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Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.

Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.

Background: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro., Methodology/principal Findings: Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay., Conclusions/significance: Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context.