2013). In support of SON regulation contributing to control of developmental programs, SON is necessary for full telomerase activity in hESCs by its promotion of exon-2 retention in the hTERT transcript (Penev et al, 2021). As pluripotency requires high fidelity cell divisions and centrosomes, SON control of centrosome assembly could contribute to stemness and provide a powerful avenue to regulate both cell division during differentiation and centrosomal function as a dominant MTOC in cell types that utilize non-centrosomal MTOCs in their differentiated state.…”

Occurrence, functionality and abundance of the TERT promoter mutations

“…37,[42][43][44][45] Most human somatic tissues and adult stem cells do not express sufficient telomerase to maintain telomere length infinitely due to repression of the reverse transcriptase subunit upon differentiation in a histone deacetylase-dependent manner and through alternative splicing of TERT. 27,46,47 Splicing out of exon 2 of TERT leads to mRNA decay in differentiated cells, and its retention promotes telomerase accumulation in pluripotent cells. 47 The age-dependent telomere attrition leading to induction of DNA damage response acts as a tumor suppressor mechanism through upregulation of checkpoint inhibitors.…”

“…27,46,47 Splicing out of exon 2 of TERT leads to mRNA decay in differentiated cells, and its retention promotes telomerase accumulation in pluripotent cells. 47 The age-dependent telomere attrition leading to induction of DNA damage response acts as a tumor suppressor mechanism through upregulation of checkpoint inhibitors. 3,[48][49][50] The unlimited replicative potential of tumor cells through stabilized telomeres constitutes one of the cancer hallmarks.…”

Occurrence, functionality, and abundance of theTERTpromoter mutations

“…The recruitment and processivity of telomerase on telomeres are assisted by the shelterin components and terminated by the heterotrimeric CTC1-STN1-TEN1 (CST) complex, followed by a Cstrand fill-in the engagement of DNA polymerase α-primase 37,[42][43][44][45] . Most human somatic tissues and adult stem cells do not express sufficient telomerase to maintain telomere length infinitely due to repression of TERT upon differentiation in a histone deacetylase-dependent manner and through alternative splicing of TERT expression 27,46,47 . Splicing out of exon 2 of TERT leads to mRNA decay in differentiated cells, and its retention promotes telomerase accumulation in pluripotent cells 47 .…”

“…Most human somatic tissues and adult stem cells do not express sufficient telomerase to maintain telomere length infinitely due to repression of TERT upon differentiation in a histone deacetylase-dependent manner and through alternative splicing of TERT expression 27,46,47 . Splicing out of exon 2 of TERT leads to mRNA decay in differentiated cells, and its retention promotes telomerase accumulation in pluripotent cells 47 . The age-dependent telomere attrition leading to induction of DNA damage response acts as a tumor suppressor mechanism through upregulation of checkpoint inhibitors 3,[48][49][50] .…”