Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN)

Abstract

FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

FIGURE 1.

The expression of FBI-1 mRNA is increased in genetically obese ob/ob C57BL/6J mice and DIO mice. The stable cells overexpressing FBI-1 or rapid growing LNCaP cells showed increased FASN expression. A, RT-PCR analysis of FBI-1 mRNA using the total RNA was isolated from the abdominal adipose tissues of lean control and obese ob/ob C57BL/6J mice. GAPDH, control. LRF is a murine counterpart of human FBI-1 (also called Pokemon, ZBTB7A). B, RT-PCR analysis of mRNA of LRF (FBI-1) and SREBP-1 using the total RNA isolated from the white adipose tissues (WAT) of control and DIO C57BL/6J mice. Norm, normal; GAPDH, control. C, RT-PCR and Western blot analysis of FBI-1, SREBP-1, and GAPDH in HEK293Trex control and stable cells overexpressing FBI-1. The stable cells were induced to express lacZ or FBI-1 gene by doxycycline treatment. Total RNA or protein was isolated from the cells and analyzed for FBI-1, SREBP-1, and FASN expression at both the mRNA and protein level. GAPDH, control. D, Western blot analysis of human colon cancer stable HCT116 cells established by transfection with lentivirus overexpressing either FBI-1 or lacZ. Cell extracts were analyzed for FBI-1, SREBP-1, and FASN proteins. GAPDH, control. E, RT-PCR and Western blot analysis of LNCaP cells treated with androgen (Andro) or EtOH. Total RNA or protein was isolated from the cells and analyzed for FBI-1, SREBP-1, and FASN expression at both the mRNA and protein level. GAPDH, control. F, Western blot analysis. Knockdown of endogenous FBI-1 decreases FASN gene expression in immortalized HEK293A and HCT116 cancer cells. The cells were transfected with two independent siRNA against FBI-1 or a control siRNA. The cells were harvested at 48 h, and the cellular extracts (35 μg) were analyzed for FASN, FBI-1, SREBP-1a, and GAPDH expression. NC, negative control.

FIGURE 2.

FBI-1 and SREBP-1a synergistically activate transcription of the pGL2-FASN-Luc via GC-box and SRE/E-box elements in immortalized or cancer cells. A, nucleotide sequence comparison of three mammalian FASN gene promoters. SRE, upstream SREBP-binding element; GC-box, Sp1-binding GC box; SRE/E-box, downstream SREBP-binding element; TRE, thyroid hormone-response element; TATA, TBP-binding element; Luc, luciferase. Numbers indicate the position of nucleotide upstream of transcription initiation (+1). B, structures of three FASN-Luc reporter promoter constructs tested to investigate the functional elements mediating transcription activation by FBI-1. Tsp, transcription start site (+1). C-F, FBI-1 and SREBP-1a synergistically activate transcription of the pGL2-FASN-Luc via GC-box and SRE/E-box elements in the mammalian cells co-transfected with three different pGL2-FASN-Luc plasmids and expression vectors of SREBP-1a (25 ng) and FBI-1 (25 ng, 125 ng, 500 ng). FBI-1 could not activate transcription of the pGL2-FASN3-luc lacking downstream SRE/E-box.

FIGURE 3.

FBI-1 and SREBP-1a or -1c synergistically activate transcription of pGL2-6x(SRE)-tk^*-Luc in immortalized or cancer cells. A, domain structures of SREBPs and FBI-1. SREBP-1c and SREBP-1a are identical except N-terminal acidic domain. The bHLH domain are similar among SREBP-1a, -1c, and -2. Acidic, acidic domain; Pro/Ser, Pro/Ser-rich domain; Ser/Gly/Pro, Gly/Pro/Ser-rich-domain; Gln, Gln-rich domain; bHLH, basic helix-loop-helix domain; POZ, POZ domain; ZF, Krüppel-like zinc fingers; NLS, nuclear localization sequence. B, structure of pGL2-6x [SRE]-tk^*-Luc plasmid. tk^* indicates the thymidine kinase minimal promoter with the mutations at the two Sp1-binding sites. C-F, FBI-1 enhances transcription activation of the pGL2-6x [SRE]-tk^*-Luc by SREBP-1 in the mammalian cells co-transfected with pGL2-6x [SRE]-tk^*-Luc plasmid and expression vectors of SREBP-1 (25 ng) and FBI-1 (25, 125, and 500 ng). Luciferase activities were normalized with the protein concentration, and data presented are the average of three independent assays. Bars represent standard deviations. ▪, SREBP-1a; □, SREBP-1c.

