Extended Data Fig. 6: SIX family proteins inhibit NF-κB activation by occupying the target gene promoters.

a, Wild-type and SIX1^−/−SIX2^−/− H1792 cells were treated with mock or 50 ng ml⁻¹ TWEAK for 24 h. Cell lysates from cytoplasmic or nuclear fractions were analysed by western blot. Neither SIX1 nor SIX2 blocked RELA phosphorylation or p100/52 processing, or restricted NF-κB translocation to the nucleus. b, Top, SIX proteins are composed of a SIX domain (SD), homeobox domain (HD), and coiled-coil (CD) region. Full-length SIX1 and SIX2 have 80% identical amino acids over the protein-coding sequence. The SD and HD domains (residues 1–183) are 96% identical⁴⁶. The indicated Flag-tagged SIX2 fragments were transfected alone into U-2 OS cells and processed for microscopy (middle, scale bars represent 20 μm) or transfected with 5 × κB-LUC into HEK293T cells and processed for κB reporter activity (bottom). The highly conserved SD–HD domain was the minimal fragment that inhibited κB reporter activity. This fragment strictly localized to the nucleus, indicating that SIX proteins inhibit nuclear activity of NF-κB. Graph data analysed as in Extended Data Fig. 5d. Data are mean ± s.d. from nine independent experiments. c, SIX2 inhibits gene activation from the IL8 promoter. pIL8-LUC plasmid composed of the 1.5-kb promoter region of IL8 cloned upstream of LUC was co-transfected with indicated plasmids into HEK293T cells. After 24 h, cells were mock-treated or treated with 25 ng ml⁻¹ TNF for 24 h. Luciferase activity was then measured and analysed as in Extended Data Fig. 5d. Data are mean ± s.d. from nine independent experiments. These data suggest that SIX proteins inhibit NF-κB activation at gene promoters. d, ChIP experiment providing additional evidence that SIX proteins bind to inflammatory gene promoters (Fig. 3a). Chromatin was prepared from stable GFP–SIX1 cell lines (HCT116 cells), mock-treated or treated with 25 ng ml⁻¹ TNF for 2 h. Anti-SIX1 antibodies (or anti-IgG control) were used to immunoprecipitate SIX1 from nuclear extracts. Co-eluted DNA was amplified using primer sets as in Fig. 3a. Relative promoter occupancy was normalized to each experimental IgG control. Bars are means of two technical replicates (shown as circles) and data are representative of three independent experiments. e, Control experiments corresponding to Fig. 3a and Extended Data Fig. 6f showing GFP–SIX1 expression. Wild-type and stable GFP–SIX1 fibroblasts were stimulated with 50 ng ml⁻¹ TWEAK for 24 h. GFP–SIX1 expression was measured by western blot. f, SIX1 expression does not affect recruitment of RELA to the IL8 promoter. Chromatin was prepared from wild-type or GFP–SIX1 stable fibroblasts and immunopreciptated with anti-RELA. Bound DNA was amplified and quantified by qPCR. Results were adjusted to ‘input DNA’ that was saved before immunopreciptation. Relative enrichment was then normalized to each group’s untreated control. Data are mean ± s.d. from three independent experiments. g, Control experiments corresponding to Fig. 3c showing that SIX2 inhibits 5 × κB-LUC activity induced by GAL4–RELA and GAL4–RELB. GAL4 –RELA or GAL4–RELB construct was co-transfected with indicated plasmids into HEK293T cells. Forty-eight hours after transfection, the luminescence units were measured. Data are mean ± s.d. from nine (left) and twelve (right) independent experiments. h, Model showing NIK-mediated reactivation of SIX protein function in a negative feedback loop to control inflammatory gene expression by targeting gene promoters and inhibiting the trans-activation function of NF-κB. In quiescent cells (top), NIK and SIX are constitutively ubiquitinated and degraded by the proteasome. Non-canonical NF-κB agonists (for example, TWEAK, LTα1β2, or BV6) promote degradation of cIAPs, loss of NIK ubiquitination and subsequent NIK protein accumulation (middle). Stabilized NIK activates non-canonical NF-κB-mediated inflammatory gene expression (middle). Under conditions of long-term cytokine exposure, NIK-mediated suppression of a currently unknown E3-ubiquitin ligase results in SIX protein accumulation (bottom). Consequently, SIX proteins suppress inflammatory gene expression by targeting gene promoters and directly inhibiting trans-activation by NF-κB in a negative feedback loop (bottom). All western blots and microscopy data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Source data