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

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

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

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

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

Results

Effect of HDAC inhibitors on interferon gene induction

Where in the STAT pathway HDAC inhibitors act

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

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

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

WHERE?

http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf

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

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

WHERE?

http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf

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

WHERE?

http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf

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

WHERE?

http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf

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

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

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

Questions?