Chapter IV Discussion
4.4 STAT3 directly suppressed type I IFN-induced gene expression
Yu’s group showed that STAT3 and DNMT1 could bind to promoter of STAT1,
which is also an ISG, in cancer cells upon tumor conditioned medium treatment (Lee
et al., 2012). Interaction of acetylated STAT3 with DNMT1 resulted in DNA
methylaton, and gene silencing. Furthermore, in our microarray data, we found that
IFIT2 was decreased in STAT3-restored DKO MEFs after IFN-α4 treatment (Fig.
18A). It implies that STAT3 could directly inhibit type I IFN-mediated gene
expression independent of STAT1. In addition to IFIT2, endonuclease domain
containing 1 (ENDOD1), which is upregulated upon type I IFN treatment in
peripheral blood mononuclear cell (PBMC) (Baechler et al., 2003), was also suppressed by STAT3 in STAT3-restored DKO MEFs (Fig. 18B). These results indicated STAT3 not only affect ISGFs binding to promoter of ISRE and indirectly
regulates some ISGs, but it also directly silences gene expression.
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36 Figures
37
Figure 1. Constructs of pLPC-FH2, pLPC-FH2-mSTAT1, and pLPC-FH2- mSTAT3 (A) The map of the pLPC-FH2 retroviral vector comprises a Flag and two HA tags (B) mSTAT1 was subcloned into a retroviral vector using XhoI site. (C)
mSTAT3 was subcloned into pLPC-FH2 using BamHI and EcoRI sites. The detailed
procedure is described in the Materials and Methods.
38
Figure 2. STAT1 and STAT3 are stably expressed and activated in restored DKO MEFs in response to IFN-α4. Empty vector (EV) or vector encoding HA- and Flag-tagged STAT1 or STAT3 stably transduced into DKO MEFs. WT and restored
DKO MEFs were treated with IFN-α4 (1000 IU/ml) for 30 minutes. Whole cell
lysates were then subjucted to immunoblotting using antibodies to pSTAT1, STAT1,
pSTAT2, STAT2, pSTAT3, STAT3, HA, Flag, and α-tubulin.
39
Figure 3. STAT3-dependent genes are induced in STAT3 and STAT1/3 restored DKO MEFs, but not in EV and STAT1 restored DKO MEFs. STAT1- and/or STAT3-restored DKO MEFs were treated with or without IFN-α4 (1000 IU/ml) for 1
or 2 hours, followed by preparing RNA for RT-QPCR using the indicated primers for
Socs3 (A) JunB (B) or β-actin. Relative mRNA was calculated by normalizing to
β-actin values.
40
41
Figure 4. STAT3 negatively regulates STAT1-dependent gene expression in STAT1/3 restored DKO MEFs. STAT1- and/or STAT3-restored DKO MEFs were stimulated with or without IFN-α4 (1000 IU/ml) for 6 hours, followed by preparing
RNA for RT-QPCR using primers for PKR (A), IP-10 (B), IRF7 (C), IFIT1 (D), IFIT2
(E) or β-actin. Relative mRNA was calculated by normalizing to β-actin values.
42
43
Figure 5. STAT3 suppresses type I IFN-induced gene expression. STAT1- and/or STAT3-restored DKO MEFs were stimulated with or without IFN-α4 for 6 hours,
followed by preparing RNA for expression microarray analysis. After normalization,
the intensity of OAS2 (A), MX1 (B), STAT2 (C), and IRF1 (D) was showed.
44
Figure 6. The relationship of gene expression compares between unstimulated and stimulated restored DKO MEFs. Empty vector (EV) (A), STAT1- (B), STAT3- (C), and STAT1/3- (D) restored DKO MEFs were treated with or without IFN-α4 for
6 hours. Total RNA of the treated cells was subjected to expression microarray
analysis. The plot was generated by R package.
45
A
B
46
Figure 7. The relationship of gene expression compares STAT1-restored with STAT1/3-restored DKO MEFs. STAT1- and STAT1/3-restored DKO MEFs were treated with IFN-α4 for 6 hours. Total RNA of the treated cells was subjected to
microarray. The plot was generated by R package (A). The result was analysis by
Excel and the induced genes were indicated (B).
47
Figure 8. STAT3 suppresses type I IFN-mediated antiviral response in STAT1 and STAT3-restored DKO MEFs. STAT1- and/or STAT3-restored DKO MEFs were stimulated with 2-fold serial dilution of IFN-α4 from 240 IU/ml for 24 hours,
followed by infection with EMCV at an MOI of 0.1. Live cells were visualized with
crystal violet.
