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![Page 1: Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1 Inna Nusinzon and Curt M. Horvath.](https://reader035.fdocuments.us/reader035/viewer/2022062417/551c325a550346ad4f8b63cc/html5/thumbnails/1.jpg)
Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone
deacetylase 1
Inna Nusinzon and Curt M. HorvathMt. Sinai School of Medicine
Presented By:Mike Waters Davidson College/VCU BBSI student
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Introduction: Acetylation
Acetylation = important modulator of cellular response/ signal transduction– HAT/HDAC– Promoter specific roles in gene expression
http://www.phy.mtu.edu/images/menu/HistoneAcetylation.jpg
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Introduction: Innate Immunity
Responsible for activation of adaptive immmunity
Toll-like receptors recognize Pathogen Associated Molecular Patterns (PAMPs)– LPS, petidoglycan,dsRNA, CpG motifs– MyD88 dependent pathway signals type-I
interferon response
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STAT Pathway
STAT= signal transduction and activator of transcription
Recognizes interferons to produce antiviral state– Upregulate genes invovled in immune response
http://www.rsc.org/images/3d_370_tcm18-68972.bmp
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This paper:
Examine the role of acetylation in the STAT pathway
Implications in gene therapy
http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture25/AMG9.11a.gif
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Results
Effect of HDAC inhibitors on interferon gene induction
Where in the STAT pathway HDAC inhibitors act
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IFN-Stimulated Gene Induction Is Blocked by HDAC Inhibitors
• Human 2fTGH’s are TSA (HDACi) challenged
•Quantitative PCR performed
•Primers specific for ISGF3 targets
•Positive control = rapid transient induction
•TSA inhibits this response
HDACi blocks transcription of ISGF3 genes
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IFN-Stimulated Gene Induction Is Blocked by HDAC Inhibitors (cont.)
•Performed the same test with NaB (HDACi)
•To rule out nonspecific effects of the compound
•To confirm inhibition is a result of HDAC inhibitory activity
It is the deacetylase activity that enables TSA to block ISGF3 target transcription
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HDAC activity as a general property of ISGF3-regulated transcription
•ISRE-luciferase reporter gene assay
•Transfection
•INF-α challenge
•Increase in Luciferase flouresence due to challenge is inhibited by TSA
Enzymes that remove acetyl groups are necessary for the induction of innate immune response genes
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WHERE?
http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf
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TSA and the IFN-JAK-STAT-ISGF3 pathway
Steps in pathway– ISGF3 phosphorylation
STAT 1 and STAT 2
– Dimerization Formation of ISGF3
– Nuclear translocation– DNA binding
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STAT 1 and STAT 2 Phosphorylation
•Immunoblotting with phosphotyrosine specific antibodies (Western Blot)
•pSTAT= tyr. Phos. STATs
•STAT= total STATs
Treatment with TSA does not affect IFN-α induced phosphorylation of STAT proteins
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WHERE?
http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf
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Heterodimerization
•Coimmunoprecipitation assay =pulls antigen out of solution
•uses specific antibody
•CO-IP= can identify interacting proteins
•STAT 2 antiserum •WB for STAT1
TSA does not affect the dimerization of STAT proteins
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WHERE?
http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf
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Nuclear Translocation
• Without IFN = no translocation
•TSA does not effect translocation
•Sakamoto et. al. confirmed in 2004
TSA does not affect nuclear translocation of ISGF3
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WHERE?
http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf
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DNA Binding
•Electrophoretic mobility shift assay (EMSA)
•P-labeled ISG15-ISRE probe
•STAT 2 antibody supershift confirmed ISGF3 identity
•TSA does not affect ISGF3-DNA binding
•ISGF3 signaling from the cytoplasm to the nucleus remains intact when exposed to TSA
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HDAC as a positive coactivator for ISGF3 transcription
• ISRE-luciferase transfection
•Fig E = RNAi test
•Fig G = Dose Response Test
HDAC acts as a coactivator for ISGF3 transcriptional response
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If you were sleeping…
HDACi inhibits innate immune response This does not occur in the ISGF3 signaling
from the cytoplasm to the nucleus HDAC act as coactivators of ISGF3
transcription
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Questions?