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Small molecule and pooled CRISPR screens investigating IL17 signaling identify BRD2 as a novel contributor to keratinocyte inflammatory responses.

08:00 EDT 2nd April 2019 | BioPortfolio

Summary of "Small molecule and pooled CRISPR screens investigating IL17 signaling identify BRD2 as a novel contributor to keratinocyte inflammatory responses."

Interleukin-17A, (IL17A) plays a critical role in the development of numerous autoimmune diseases including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 (Th17) cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of ~22,000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins, and bromodomain containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2 deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2 deficient keratinocytes. In IL17A/TNF/IL22 stimulated 3D organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.

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This article was published in the following journal.

Name: ACS chemical biology
ISSN: 1554-8937
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Medical and Biotech [MESH] Definitions

Protein components of the CRISPR-CAS SYSTEMS for anti-viral defense in ARCHAEA and BACTERIA. These are proteins that carry out a variety of functions during the creation and expansion of the CRISPR ARRAYS, the capture of new CRISPR SPACERS, biogenesis of SMALL INTERFERING RNA (CRISPR or crRNAs), and the targeting and silencing of invading viruses and plasmids. They include DNA HELICASES; RNA-BINDING PROTEINS; ENDONUCLEASES; and RNA and DNA POLYMERASES.

Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS.

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Part of a MESSENGER RNA molecule that undergoes a conformation change upon binding a specific metabolite or other small molecule thereby regulating the messenger RNA's transcription, post-transcriptional processing, transport, translation, or stability in response to varying levels of the metabolite or other small molecule.

A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)

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