FIGURE 4.

The FBI-1 interacts with SREBP-1a in immortalized human HEK293A and HCT116 colon cancer cells. A, Western blot (WB) analysis of co-immunoprecipitates of endogenous FBI-1 and SREBP-1. The HEK293A cell lysates (500 μg) were immunoprecipitated (IP) with anti-FBI-1 antibody and analyzed by Western blot using an antibody against SREBP-1a. The same extracts were also immunoprecipitated with anti-SREBP-1a antibody and analyzed by Western blot using an anti-FBI-1 antibody. B, Western blot analysis of the co-immunoprecipitates of endogenous FBI-1 and SREBP-1a in HCT116 cells. The procedures are the same as above. C, immunocytochemistry and cellular co-localization of SREBP-1a and FBI-1. The Alexander cells transfected with the expression vectors of FLAG-FBI-1 and His-tagged SREBP-1a were analyzed by immunocytochemical staining using both mouse anti-FLAG antibody and rabbit anti-His antibody. Fluorescein isothiocyanate-conjugated anti-mouse IgG or rhodamine-conjugated anti-rabbit IgG antibody were used as secondary antibodies.

FIGURE 5.

The ZFDBD of FBI-1 interacts with the bHLH domain of SREBP-1a directly in vitro, and the ZFDBD of FBI-1 is sufficient to activate transcription of SREBP-1-responsive promoter, pGL2-6x(SRE)-tk^*-Luc, by SREBP-1a. A, structures of in vitro translated [³⁵S]methionine-labeled FBI-1 fragments and recombinant GST fusion bHLHSREBP-1a used for pulldown assay. bHLH, basic helix-loop-helix domain (aa 323-394); POZ, POZ domain; ZF, Krüppel-like zinc fingers; NLS, nuclear localization sequence. B, GST fusion protein pulldown assays. Recombinant GST and GST-bHLHSREBP-1 fusion proteins were incubated with the in vitro synthesized [³⁵S]methionine-labeled FBI-1 polypeptides and then were pulled down. The precipitated samples were resolved by 12% SDS-PAGE and exposed to x-ray film. Input, 10% of the labeled polypeptides added in the binding reactions. C, FBI-1 activates transcription of pGL2-6x(SRE)-tk^*-Luc by SREBP-1a. The ZFDBD of FBI-1 is sufficient to activate transcription. Alexander or HEK 293A cells were co-transfected with mixtures of pGL2-6x(SRE)-tk^*-Luc and the expression vectors of SREBP-1a and FBI-1 full-length or the ZFDBD of FBI-1 in various combinations. Luciferase activities were normalized with the protein concentration, and data presented are the average of three independent assays. Bars represent standard deviations. ▪, Alexander cells; □, 293A cells.

FIGURE 6.

FBI-1 and SREBP-1a activate transcription of pGL2-FASN-Luc (-2.7 kb) via the SRE/E-box elements in immortalized or cancer cells. A, structures of pGL3-FASN-Luc WT or Mt constructs tested to investigate the functional role of SRE/E-box in transcription activation by FBI-1. SRE, SREBP-binding element; GC, Sp1-binding GC box; SRE/E-box, downstream SREBP-binding element; Tsp, transcription start site (+1). In the mutant FASN constructs, two SREBP-binding nucleotide sequences of SRE/E-box were mutated by site-directed mutagenesis. B-F, five mammalian cells were co-transfected with pGL3-FASN-Luc WT or Mt (-2.7 kb) and expression vectors of SREBP-1 (25 ng) and FBI-1 (25, 125, and 375 ng) in various combination indicated. Luciferase activities were normalized with the protein concentration, and data presented are the average of three independent assays. Bars represent standard deviations.

