Aberrant T cell differentiation in the absence of Dicer

Dicer is an RNaseIII-like enzyme that is required for generating short interfering RNAs and microRNAs. The latter have been implicated in regulating cell fate determination in invertebrates and vertebrates. To test the requirement for Dicer in cell-lineage decisions in a mammalian organism, we have generated a conditional allele of dicer-1 (dcr-1) in the mouse. Specific deletion of dcr-1 in the T cell lineage resulted in impaired T cell development and aberrant T helper cell differentiation and cytokine production. A severe block in peripheral CD8+ T cell development was observed upon dcr-1 deletion in the thymus. However, Dicer-deficient CD4+ T cells, although reduced in numbers, were viable and could be analyzed further. These cells were defective in microRNA processing, and upon stimulation they proliferated poorly and underwent increased apoptosis. Independent of their proliferation defect, Dicer-deficient helper T cells preferentially expressed interferon-γ, the hallmark effector cytokine of the Th1 lineage

MicroRNAs Are Indispensable for Reprogramming Mouse Embryonic Fibroblasts into Induced Stem Cell-Like Cells

MicroRNAs play a pivotal role in cellular maintenance, proliferation, and differentiation. They have also been implicated to play a key role in disease pathogenesis, and more recently, cellular reprogramming. Certain microRNA clusters can enhance or even directly induce reprogramming, while repressing key proteins involved in microRNA processing decreases reprogramming efficiency. Although microRNAs clearly play important roles in cellular reprogramming, it remains unknown whether microRNAs are absolutely necessary. We endeavored to answer this fundamental question by attempting to reprogram Dicer-null mouse embryonic fibroblasts (MEFs) that lack almost all functional microRNAs using a defined set of transcription factors. Transduction of reprogramming factors using either lentiviral or piggyBac transposon vector into two, independently derived lines of Dicer-null MEFs failed to produce cells resembling embryonic stem cells (ESCs). However, expression of human Dicer in the Dicer-null MEFs restored their reprogramming potential. Our study demonstrates for the first time that microRNAs are indispensable for dedifferentiation reprogramming

Aberrant T cell differentiation in the absence of Dicer.

Dicer is an RNaseIII-like enzyme that is required for generating short interfering RNAs and microRNAs. The latter have been implicated in regulating cell fate determination in invertebrates and vertebrates. To test the requirement for Dicer in cell-lineage decisions in a mammalian organism, we have generated a conditional allele of dicer-1 (dcr-1) in the mouse. Specific deletion of dcr-1 in the T cell lineage resulted in impaired T cell development and aberrant T helper cell differentiation and cytokine production. A severe block in peripheral CD8(+) T cell development was observed upon dcr-1 deletion in the thymus. However, Dicer-deficient CD4(+) T cells, although reduced in numbers, were viable and could be analyzed further. These cells were defective in microRNA processing, and upon stimulation they proliferated poorly and underwent increased apoptosis. Independent of their proliferation defect, Dicer-deficient helper T cells preferentially expressed interferon-gamma, the hallmark effector cytokine of the Th1 lineage

Efficiency of RNA interference in the mouse hematopoietic system varies between cell types and developmental stages

RNA interference (RNAi) is a naturally occurring posttranscriptional gene-silencing mechanism that has been adapted as a genetic tool for loss-of-function studies of a variety of organisms. It is more widely applicable than classical gene targeting and allows for the simultaneous inactivation of several homologous genes with a single transgene. Recently, RNAi has been used for conditional and conventional gene inactivation in mice. Unlike gene targeting, RNAi is a dynamic process, and its efficiency may vary both between cell types and throughout development. Here we demonstrate that RNAi can be used to target three separately encoded isoforms of the bcl-2 family gene bfl-1/A1 in a conditional manner in mice. The extent of gene inactivation varies between different cell types and is least efficient in mature lymphocytes. Our data suggest that RNAi is affected by factors beyond small interfering RNA-mRNA stoichiometry

Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing

Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21–25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. We disrupted the dicer-1 (dcr-1) gene in mouse embryonic stem (ES) cells by conditional gene targeting and generated Dicer-null ES cells. These cells were viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells displayed severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs were markedly reduced. Re-expression of Dicer in the knockout cells rescued these phenotypes. Our data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing

Reprogramming of Polycomb-Mediated Gene Silencing in Embryonic Stem Cells by the miR-290 Family and the Methyltransferase Ash1l

Members of the miR-290 family are the most abundantly expressed microRNAs (miRNAs) in mouse embryonic stem cells (ESCs). They regulate aspects of differentiation, pluripotency, and proliferation of ESCs, but the molecular program that they control has not been fully delineated. In the absence of Dicer, ESCs fail to express mature miR-290 miRNAs and have selective aberrant overexpression of Hoxa, Hoxb, Hoxc, and Hoxd genes essential for body plan patterning during embryogenesis, but they do not undergo a full differentiation program. Introduction of mature miR-291 into DCR−/− ESCs restores Hox gene silencing. This was attributed to the unexpected regulation of Polycomb-mediated gene targeting by miR-291. We identified the methyltransferase Ash1l as a pivotal target of miR-291 mediating this effect. Collectively, our data shed light on the role of Dicer in ESC homeostasis by revealing a facet of molecular regulation by the miR-290 family

Human <i>Dicer</i> expression in <i>Dicer</i>-null MEFs allows generation of iPSCs.

