Figure 5. PARP9-DTX3L regulates STAT1's control of ISG expression.

(a) Domains of PARP and DTX3L (top; amino acid positions above and right), and structure (below) of the two macro domains (Macro1 and Macro2) in PARP9, for binding of ADP-ribose (left), and the RING domain in DTX3L, for E3 ligase activity (right), based on homology models, identifying glycine residues (left) or cysteine zinc-chelating residues (right) targeted for loss-of-function substitution. Models based on the crystal structure of histone macroH2A1.1 (Protein Data Bank accession code, 1ZR3) with bound ADP-ribose (left) or the structure of the equine herpesvirus-1 RING domain (Protein Data Bank accession code, 1CHC) (right). (b) Gene expression (log₂ normalized) in U3A-STAT1-PARP9-DTX3L cells versus U3A-STAT1-PARP9M-DTX3LM cells, from whole-genome array, presented relative to expression in U3A-STAT1 cells; red indicates genes expressed differentially here whose expression was increased by treatment with IFN-β (as reported in Fig. 2c). (c) ISG expression in U3A-STAT1, U3A-STAT1-PARP9-DTX3L, U3A-STAT1-PARP9M-DTX3LM, U3A-STAT1-PARP9-DTX3LM and U3A-STAT1-PARP9M-DTX3L cells (key) left untreated (− IFN-β) or treated (+ IFN-β) for 12 h with IFN-β (10 U/ml). (d) CHART PCR assays of cells as in c. (e) Viral RNA (as in Fig. 2d) in U3A-STAT1 cell lines given no pretreatment (− IFN-β) or pretreated with various concentrations (horizontal axes) of IFN-β (+ IFN-β) and then inoculated with EMCV (left), IAV (middle) or SINV (right). (f) Significance of histone ubiquitination (P value) versus ubiquitination of various histones (key) with DTX3L relative to their ubiquitination without DTX3L (horizontal axis), assessed by ubiquitin proteome array with DTX3L as the E3 ligase; dashed lines indicate a P value of 0.05 and a change in ubiquitination of '0-fold', so results in the top right quadrant are both statistically significant (P < 0.05) and biologically relevant (change in ubiquitination of >0-fold). (g) Native ChIP assay of the binding of ubiquitinated histone H2B (H2B-Ub), histone H3 trimethylated at Lys4 (H3K4me3) or H2B to the IFIT1 promoter in various U3A cell lines (as in c) left untreated (− IFN-β) or treated (+ IFN-β) for 16 h with IFN-β (1 U/ml), followed by incubation of nucleosomes from the cells with antibody to ubiquitinated H2B, trimethylated H3K4 or H2B, then real-time PCR analysis of the IFIT1 promoter sequence in the immunoprecipitated DNA; results were normalized to those of the corresponding input DNA and are presented relative to those of U3A-STAT1 cells, set as 1. Results obtained with control antibody (mouse IgG) were similar to those of cells transfected with empty vector only (data not shown). *P < 0.05, versus U3A-STAT1 cells (unpaired t-test). Data from one experiment with pooling of three cell samples (b,f) or are representative of three independent experiments (c–e,g; mean and s.e.m.).