α4-Integrin deficiency in human CD34+ cells engenders precocious erythroid differentiation but inhibits enucleation.

The functional impact of integrin expression in erythropoiesis has been previously emphasized through its decisive influence on erythroid cell-microenvironment (matrix and cellular) interactions, especially under conditions of stress. Beyond that, in several in vitro studies the relationship between the two erythroid integrins, α4 and α5, has been incongruous in terms of a proliferative support, either synergistic or antagonistic, whereas a dominant influence of α4 integrin on terminal erythropoiesis in vitro and in vivo has been consistently emphasized. However, the specific cellular and molecular details of this effect have not been defined, especially for human cells. In the study described here, we cultured human CD34+ progenitor cells with induced deficiency of α4 integrin (shRNAα4) under erythroid differentiation conditions, in which expanded erythroid progenitor cells are directed to terminal erythroid maturation stages in the absence of any microenvironmental influence. Our data indicate that early proliferative expansion in cells lacking α4 expression is significantly limited, but although erythroid cell differentiation can proceed normally to terminal stages, their enucleation is drastically impaired. This novel aspect of α4 integrin participation in the enucleation process in vitro resonates on the lack of in vivo enucleation of primitive erythroid cells lacking any integrin expression but affecting adult cells only under stress conditions.

Cotargeting among microRNAs in the brain.

MicroRNAs (miRNAs) play roles in diverse developmental and disease processes. Distinct miRNAs have hundreds to thousands of conserved mRNA binding sites but typically direct only modest repression via single sites. Cotargeting of individual mRNAs by different miRNAs could potentially achieve stronger and more complex patterns of repression. By comparing target sets of different miRNAs, we identified hundreds of pairs of miRNAs that share more mRNA targets than expected (often by twofold or more) relative to stringent controls. Genetic perturbations revealed a functional overlap in neuronal differentiation for the cotargeting pair miR-138/miR-137. Clustering of all cotargeting pairs revealed a group of nine predominantly brain-enriched miRNAs that share many targets. In reporter assays, subsets of these miRNAs together repressed gene expression by five- to 10-fold, often showing cooperative repression. Together, our results uncover an unexpected pattern in which combinations of miRNAs collaborate to robustly repress cotargets, and suggest important developmental roles for cotargeting.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins.

RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3' UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3' UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3' UTR isoforms was altered following CELF1 induction, with 3' UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes.

Genetic engineering of human pluripotent cells using TALE nucleases.

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.

The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.