Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

Lebensohn, Andres M.; Dubey, Ramin; Neitzel, Leif R.; Tacchelly-Benites, Ofelia; Yang, Eungi; Marceau, Caleb; Davis, Eric M.; Patel, Bansari; Bahrami-Nejad, Zahra; Travaglini, Kyle J.; Ahmed, Yashi; Lee, Ethan; Carette, Jan E.; Rohatgi, Rajat

doi:10.7554/elife.21459

Cited by 52 publications

(150 citation statements)

References 61 publications

(71 reference statements)

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“…Interestingly, LAPTM4B was shown to also regulate AP4 expression via its effects on c‐myc, resulting in a circular positive feedback loop. Although some research has shown that c‐myc works together with AP4 to regulate tumour pathogenesis (Xue et al ., ) or that AP4 regulates the Wnt signalling pathway (Lebensohn et al ., ), which also involves c‐myc, our report is the first to show that AP4 also regulates c‐myc by affecting LAPTM4B expression. As summarized in Fig.…”

Section: Resultsmentioning

confidence: 99%

AP4 positively regulates LAPTM4B to promote hepatocellular carcinoma growth and metastasis, while reducing chemotherapy sensitivity

Yue

Wang

et al. 2018

Molecular Oncology

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Polymorphisms of the lysosomal‐associated protein transmembrane‐4 beta (LAPTM4B) gene are related to various forms of tumour susceptibility, which led us to hypothesize that some unique transcription factors targeting this polymorphism region may affect the biological function of LAPTM4B in tumour progression. In this study, we found that the transcription factor AP4 directly binds to the polymorphism region of the LAPTM4B gene promoter and induces its transcription. In addition, we demonstrated that AP4 promotes hepatocellular carcinoma (HCC) cell proliferation and metastasis and depresses chemotherapy sensitivity via LAPTM4B by activating the PI3K/AKT signalling pathway and caspase‐dependent pathway. Interestingly, we found that AP4 could not only regulate LAPTM4B by directly binding to the promoter, but also be regulated via a positive feedback mechanism involving LAPTM4B acting on c‐myc. Finally, we showed that AP4 and LAPTM4B are highly coexpressed in HCC tissues, and their coexpression may be a marker of poor prognosis. These findings provide evidence of the expression and functional coupling between AP4 and LAPTM4B and shed light on the regulation of LAPTM4B and its function in liver cancer.

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Section: Resultsmentioning

confidence: 99%

AP4 positively regulates LAPTM4B to promote hepatocellular carcinoma growth and metastasis, while reducing chemotherapy sensitivity

Yue

Wang

et al. 2018

Molecular Oncology

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“…WT HAP1-7TGP cells and genetically modified clonal derivatives were grown as described in the “Cell lines and growth conditions” section of Materials and methods in [3].…”

Section: Methodsmentioning

confidence: 99%

Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens

Patel

Lebensohn

Carette

et al. 2018

Preprint

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28The systematic identification of regulatory elements that control gene expression remains a 29 challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-30 coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused 31 on identifying the former two. To identify regulatory elements, we conducted a new 32 bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in 33 haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used 34 as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions 35 upstream of genes. We uncovered atypical patterns of gene trap insertions that were not 36 predicted to disrupt coding sequences, but caused changes in the expression of two key 37 regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our 38 methodology extends the scope of haploid genetic screens by enabling the identification of 39 regulatory elements that control gene expression. 40

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“…Our reporter-based screening platform has been described previously in detail (Lebensohn et al 2016;Pusapati, Kong, Patel, Krishnan, et al 2018) . NIH/3T3-CG cells were used because they respond to SHH in a concentration-dependent manner, carry stably integrated Cas9, and carry fluorescence-based, quantitative reporter of HH signaling (GLI-GFP) (Pusapati, Kong, Patel, Krishnan, et al 2018) .…”

Section: Crispr/cas9 Screen Targeting Lipid-related Genesmentioning

confidence: 99%

Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

Kinnebrew

Iverson

Patel

et al. 2019

Preprint

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Transmission of the Hedgehog signal across the plasma membrane by Smoothened is proposed to be triggered by its direct interaction with cholesterol. But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find here that Smoothened activation and Hedgehog signaling are driven by a biochemically defined fraction of membrane cholesterol, termed accessible cholesterol. Increasing accessible cholesterol levels by depletion of sphingomyelin, which sequesters cholesterol in complexes, potentiates Hedgehog signaling. By inactivating the transporter-like protein Patched 1, Hedgehog ligands trigger an increase in cholesterol accessibility in the ciliary membrane, the subcellular location for Smoothened signaling. Thus, compartmentalization of Hedgehog signaling in the primary cilium may allow cholesterol accessibility to be used as a second messenger to mediate the communication between Patched 1 and Smoothened, without causing collateral effects on other cellular processes. We thank Kyle Travaglini and Onn Brandman for help with the MATLAB code for automated quantitation of probe fluorescence at primary cilia, Xiaohui Zha and Kevin Courtney for helpful discussions and protocols for sphingomyelin assays, and Suzanne Pfeffer for comments on the manuscript. Author contributionsRR and AR designed the project. BP analyzed the screen data, with the exception of the KEGG analysis which was performed by MK. BP and GP designed and cloned the CRISPR library focused on lipid-related genes. BP, GP and JK generated the mutant cell library and performed the HiSHH-Bottom10% screen. MK performed the LoSHH-Top5% screen. MK performed the experiments related to cholesterol genes and MK and EJI performed experiments related to sphingomyelin genes. MK, GL and KAJ performed the lipid probe staining experiments. MK and BP designed the computational pipeline for probe quantitation at primary cilia. CS and DFC contributed key conceptual and structural insights and experimental suggestions. RR and MK wrote the paper, with input from all authors.

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Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

Cited by 52 publications

References 61 publications

AP4 positively regulates LAPTM4B to promote hepatocellular carcinoma growth and metastasis, while reducing chemotherapy sensitivity

AP4 positively regulates LAPTM4B to promote hepatocellular carcinoma growth and metastasis, while reducing chemotherapy sensitivity

Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens

Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

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