Telomere dysfunction activates p53 and represses HNF4α expression leading to impaired human hepatocyte development and function.

Telomere attrition is a major risk factor for end-stage liver disease. Due to a lack of adequate models and intrinsic difficulties in studying telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver disease in patients with telomere syndromes remain elusive. To circumvent that, we used genome editing to generate isogenic human embryonic stem cell lines (hESCs) harboring clinically relevant mutations in telomerase and subjected them to an in vitro, stage-specific hepatocyte differentiation protocol that resembles hepatocyte development in vivo. Utilizing this platform we observed that while telomerase is highly expressed in hESCs, it is quickly silenced, specifically due to TERT down-regulation, immediately after endoderm differentiation, and completely absent in in vitro derived hepatocytes, similar to what is observed in human primary hepatocytes. While endoderm derivation is not impacted by telomere shortening, progressive telomere dysfunction impaired hepatic endoderm formation. Consequently, hepatocyte-derivation, as measured by expression of specific hepatic markers, as well by albumin expression and secretion, is severely compromised in telomerase mutant cells with short telomeres. Interestingly, this phenotype was not caused by cell death induction or senescence. Rather, telomere shortening prevents the up-regulation and activation of the human hepatocyte nuclear factor 4α (HNF4α), in a p53-dependent manner. Both reactivation of telomerase and silencing of p53 rescued hepatocyte formation in telomerase mutants. Likewise, the conditional expression (doxycycline-controlled) of HNF4α even in cells that retained short telomeres, accrued DNA damage, and exhibited p53 stabilization, successfully restored hepatocyte formation from hESCS. In conclusion, our data shows that telomere dysfunction acts as a major regulator of HNF4α during hepatocyte development, pointing to a novel target in the treatment of liver disease in telomere-syndrome patients.