Amy Heidersbach

Chd1 regulates open chromatin and pluripotency of embryonic stem cells

An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.

Up-regulation of miR-21 by HER2/neu signaling promotes cell invasion

The cell surface receptor tyrosine kinase HER2/neu enhances tumor metastasis. Recent studies suggest that deregulated microRNA (miRNA) expression promotes invasion and metastasis of cancer cells; we therefore explored the possibility that HER2/neu signaling induces the expression of specific miRNAs involved in this process. We identified a putative oncogenic miRNA, miR-21, whose expression is correlated with HER2/neu up-regulation and is functionally involved in HER2/neu-induced cell invasion. We show that miR-21 is up-regulated via the MAPK (ERK1/2) pathway upon stimulation of HER2/neu signaling in breast cancer cells, and overexpression of other ERK1/2 activators such as RASV12 or ID-1 is sufficient to induce miR-21 up-regulation in HER2/neu-negative breast cancer cells. Furthermore, the metastasis suppressor protein PDCD4 (programmed cell death 4) is down-regulated by miR-21 in breast cancer cells expressing HER2/neu. Our data reveal a mechanism for HER2/neu-induced cancer cell invasion via miRNA deregulation. In addition, our results identify miR-21 as a potential therapeutic target for the prevention of breast cancer invasion and metastasis.

microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart

microRNA-1 (miR-1) is an evolutionarily conserved, striated muscle-enriched miRNA. Most mammalian genomes contain two copies of miR-1, and in mice, deletion of a single locus, miR-1-2, causes incompletely penetrant lethality and subtle cardiac defects. Here, we report that deletion of miR-1-1 resulted in a phenotype similar to that of the miR-1-2 mutant. Compound miR-1 knockout mice died uniformly before weaning due to severe cardiac dysfunction. miR-1-null cardiomyocytes had abnormal sarcomere organization and decreased phosphorylation of the regulatory myosin light chain-2 (MLC2), a critical cytoskeletal regulator. The smooth muscle-restricted inhibitor of MLC2 phosphorylation, Telokin, was ectopically expressed in the myocardium, along with other smooth muscle genes. miR-1 repressed Telokin expression through direct targeting and by repressing its transcriptional regulator, Myocardin. Our results reveal that miR-1 is required for postnatal cardiac function and reinforces the striated muscle phenotype by regulating both transcriptional and effector nodes of the smooth muscle gene expression network. DOI: http://dx.doi.org/10.7554/eLife.01323.001

The RNA Binding Protein TDP-43 Selectively Disrupts MicroRNA-1/206 Incorporation into the RNA-Induced Silencing Complex

Background: Regulation of microRNA activity independent of processing and biogenesis has not been demonstrated. Results: The RNA-binding protein, TDP-43, interacts with mature miR-1/miR-206, limiting their RNA-induced silencing complex (RISC) association and activity. Conclusion: RNA-binding proteins can selectively control microRNA activity by disrupting RISC incorporation. Significance: This is the first known microRNA-protein interaction that controls microRNA activity independent of processing. MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity.

Duox2 exhibits potent heme peroxidase activity in human respiratory tract epithelium

The dual oxidase isozymes Duox1 and Duox2 exhibit functional NADPH:O2 oxidoreductase activity in thyroid and respiratory tract cells and are thought to be essential for H2O2 generation in these tissues. However, it is not universally accepted that the heme peroxidase domains of the Duox isozymes are functional. To address this question, we modulated Duox2 expression in human tracheobronchial epithelial (TBE) cell culture systems and quantified peroxidase activity. We discovered that interferon‐gamma (IFN‐γ) induced robust peroxidase activity in TBE cells that paralleled Duox2 expression. IFN‐γ‐induced peroxidase activity was abolished in the presence of sodium azide, which implicated the activation of a heme peroxidase. IFN‐γ‐induced peroxidase activity was abolished in TBE cell lines expressing anti‐Duox2 short hairpin RNA transcripts. Together, these data unequivocally demonstrated that Duox2 contains a functional heme peroxidase in intact respiratory tract epithelium.

MicroRNA-155 Reinforces HIV Latency.

