1. 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

2. 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

3. 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

4. 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

5. 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

6. Results Effect of HDAC inhibitors on interferon gene induction Where in the STAT pathway HDAC inhibitors act

7. 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

8. 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

9. 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

10. WHERE? http://www.nature.com/ nri/journal/v5/n9/pdf/nri 1684.pdf

11. 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

12. 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

13. WHERE? http://www.nature.com/ nri/journal/v5/n9/pdf/nri 1684.pdf

14. 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

15. WHERE? http://www.nature.com/ nri/journal/v5/n9/pdf/nri 1684.pdf

16. 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

17. WHERE? http://www.nature.com/ nri/journal/v5/n9/pdf/nri 1684.pdf

18. 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

19. 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

20. 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

21. Questions?