Identification of targets of tumor suppressor microRNA-34a using a reporter library system.

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3' UTR of a luciferase gene.

Altered lymphopoiesis and immunodeficiency in miR-142 null mice.

MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes.

miR-146a controls the resolution of T cell responses in mice.

T cell responses in mammals must be tightly regulated to both provide effective immune protection and avoid inflammation-induced pathology. NF-κB activation is a key signaling event induced by T cell receptor (TCR) stimulation. Dysregulation of NF-κB is associated with T cell-mediated inflammatory diseases and malignancies, highlighting the importance of negative feedback control of TCR-induced NF-κB activity.

MicroRNA-125b potentiates macrophage activation.

MicroRNA (miR)-125b expression is modulated in macrophages in response to stimulatory cues. In this study, we report a functional role of miR-125b in macrophages. We found that miR-125b is enriched in macrophages compared with lymphoid cells and whole immune tissues.

NF-kappaB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies.

MicroRNA miR-146a has been implicated as a negative feedback regulator of NF-κB activation. Knockout of the miR-146a gene in C57BL/6 mice leads to histologically and immunophenotypically defined myeloid sarcomas and some lymphomas. The sarcomas are transplantable to immunologically compromised hosts, showing that they are true malignancies.

miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice.

Excessive or inappropriate activation of the immune system can be deleterious to the organism, warranting multiple molecular mechanisms to control and properly terminate immune responses. MicroRNAs (miRNAs), ∼22-nt-long noncoding RNAs, have recently emerged as key posttranscriptional regulators, controlling diverse biological processes, including responses to non-self. In this study, we examine the biological role of miR-146a using genetically engineered mice and show that targeted deletion of this gene, whose expression is strongly up-regulated after immune cell maturation and/or activation, results in several immune defects.

Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses.

Foxp3(+) regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in fatal IFNγ-dependent immune-mediated lesions in a variety of organs.

MicroRNAs: new regulators of immune cell development and function.

Decades of research went into understanding immune cell development and function without awareness that consideration of a key element, microRNA (miRNA), was lacking. The discovery of miRNAs as regulators of developmental events in model organisms suggested to many investigators that miRNA might be involved in the immune system. In the past few years, widespread examination of this possibility has produced notable results.

Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder.

Mammalian microRNAs are emerging as key regulators of the development and function of the immune system. Here, we report a strong but transient induction of miR-155 in mouse bone marrow after injection of bacterial lipopolysaccharide (LPS) correlated with granulocyte/monocyte (GM) expansion. Demonstrating the sufficiency of miR-155 to drive GM expansion, enforced expression in mouse bone marrow cells caused GM proliferation in a manner reminiscent of LPS treatment.

NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes.

Genome-wide analyses of avian sarcoma virus integration sites.

The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected.

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair.

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions.

Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway.

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK(CS)) and the damage-signaling kinases (ATM and ATR) have been implicated in this process.

Integrase-specific enhancement and suppression of retroviral DNA integration by compacted chromatin structure in vitro.

Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1.