Background: Curing HIV will require suppression of viral replication and clearance of the transcriptionally silent latent reservoir. Results: We have discovered a novel cellular pathway involving a latency-promoting host miRNA and HIV-activating TRIM protein. Conclusion: miR-155 inhibits the HIV-activating effects of TRIM32 and thus may promote a return to latency in reservoir cells transiently expressing HIV. Significance: Preventing reversion to latency could provide a novel approach for eliminating the latent reservoir. The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation.

RNA interference in embryonic stem cells and the prospects for future therapies.

In 1998, two distinct and exciting scientific fields emerged which have profoundly shaped the current direction of biomedical research. The discovery of RNA interference (RNAi) and the derivation of human embryonic stem (ES) cells have yielded exciting new possibilities for researchers and clinicians alike. While fundamentally different, aspects from these two fields may be combined to yield extraordinary scientific and medical benefits. Here, we review the prospects of combining RNAi and ES cell manipulation for both basic research and future therapies, as well as current limitations and obstacles that need to be overcome.

Lentiviral Strategies for RNAi Knockdown of Neuronal Genes

RNA interference (RNAi) refers to the process by which 21‐ to 23‐nucleotide short interfering RNAs (siRNAs) mediate post‐transcriptional degradation of homologous mRNA transcripts. This process is carried out by an endogenous pathway that centers on the use of endogenously encoded small RNAs, and can be hijacked to knock down the expression of any target protein by introducing a specific siRNA into a cell. Stable knockdown can be obtained by constitutive expression of the siRNA from the host chromosome. Retroviruses, such as lentivirus, provide a convenient vector by which to integrate RNAi expression constructs. Lentiviruses can infect nondividing cells, thereby allowing knockdown in cells such as mature neurons. This unit provides methods to design and clone siRNAs into a lentiviral vector. Additional protocols describe production and titering of the lentivirus, as well as safety testing. Finally, methods are provided for infecting neurons in culture and in vivo with RNAi lentivirus.

Small Solutions to Big Problems MicroRNAs for Cardiac Regeneration

Heart disease is a major cause of morbidity and mortality in the developed world. Patient recovery after cardiac injury is hampered by the extremely limited regenerative capacity of the adult mammalian heart. In addition to cell-based approaches and in situ cardiac reprogramming, significant interest has been focused on genetic and small molecule–based strategies to enhance endogenous cardiomyocyte proliferative potential. A recent study in Nature suggests, for the first time, that microRNAs may have the ability to induce cardiomyocyte proliferation and cardiac regeneration in adult mice. The overwhelming majority of cardiomyocytes in the mammalian heart lose their proliferative capacity shortly after birth. Acute or chronic injury to the myocardium often results in extensive loss of cardiomyocytes and a nonregenerative healing response characterized by scar formation, both of which contribute to a permanent loss of cardiac function. Despite some evidence that the rate of cardiomyocyte renewal may increase slightly after injury,1 this response is insufficient to replace the ≈1 billion cardiomyocytes that may be lost during a typical myocardial infarction. The insufficiency of the cardiac repair response results in progressive cardiac dysfunction, and individuals suffering from end-stage heart failure are currently limited to orthotopic cardiac transplant. It is, therefore, of great clinical importance to develop therapeutic strategies that could enhance the normal regenerative potential of the adult mammalian heart. Recently, a study published in Nature by Eulalio et al2 took a novel approach to this problem. Using a screening approach, the authors interrogated the potential of a class of genes called microRNAs (miRNAs) to induce cell-cycle reentry in postnatal cardiomyocytes. miRNAs are small noncoding RNAs that negatively regulate the translation or stability of their target mRNAs. Although miRNA targeting of mRNAs occurs in a sequence-specific manner, perfect base-pair complementarity is not required for effective silencing. Thus, a single miRNA may …

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1.

miR-1 is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. Here, we show that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3′UTR of the E3 ubiquitin ligase Nedd4. Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila. Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, we have found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart. Summary: The cardiac-enriched microRNA miR-1 negatively regulates the E3 ubiquitin ligase Nedd4L, which ubiquitylates many proteins and controls heart development in Drosophila.