FBI-1 deacetylates histones Ac-H3 and Ac-H4 at the proximal promoter of the endogenous Rb gene promoter. ChIP assays of H3 and H4 histone modification at the proximal promoter (bp –131 to +167) of the endogenous Rb gene using antibodies against Ac-H3 and Ac-H4. HEK293A cells were transfected with FLAG-FBI-1 expression vector, and immunoprecipitated with the antibodies indicated. A histogram of the ChIP assay is shown below.

FBI-1 binds to the proximal Sp1-binding GC-box 2, and Sp1 binds to the distal FRE3 element in vitro. FBI-1 and Sp1 compete with each other for binding to FRE3 and GC-box 2. A, comparisons of the nucleotide sequences of the FREs and Sp1-binding GC-boxes with their consensus sequences. B, EMSA. FBI-1ZFDBD was incubated with FRE4 or the typical Sp1 GC-box probe. FBI-1 binds to the Sp1-binding GC-box probe. FRE4, control FBI-1 binding probe. Sp1 GC-box, a typical Sp1-binding oligonucleotide probe with 5′-GGGCGGGG-3′ core sequence. C, EMSA. Recombinant Sp1ZFDBD or FBI-1ZFDBD was incubated with the GC-box probes. Sp1 binds both GC-box 1 and 2, but binds more strongly to GC-box 2 (lanes 2 and 7). FBI-1 binds to the Sp1 GC-box 2 probe only. D, EMSA and identification of a new Sp1 binding element, FRE3. Recombinant Sp1ZFDBD (0.1 μg) was incubated with the FRE probes or the GC-box probes. Sp1 significantly binds to FRE3 and GC-box 2 probes. E, EMSA. FBI-1 and Sp1 compete for binding to FRE3 and GC-box 2 in vitro. The probes were incubated with Sp1ZFDBD (0.1 μg) in the presence of increasing amounts of FBI-1ZFDBD (0.1, 0.4, and 0.8 μg). FBI-1 competes with Sp1 for binding to FRE3 and GC-box 2 in vitro.

Hypothetical regulatory mechanism for the transcriptional regulation of Rb gene by FBI-1 and Sp1. When FBI-1 is highly expressed as in cancer cells, FBI-1 binds to the distal FREs and GC-box 2 on the Rb promoter by competing with Sp1. FBI-1 preferentially binds to FRE2 (⋆⋆). After FBI-1 binding, the co-repressor-HDAC complex is recruited by the POZ-domain of FBI-1 and represses transcription by deacetylating histones H3 and H4 of the nucleosomes around the proximal promoter. When FBI-1 is low, as in normal cells, Sp1 binds to FRE3 (††), a newly identified strong Sp1-binding element, GC-box 1, and GC-box 2 to activate transcription. FRE, FBI-1 binding element; GC-box, Sp1-binding GC-rich element; ⋆⋆, high affinity FBI-1 binding element; ⋆, high affinity FBI-1 binding GC-box2 element; ††, a newly identified high affinity Sp1-binding element; Tsp (+1), transcription start site; ⊥ transcription repression by histone modification at the proximal promoter.

ChIP assays. FBI-1 and Sp1 compete with each other for binding to FRE3 and GC-box 2 in vivo. A, structure of human Rb gene. Arrows, primers used in ChIP; Tsp (+1), transcription start site. Circles with number, FBI-1 binding FREs; filled black circles with number, GC-box that binds Sp1; open boxes, exons; 3′-UTR, 3′-untranslated region with no known binding sites for either Sp1 or FBI-1. B, ChIP assays of binding competition between Sp1 and FBI-1 for the distal (bp –370 to –147), proximal promoter (bp, –131 to +93), and 3′-UTR elements (bp +176326 to +176626) of the endogenous Rb gene promoter in HEK293A cells. Human HEK293A cells were transfected with increasing amount of FLAG-FBI-1 expression vector and analyzed for Sp1 and FBI-1 binding using the antibodies indicated. Sp1 and FBI-1 compete with each other both on the distal and proximal promoter regions. C, histogram of the ChIP assay shown in B. D, ChIP assays on the endogenous Rb gene promoter after knockdown of endogenous FBI-1 expression in HEK293A cells. Knockdown of FBI-1 resulted in an increase in Sp1 binding to the endogenous Rb gene promoter, both on the proximal and distal promoters. E, histogram of the ChIP assay shown in C.

