Recent evidence has shown that deregulated expression of members of the microRNA-29 (miR-29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR-29 in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T-ALL) has not been investigated. Here, we show that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T-ALL patients. Furthermore, miR-29a levels are extremely reduced in T-ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR-29a mimics into Jurkat cells revealed the downregulation of several predicted targets (CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR-29a levels in Jurkat and Molt-4 T-ALL cells led to the demethylation of many genes commonly methylated in T-ALL. Overall, our results suggest that reduced miR-29a levels may contribute to the altered epigenetic status of T-ALL, highlighting its relevance in the physiopathology of this disease.

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Somatic cell reprogramming by transcription factors and other modifiers such as microRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP-colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied components of two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wnt-signaling. We show that inhibition of Tet1, Tet2 and Tet3 as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of pre-miR-29 resulted in elevated expression of β-Catenin transcriptional target sFRP2 and increased TCF/LEF-promoter activity. Finally, we report that Gsk3-β is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR-29 influences reprogramming.