48
Figure 9. STAT3 does not to affect IFN-α4-activated STAT1 or STAT2. STAT1- and/or STAT3-restored DKO MEFs were treated with or without IFN-α4 (1000 IU/ml)
for the indicated durations. Total cell lysates were subjucted to immunoblotting using
antibodies to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, STAT3, HA, and α-tubulin.
49
Figure 10. STAT3 does not affect IFN-α4-induced nuclear translocation of STAT1 or STAT2. STAT1- and/or STAT3-restored DKO MEFs were treated with or without IFN-α4 (1000 IU/ml) for 0.5 hours. Cytosolic extracts (left) and nuclear
extracts (right) were subjucted to immunoblotting using antibodies to pSTAT1,
pSTAT2, pSTAT3, STAT1, STAT2, STAT3, HA, α-tubulin, and laminB.
50
Figure 11. STAT3 decreases the recruitment of ISGF3 complex to the promoter of ISRE. STAT1- and/or STAT3-restored DKO MEFs were treated with or without IFN-α4 for 30 minutes, followed by performing ChIP assay using antibody against
STAT1. QPCR analysis was performed using a ChIP-specific primer for the ISRE of
MDA5 (A) or IFIT1 (B) promoter. Relative abundance was calculated by normalizing
to input control.
51
52
Figure 12. HDAC inhibitor blocks suppressive effect of STAT3 on type I IFN-induced gene expression. STAT1- and STAT1/STAT3-restored DKO MEFs were stimulated with or without IFN-α4 alone or simultaneously with the indicated
concentration of SAHA for 6 hours. Total RNA of the treated cells was subjected to
RT-QPCR using primers for STAT1-mediated genes such as IP-10 (A), IRF7 (B),
IFIT1 (C) or β-actin. Relative mRNA was calculated by normalizing to β-actin values.
53
Figure 13. Comparable expression and activation of WT and mutant STAT3s in STAT3KO MEFs. Empty vector (EV), WT STAT3, STAT349Q, STAT387Q, STAT3QQ (A), STAT349R, STAT387R, or STAT3RR (B)was transfected into STAT3KO MEFs for
48 hours. Cells were treated with or without IFN-α4 (1000 IU/ml) for 30 minutes.
Whole cell lysates were then subjucted to immunoblotting using antibodies to
pSTAT3, HA, and α-tubulin.
54
Figure 14. IFNα-dependent acetylation of STAT3 is abolished in RR mutant STAT3. WT STAT3, STAT3RR,and STAT3QQ was transfected into STAT3KO MEFs for 48 hours. Cells were treated with or without IFN-α4 (1000 IU/ml) for 30 minutes.
Whole cell lysates were then subjucted to immunopreciptation using anti-HA
antibodies, and immunoblotting of acetylated-lycine and STAT3. Third raw expressed
western blotting for whole cell lysate using anti-STAT3 antibody.
55
Figure 15. Mutations in Lys49 and Lys87 of STAT3 at NTD affect STAT3 downstream gene induction. Empty vector (EV), WT STAT3, STAT3 acetylation- deficient (49R, 87R, or RR), or STAT3 acetylation mimics (49Q, 87Q, or QQ) was
transfected into STAT3KO MEFs. After 48 hours, MEFs were treated with or without
IFN-α4 (1000 IU/ml) for 1 hour. Total RNA of the treated cells was subjected to
RT-QPCR using primers for Socs3 (A), JunB (B), or β-actin. Relative mRNA was
calculated by normalizing to β-actin values.
56
57
Figure 16. Negative regulation of type I IFN responses by STAT3 is abrogated in Lys49 and/or Lys87 STAT3 mutants. Empty vector (EV), WT STAT3, STAT3 acetylation-deficient, or STAT3 acetylation mimics was transfected into STAT3KO
MEFs for 48 hours, followed by treating the cells with or without IFN-α4 (1000 IU/ml)
for 1 hour. Expression of IP-10 (A), IRF7 (B), IFIT1 (C), or β-actin was monitored by
RT-QPCR. Relative mRNA was normalized with β-actin values.
58
59
Figure 17. Acetylation of STAT3 at Lys685 affects negative effect of STAT3 on type I IFN-mediated gene expression. Empty vector (EV), WT STAT3, STAT3RR, or STAT3685R was transfected into STAT3KO MEFs. After 48 hours, MEFs were treated
with or without IFN-α4 for 6 hour. Total RNA of the treated cells was subjected to
RT-QPCR using primers for IP-10 (A), IRF7 (B), IFIT1 (C) or β-actin. Relative
mRNA was calculated by normalizing to β-actin values.
60
Figure 18. STAT3 directly suppresses gene expression. Restored DKO MEFs were stimulated with or without IFNα4 for 6 hours, followed by preparing RNA for
microarray. After analysis, intensity of OAS2 (A), MX1 (B), STAT2 (C), and IRF1 (D)
was showed.