<p>(<b>A</b>) Timeline of reprogramming <i>Dicer</i>-null MEFs rescued with human <i>Dicer</i>. Once human <i>Dicer</i> cDNA integrated into the <i>Dicer</i>^Δ/Δ MEF genome, reprogramming became possible even when factors were transduced 6 days after Cre induction. (<b>B</b>) Rescued iPSCs (ResDcr iPSC) lacked mouse <i>Dicer</i> (<i>mDcr</i>), but instead expressed human <i>Dicer</i> (<i>hDcr</i>) gene, verified by RT-PCR. (<b>C, D, E</b>) <i>Dicer</i>^Δ/Δ MEFs expressing human <i>Dicer</i> can reprogram to become iPSCs. Rescued iPSCs expressing human <i>Dicer</i> showed ESC morphology and stained for alkaline phosphatase (boxed areas represent magnified view) (<b>C</b>), and expressed stem cell markers tested by RT-PCR (<b>D</b>) and immunofluorescence (<b>E</b>). (<b>F</b>) Rescued iPSC promoters for stem cell genes <i>Oct4</i> and <i>Nanog</i> became demethylated, resembling wild-type ESCs. (<b>G</b>) Upon subcutaneous injection into SCID mice, rescued iPSCs formed teratomas that showed differentiation into all three germ layers. (<b>H</b>) Human Dicer can cleave mouse pre-miRNAs into mature miRNAs. qPCR for a panel of mature miRNAs in rescued iPSCs (ResDcr) lacking mouse <i>Dicer</i> demonstrated comparable expression levels to that of wild-type ESCs (W4). In contrast, mature miRNAs were completely depleted in <i>Dicer</i>-null ESCs (Dicer^Δ/Δ). Each value is represented relative to an assigned W4 value of 1.0 for that miRNA. Data are presented as mean +/− SD.</p

<i>Dicer</i>-null MEFs lacking miRNAs fail to reprogram.

<p>(<b>A, B</b>) Timelines of attempt at reprogramming <i>Dicer</i>-null MEFs. The main difference between the two strategies is that (<b>A</b>) transduces reprogramming transcription factors (TFs) 6 days post induction with Cre, while (<b>B</b>) transduces TFs 1 day post induction with Cre. <i>Dicer</i>-null MEFs could not be reprogrammed when reprogramming factors were transduced 6 days after induction. However, reprogramming <i>Dicer</i>-null MEFs was possible when reprogramming factors were transduced 1 day post induction with Cre. (<b>C</b>) <i>Dicer</i>^+/+, <i>Dicer</i>^Δ/+, and <i>Dicer</i>^f/f MEFs consistently reprogrammed into iPSCs with reprogramming factors. These iPSCs stained for alkaline phosphatase. <i>Dicer</i>^Δ/Δ MEFs reprogrammed to form induced stem cell-like cell colonies that stained for alkaline phosphatase when reprogramming factors were transduced 1 day post induction with Cre (Δ/Δ-1dpi). Transducing reprogramming factors 6 dpi (Δ/Δ-6dpi) or not transducing any factors (No TF) never reprogrammed <i>Dicer</i>-null MEFs. Boxed areas represent magnified view. (<b>D</b>) Genomic PCR confirmed induced stem cell-like cell colonies formed by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) having <i>Dicer</i>^Δ/Δ genotype (floxed DNA band). Control iPSC colonies (f/f iPSC) formed without Cre induction had <i>Dicer</i>^f/f genotype (flox DNA band). (<b>E</b>) Residual Dicer protein is still present 1 day after deletion of <i>Dicer</i> gene. By 6 days post induction (dpi) with Cre, residual Dicer protein is completely degraded, inhibiting cellular reprogramming. (<b>F, G</b>) Wild-type ESCs, <i>Dicer</i>^f/f iPSCs (f/f iPSC), and <i>Dicer</i>^Δ/Δ induced stem cell-like cells generated by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) expressed all stem cell markers tested by RT-PCR (<b>F</b>), and immunofluorescence (<b>G</b>). (<b>H</b>) <i>Dicer</i>^f/f iPSCs and <i>Dicer</i>^Δ/Δ induced stem cell-like cells acquired ESC methylation patterns in <i>Oct4</i> and <i>Nanog</i> promoters.</p