Figure 7.

H2A.Z Suppresses ISGF3 Occupancy, ISG Expression, and IFN-Mediated Antiviral Protection

HeLa cells were transduced with an shRNA vector targeting H2A.Z or a non-targeting control.

(A) Immunoblot of H2A.Z, STAT1, phosphotyrosine 701 STAT1, STAT2, phosphotyrosine 690 STAT2, and GAPDH protein expression in control or H2A.Z knockdown HeLa cells with or without 1-hr IFNα treatment. H2A.Z expression level normalized to GAPDH indicated as % of control.

(B) ChIP analysis of STAT2 occupancy in H2A.Z knockdown or control HeLa cells with or without 1-hr IFNα stimulation at promoters of IFIT1/ISG56, IFIT2/ISG54, IFITM1/9–27, OAS3, ISG15, and LOC100419583. % Indicates the increased percentage of STAT2 occupation in shH2A.Z cells compared with non-targeting control cells. Error bars denote mean ± SD of a representative experiment with technical triplicates. Statistical analysis was computed using Student's t test with n ≥ 2 (*p < 0.05, **p < 0.005.).

(C) H2A.Z mRNA levels were quantified by RT-qPCR in unstimulated and 10-hr IFNα-stimulated H2A.Z knockdown or control cells. Error bars denote mean ± SD of a representative experiment with technical triplicates.

(D) Levels of ISG mRNAs, IFIT1/ISG56, IFIT2/ISG54, IFITM1/9–27, OAS3, ISG15, and LOC100419583 were measured as in (C). Statistical analysis was computed using Student's t test with n ≥ 3 (*p < 0.05, **p < 0.005, ***p< 0.0005).

(E) Plaque assay in HeLa cells harboring control shRNA or H2A.Z shRNA. Cells were treated for 9 hr with IFNα, followed by 1.5 hr inoculation with a titration of vesicular stomatitis virus (VSV), and then overlaid with DMEM-agar at 37°C for 72 hr before staining with crystal violet. TMTC, too many to count.

(F) Same as E, but in control or H2A.Z-deficient 2fTGH cells.

See also Figure S4.