Identification of proteins binding to E-Box/Ku86 sites and function of the tumor suppressor SAFB1 in transcriptional regulation of the human xanthine oxidoreductase gene

Abstract

The xanthine oxidoreductase gene (XOR) encodes an important source of reactive oxygen species and uric acid, and its expression is associated with various human diseases including several forms of cancer. We previously reported that basal human XOR (hXOR) expression is restricted or repressed by E-box and TATA-like elements and a cluster of transcriptional proteins, including AREB6-like proteins and DNA-dependent protein kinase (DNA-PK). We now demonstrate that the cluster contains the tumor suppressors SAFB1, BRG1, and SAF-A. We further demonstrate that SAFB1 silencing increases hXOR expression and that SAFB1 directly binds to the E-box. Multiple studies in vitro and in vivo including pulldown, immunoprecipitation and chromatin immunoprecipitation analyses indicate that SAFB1, Ku86, and BRG1 associate with each other. The results suggest that the SAFB1 complex binds to the hXOR promoter in a chromatin environment and plays a critical role in restricting hXOR expression via its direct interaction with the E-box, DNA-PK, and tumor suppressors. Moreover, we demonstrate that the cytokine, oncostatin M (OSM), induces the phosphorylation of SAFB1 and that the OSM-induced hXOR mRNA expression is significantly inhibited by silencing the DNA-PK catalytic subunit or SAFB1 expression. The present studies for the first time demonstrate that hXOR is a tumor suppressor-targeted gene and that the phosphorylation of SAFB1 is regulated by OSM, providing a molecular basis for understanding the role of SAFB1-regulated hXOR transcription in cytokine stimulation and tumorigenesis.

FIGURE 1.

Magnetic DNA affinity purification of nuclear proteins binding to probe 3× EG. The PFSK-1 nuclear proteins binding to the probe were eluted after purification with probe 3× EG-conjugated DYNAL magnetic beads and analyzed by electrophoresis. Six bands with sizes of around 350, 200, 150, 120, 86, and 70 kDa were observed.

FIGURE 2.

A, detection of the eluted proteins from probe 3× EG-conjugated DYNAL magnetic beads using AREB6-C, SAFB1, and BRG1 antibodies. B, detection of SAFB1 was blocked by preabsorption of SAFB1 antibodies with AREB6-C extracts but not AREB6-H.

FIGURE 3.

A, a representative image showing that SAFB1 expression is inhibited by SAFB1-targeted siRNA (SAFB1 siRNA) but not by the nonspecific siRNA (control). In addition, the expression of hXOR transcripts was greater in the cells with the silenced SAFB1 expression than that in the control cells. B, statistical results of three independent experiments (n = 3 for each experiment). *, indicates p < 0.05.

FIGURE 4.

SAFB1C binds to the E-box. A, EMSA were performed using recombinant SAFB1C proteins. Probe EG contains both E-box and putative Ku86 sites. Binding to the E-box is indicated by an arrow and was specifically competed by the unlabeled probes EG (lane 6) or Kum1 (probe with mutant Ku86 site, lane 7), whereas the mutations of the E-box (probe EmG, lane 5) lost the ability to specifically compete. In comparison, Ku86 (lane 3) and Ku70 (lane 2) did not bind to the probe EG. B, lane 4 shows binding of SAFB1C to probe EG, as indicated by an arrow. The binding was blocked by SAFB1 antibodies (lanes 2) but not by nonspecific antibodies (lane 3).

FIGURE 5.

Representative immunoblots showing pulldown analysis of protein-protein interaction between SAFB1 and Ku86 proteins. A, nuclear proteins immunoreactive to SAFB1 antibodies, as indicated by an arrow, were pulled down by GST-Ku86 proteins. B, nuclear proteins immunoreactive to Ku86 antibodies, as indicated by an arrow, were pulled down by GST-SAFB1C proteins. Map2 and GST were used as negative controls.

FIGURE 6.

Coimmunoprecipitation analysis of SAFB1, Ku86, and BRG1. A, nuclear proteins immunoreactive to SAFB1 and BRG1 antibodies, as indicated by the arrows, were present in the coimmunoprecipitates with Ku86 antibodies. B, nuclear proteins immunoreactive to Ku86 and BRG1 antibodies, as indicated by the arrows, were present in the coimmunoprecipitates with SAFB1 antibodies. GST was used as a negative control. SAFB1, Ku86, and BRG1 in the nuclear extracts (NE) were used as the positive controls.

FIGURE 7.

Two independent representative ChIP assays of DNA-PKcs, Ku70, Ku86, and SAFB1 interactions with hXOR promoter in PFSK-1 cells. Bands are the PCR products (a 286-bp fragment of the hXOR promoter). A and B are images showing two independent results. In A each antibody essay was performed in triplicate (1–3, 4–6, 7–9, 10–12), whereas in B each was performed in duplicate (1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10).

FIGURE 8.

Western blot images demonstrating that the phosphorylation of SAFB1 is up-regulated by OSM. A, the representative images showing that OSM (100 nm) increased the phosphorylated SAFB1 (P) that was dephosphorylated by calf intestinal alkaline phosphatase (CIAP) treatment but not the non-phosphorylated CIAP (non-P). B, the representative images showing that phosphorylated and nonphosphorylated SAFB1 were reduced by SAFB1-silencing (siRNA) but not by the nonspecific silencing (control). β-Actin was detected as a loading control.

FIGURE 9.

A model illustrating the molecular basis for regulation of the hXOR promoter. In this model SAFB1 binds to the consensus E-box (ACAGGTG) and restricts hXOR promoter activity and interacts with other co-repressors, such as Ku86, SAFA, and BRG1. Ku86 is associated with Ku70 and DNA-PKcs. Based on EMSA, other unknown transcription factors that may be involved in the regulation of hXOR are labeled with capital letters (A–C).