FBI-1 binds to the proximal Rb promoter and may repress transcription via the GC-box 2 in vivo. A, ChIP assays of FBI-1 binding at the proximal promoter (bp –131 to +93) of the endogenous Rb gene. Structure of the Rb gene promoter and locations of PCR primer sets used for ChIP assays. pcDNA3-FLAG-FBI-1 expression plasmid was transfected into HeLa cells and chromatin was immunoprecipitated with anti-FLAG antibody or control IgG. Arrows, primers used in ChIP; Tsp (+1), transcription start site. B, structures of pGL2-Rb-Luc Wt and mutant plasmids and transcription analysis. Mutations were introduced into the proximal promoter. Open circles, FBI-1 binding sites; filled black circles, Sp1-binding GC-boxes; X, mutation introduced. Mutation of the GC-box 1 decreased transcription, but mutation of GC-box 2 increased Rb gene transcription compared with the Wt promoter, which suggests that FBI-1 may bind to GC-box 2 and repress transcription. C, Western blot analysis of Sp1 and FBI-1 in the HeLa cells transfected and described in B.

Inhibition of mouse C2C12 myoblast differentiation by FBI-1 can be rescued by transfection with recombinant adenovirus overexpressing Rb gene. A, differentiation of control mouse C2C12 myoblasts and stable C2C12 myoblasts overexpressing FLAG-FBI-1, by horse serum treatment. Pictures of the cells were taken at days 0, 2, and 4 after treatment with 2% horse serum differentiation medium. Differentiation into myotubes was observed only in control cells at day 4. Stable C2C12 myoblast cells overexpressing FBI-1 were differentiation into myotubes only when transfected with recombinant adenovirus overexpressing Rb gene at day 4, but not in the cells rescued with control adenovirus. Ad, recombinant adenovirus. B, Western blot analysis of the C2C12 control or C2C12-FBI-1 cell lysates using the antibodies indicated. Rb expression was increased in the control cells with concomitant decrease in FBI-1 (LRF). LRF is a mouse homologue of human FBI-1. In the stable cells, although endogenous LRF is decreased, FLAG FBI-1 is stably expressed and repressed Rb expression. Transfection with recombinant adenovirus increases Rb expression both in control and stable cells. Cell extracts (40 μg) were analyzed by Western blot analysis. GAPDH, control; Rb, retinoblastoma protein.

FBI-1 inhibits mouse C2C12 myoblast differentiation by inhibiting Rb gene expression. A, differentiation of control mouse C2C12 myoblasts and stable C2C12 myoblasts overexpressing FLAG-FBI-1, by horse serum treatment. Pictures of the cells were taken at days 0–4 after treatment with 2% horse serum differentiation medium. Differentiation into myotubes was observed only in control cells at days 3 and 4. Stable C2C12 myoblast cells overexpressing FBI-1 were prepared by transfection with either control lentri virus or recombinant Lenti-M1.4-FLAG-FBI-1 virus. B, Western blot analysis of the C2C12 cell lysates using the antibodies indicated. Rb expression was gradually increased in the control cells with concomitant decrease in FBI-1 (LRF). LRF is a mouse homologue of human FBI-1. In the stable cells, although endogenous LRF is decreased, FLAG FBI-1 is stably expressed and repressed Rb expression. Cell extracts (40 μg) were analyzed by Western blot analysis. GAPDH, control; Rb, retinoblastoma protein. C, myogenic differentiation is inhibited by FBI-1 in the stable C2C12-FLAG-FBI-1 cells. However, control cells underwent myogenic differentiation and showed multinuclear myotubes at days 3–4.

