Transcript Interferon-stimulated transcription and innate antiviral immunity

Interferon-stimulated transcription and
innate antiviral immunity require
deacetylase activity and histone
deacetylase 1
Inna Nusinzon and Curt M. Horvath
Mt. 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/HistoneAce
tylation.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

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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
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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/nri
1684.pdf
TSA and the IFN-JAK-STAT-ISGF3
pathway

Steps in pathway
–
ISGF3 phosphorylation

–
Dimerization

–
–
STAT 1 and STAT 2
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/nri
1684.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/nri
1684.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/nri
1684.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…
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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?