CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic ...screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with an optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts.
Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ...ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.
MicroRNA-155 Reinforces HIV Latency Ruelas, Debbie S.; Chan, Jonathan K.; Oh, Eugene ...
Journal of biological chemistry/The Journal of biological chemistry,
05/2015, Volume:
290, Issue:
22
Journal Article
Peer reviewed
Open access
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.
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.
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.
Abstract
CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (
dCas9a-SAM
KI
) ...for inducing gene expression in vivo and in vitro. Using
dCas9a-SAM
KI
primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in
Eµ-Myc
T/+
;dCas9a-SAM
KI/+
haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard
Eµ-Myc
lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.
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 (programmedcell 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.
is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of
produces defects in somatic muscle and embryonic heart development, which have been partly ...attributed to
directly targeting Delta to decrease Notch signaling. Here, we show that overexpression of
in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential
targets revealed that
directly regulates the 3'UTR of the E3 ubiquitin ligase
Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in
Larval fly hearts overexpressing
have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that
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
and the mammalian ortholog Nedd4-like (
) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus,
-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart.
Chinese hamster ovary (CHO) cells have been adapted to grow in serum‐free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates ...while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti‐cell‐aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM‐1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM‐1 gene significantly decreased cell aggregate formation in the culture media without anti‐cell‐aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM‐1 KO and wild‐type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM‐1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.
PDF