FBI-1 interacts with the co-repressors via its POZ-domain, and BCoR is recruited to the promoter by FBI-1. A, diagram of the mammalian two-hybrid assay and protein-protein interaction between the POZ-domain of FBI-1 and the co-repressors. Upstream activating sequence (UAS), binding site for Gal4DBD (B) GST fusion protein pulldown assays and structure of the co-repressors SMRT, NCoR, BCoR, and FBI-1. GST and GST-POZFBI-1 fusion proteins were incubated with in vitro synthesized [³⁵S]methionine-labeled co-repressor polypeptides and pulled down. RD, repression domain; N3-1 and S1–2, domains involved in interaction with nuclear receptors; ZF, zinc finger domain; POZ, POZ-domain of FBI-1. Domains of co-repressors used for in vitro pulldown assays are indicated below by light gray filled bars. C, co-immunoprecipitation of FBI-1 and BCoR. Cell lysates prepared from HEK293A cells transfected with FLAG-FBI-1 and Myc-BCoR expression vectors were immunoprecipitated using anti-Myc antibody and analyzed by Western blotting using anti-FLAG antibody. D, ChIP assays of BCoR and FBI-1 binding at the distal FRE elements and 3′-UTR of Rb gene. HEK293A cells were transfected with FLAG-FBI-1 and/or Myc-BCoR expression vector and immunoprecipitated with the antibodies indicated. More BCoR is bound to the FRE cluster in the presence of FBI-1. A histogram of the ChIP assays is shown on the right.

FBI-1 represses transcription of the Rb gene. A, structure of the Rb gene promoter and sequence comparison of four potential FBI-1 binding sites (FRE). Transcription factor binding sites are indicated by circles. The FREs are located in the –180 to –308 bp region. FRE; FBI-1 binding site; Tsp (+1), transcription start site. B, structures of FBI-1 and the FBI-1 with a POZ-domain deletion. FBI-1 repressed transcription of the Rb gene by more than 50% in HeLa, HCT116, and HEK 293A cells. POZ, POZ-domain; ZF, Krüppel-like zinc fingers; NLS, nuclear localization sequence. C and D, RT-PCR and Western blot analysis of Rb gene repression by FBI-1 at mRNA and protein levels in stable HeLa and HCT116 cells overexpressing FBI-1 or β-galactosidase. GAPDH, control for RT-PCR and Western blot analysis. Histogram of the qRT-PCR analysis of FBI-1 and Rb mRNA is shown below. NC, negative control siRNA. E, transient transcription assays in HEK293A cells transfected with pGL2-Rb-Luc and siRNA designed to knock down endogenous FBI-1. Mock, negative control siRNA; GAPDH, positive control siRNA against GAPDH mRNA; FBI-1#1 and #2, siRNA designed to knock down endogenous FBI-1. F and G, RT-PCR and Western blot analysis of RNA interference of endogenous FBI-1 mRNA and protein expression. Knockdown of FBI-1 mRNA by siRNA increased Rb gene expression both at the mRNA and protein levels in HEK293A cells.

FBI-1 binds to the distal FRE clusters of the endogenous Rb promoter. Characterization of the functional role of the FRE elements in transcription. A, EMSA. Four ³²P-labeled FRE1–4 oligonucleotide probes (bp –308 to –300, –298 to –290, –244 to –236, and –188 to –180) were incubated with recombinant FBI-1ZFDBD (aa 382–490) and separated on a 4% non-denaturating polyacrylamide gel. B, ChIP assays of FBI-1 binding at the FRE cluster on the endogenous Rb gene. Structure of the Rb gene promoter and locations of PCR primer sets used for ChIP assays. pcDNA3-FLAG-FBI-1 expression plasmid was transfected into HeLa cells, and chromatin was immunoprecipitated with anti-FLAG antibody or control IgG. Arrows, primers used in ChIP; Tsp (+1), transcription start site. C, EMSA. Sequence comparisons of the FRE consensus sequence and mutant FRE probes. The core GGG sequence of the FREs was substituted with AAA in mutant probes. EMSA was carried out as in A. FBI-1ZFDBD was able to bind to the wild type, but not to any of the mutant probes. D, functional characterization of FBI-1 binding to FRE elements by transient transcription analysis in HeLa cells. Also shown are the structures of the pGL2-Rb-Luc Wt and mutant plasmids tested. Open circles, FBI-1 binding sites; black circles, Sp1-binding GC-boxes. X, mutation introduced. Mutations of the FREs increased transcription of Rb gene by 20–130% compared with the Wt promoter. E, Western blot analysis of Sp1 and FBI-1 in the HeLa cells transfected and described in D.