scholarly journals Genome-wide screens identify specific drivers of mutant hTERT promoters

2022 ◽  
Vol 119 (3) ◽  
pp. e2105171119
Author(s):  
Raghuvaran Shanmugam ◽  
Mert Burak Ozturk ◽  
Joo-Leng Low ◽  
Semih Can Akincilar ◽  
Joelle Yi Heng Chua ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Regulatory Region ◽  
Telomerase Activity ◽  
Chromatin Interaction ◽  
Mediator Complex ◽  
Wild Type ◽  
Distal Regulatory Region ◽  
Genome Wide ◽  
Promoter Mutations ◽  
Interfering Rna

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

scholarly journals Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

2014 ◽  
Vol 88 (15) ◽  
pp. 8565-8578 ◽  
Author(s):  
R. Meier ◽  
A. Franceschini ◽  
P. Horvath ◽  
M. Tetard ◽  
R. Mancini ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Host Factor ◽  
Genome Wide ◽  
Novel Host ◽  
Interfering Rna

Intra-airway administration of small interfering RNA targeting plasminogen activator inhibitor-1 attenuates allergic asthma in mice

2011 ◽  
Vol 301 (6) ◽  
pp. L908-L916 ◽  
Author(s):  
Shintaro Miyamoto ◽  
Noboru Hattori ◽  
Tadashi Senoo ◽  
Yojiro Onari ◽  
Hiroshi Iwamoto ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Plasminogen Activator Inhibitor ◽  
Healthy Controls ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Asthma Model ◽  
Interfering Rna ◽  
Deficient Mice ◽  
Activator Inhibitor ◽  
Pai 1

Recent studies suggest that plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of the fibrinolytic system, may promote the development of asthma. To further investigate the significance of PAI-1 in the pathogenesis of asthma and determine the possibility that PAI-1 could be a therapeutic target for asthma, this study was conducted. First, PAI-1 levels in induced sputum (IS) from asthmatic subjects and healthy controls were measured. In asthmatic subjects, IS PAI-1 levels were elevated, compared with that of healthy controls, and were significantly higher in patients with long-duration asthma compared with short-duration asthma. PAI-1 levels were also found to correlate with IS transforming growth factor-β levels. Then, acute and chronic asthma models induced by ovalbumin were established in PAI-1-deficient mice and wild-type mice that received intra-airway administrations of small interfering RNA against PAI-1 (PAI-1-siRNA). We could demonstrate that eosinophilic airway inflammation and airway hyperresponsiveness were reduced in an acute asthma model, and airway remodeling was suppressed in a chronic asthma model in both PAI-1-deficient mice and wild-type mice that received intra-airway administration of PAI-1-siRNA. These results indicate that PAI-1 is strongly involved in the pathogenesis of asthma, and intra-airway administration of PAI-1-siRNA may be able to become a new therapeutic approach for asthma.

Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ: importance of kinase activity and binding to AP2α

2007 ◽  
Vol 409 (2) ◽  
pp. 471-479 ◽  
Author(s):  
Takumi Kawasaki ◽  
Takeshi Kobayashi ◽  
Takehiko Ueyama ◽  
Yasuhito Shirai ◽  
Naoaki Saito
Keyword(s):  
Catalytic Domain ◽  
Intracellular Localization ◽  
Adaptor Protein ◽  
Diacylglycerol Kinase ◽  
Wild Type ◽  
Coated Pits ◽  
Genome Wide ◽  
A Subunit ◽  
Transferrin Uptake ◽  
Interfering Rna

DGKδ (diacylglycerol kinase δ), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGKδ-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKδ in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGKδ2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKδ2 and its interaction with some endocytosis-regulatory proteins. DGKδ2 contained the DXF-type binding motifs, and DGKδ2 bound to AP2α, a subunit of the AP-2 complex. DGKδ2 interacted with the platform subdomain in the AP2α ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKδ2. For further insight into the role for DGKδ2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKδ2 under knockdown of endogenous DGKδ. Mutants lacking binding ability to AP2α as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGKδ2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKδ2 and AP2α is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.

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