Person:

Daley, George

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodeling. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used shRNAs to target genes in DNA and histone methylation pathways, and have identified positive and negative modulators of iPSC generation. While inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency, suppression of SUV39H1, YY1, and Dot1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase Dot1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc. Inhibition of Dot1L early in the reprogramming process is associated with a marked increase in two alternative factors, Nanog and Lin28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

The Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3′ terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression.

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a sub-group of frontotemporal lobar degeneration (FTLD-TDP). Identification of TARDBP mutations in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts using induced pluripotent stem cells (iPSC). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation, and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent- resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the phosphoinositide 3-kinase pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening.

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

MYC is a major oncogenic driver of Multiple Myeloma (MM) and yet almost no therapeutic agents exist that target MYC in MM. Here we report that the let-7 biogenesis inhibitor LIN28B correlates with MYC expression in MM and is associated with adverse outcome. We also demonstrate that the LIN28B/let-7 axis modulates the expression of MYC, itself a let-7 target. Further, perturbation of the axis regulates the proliferation of MM cells in vivo in a xenograft tumor model. RNA sequencing and gene set enrichment analyses of CRISPR-engineered cells further suggest that the LIN28/let-7 axis regulates MYC and cell cycle pathways in MM. We provide proof-of-principle for therapeutic regulation of MYC through let-7 with an LNA-GapmeR containing a let-7b mimic in vivo, demonstrating that high levels of let-7 expression repress tumor growth by regulating MYC expression. These findings reveal a novel mechanism of therapeutic targeting of MYC through the LIN28B/let-7 axis in MM that may impact other MYC dependent cancers as well.

Background: Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear. Design and Methods: We inactivated Cdx4 in mice through either a germline or conditional knockout approach and analyzed requirement for Cdx4 in both normal adult hematopoiesis and leukemogenesis initiated by the MLL-AF9 fusion oncogene. Results: Here, we report that loss of Cdx4 had a minimal effect on adult hematopoiesis. Indeed, although an increase in white blood cell counts was observed, no significant differences in the distribution of mature blood cells, progenitors or stem cells were observed in Cdx4-deficient animals. In addition, long-term repopulating activity in competitive transplantation assays was not significantly altered. In vitro, B-cell progenitor clonogenic potential was reduced in Cdx4-deficient animals but no significant alteration of mature B cells was detected in vivo. Finally, induction of acute myeloid leukemia in mice by MLL-AF9 was significantly delayed in the absence of Cdx4 in a retroviral transduction/bone marrow transplant model. Conclusions: These observations indicate that Cdx4 is dispensable for the establishment and maintenance of normal hematopoiesis in adult mammals. These results, therefore, outline substantial differences in the Cdx-Hox axis between mammals and zebrafish and support the hypothesis that Cdx factors are functionally redundant during mammalian hematopoietic development under homeostatic conditions. In addition, our results suggest that Cdx4 participates in MLL-AF9-mediated leukemogenesis supporting a role for Cdx factors in the pathogenesis of myeloid leukemia.

Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases (gata1^+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic (cdx4^{−/−}) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.

We read with great interest the excellent review by Maherali and Hochedlinger (2008) that recommends standards for characterization of pluripotent stem cell lines, especially the many new lines being generated using factor-based reprogramming techniques (induced pluripotent stem cells, or iPSCs). Of note was the suggestion that iPSCs should be assessed for “functional differentiation through the highest-stringency test acceptable.” For murine iPSCs, this means germline transmission following blastocyst chimerism, and for human iPSCs this means assessment of teratoma pathology.