FIGURE 7.

EMSA of the proximal promoter elements, SRE, GC-box, and SRE/E-box of FASN gene. A, EMSA of the GC-box probe. The probes were incubated with recombinant SREBP-1 (200 ng), Sp1 (70 ng), and GST-FBI-1 ZFDBD (150 ng). B, EMSA of the SRE/E-box probe. The probes were incubated with recombinant SREBP-1 (20 ng), Sp1 (200 ng), and GST-FBI-1 ZFDBD (150 ng). C and D, EMSA of the GC-box and SRE/E-box probe. Cold probe competition. E, EMSA. Effect of excess SREBP-1a and FBI-1 proteins on Sp1 binding to the GC-box probe. The probes were incubated with recombinant Sp1 (50 ng), in the presence of increasing amount of SREBP-1 (200, 600, and 1800 ng) or GST-FBI-1 ZFDBD (150, 450, and 1350 ng). F, EMSA. Effect of excess Sp1 and FBI-1 proteins on SREBP-1a binding to the SRE/E-box probe. The probes were incubated with recombinant SREBP-1 (20 ng), in the presence of increasing amount of Sp1 (200, 600, and 1800 ng), and GST-FBI-1 ZFDBD (150, 450, and 1350 ng). Arrowheads (▸), retarded protein-probe complexes; arrowheads with star, supershifted probe-protein-antibody complex. All three recombinant proteins bind to the probes but with different affinity.

FIGURE 8.

ChIP assays and the molecular binding interaction among FBI-1, SREBP-1, Sp1, and proximal FASN promoter. A, structures of the pGL2-FASN-Luc reporter promoter constructs tested to study in vivo transcription factor binding by SREBP-1, FBI-1, and Sp1. B, ChIP assays of the HEK293A cells transfected with either pGL2-FASN2-Luc or pGL2-FASN3-Luc. C, ChIP assays of the HEK293A cells transfected with pGL3-FASN-Luc WT or Mt (-2.7 kb) plasmid. The HEK293A cells were co-transfected with FASN promoter reporter fusion plasmid, pcDNA3.1-SREBP-1a and/or pcDNA3-FLAG-FBI-1. Sheared chromatin was immunoprecipitated (IP) using antibodies indicated. PCR amplification primers were designed to bind to the plasmid vector sequences, not to the endogenous FASN gene promoter in case of FASN2 and -3. SRE, SREBP-binding element; GC, Sp1-binding GC box; SRE/E-box, downstream SREBP-binding element; Tsp, transcription start site (+1). D, histogram showing the average of independent ChIP assays shown in C. ChIP binding band intensities by SREBP-1, FBI-1 and Sp1, were divided by input band intensities.

FIGURE 9.

Hypothetical model on the transcription and changes in binding dynamic of three transcription factors (FBI-1, Sp1, SREBP-1) on the SRE, GC-box, and SRE/E-box on the proximal promoter of FASN gene. A, in the control HEK293A cells where Sp1 is abundant and SREBP-1 is uninduced and low, the promoter elements are mainly bound by Sp1, and transcription is at basal level. No FBI-1 binding is detected. B, upon induction of SREBP-1, Sp1 binding is significantly reduced, and SREBP-1 binding is high, which resulted in potent transcription activation. C, upon introduction of FBI-1, Sp1 and SREBP-1 binding are reduced, and FBI-1 binding is high. The binding pattern with respect to Sp1 and SREBP-1 is similar to that of the control, although at low level. Transcription is at basal level. D, synergistic activation of FASN gene transcription by FBI-1 and SREBP-1. In the presence of high FBI-1 and SREBP-1, moderate level of SREBP-1 binding and high Sp1 binding close to the control is achieved. This is probably because of protein interaction between SREBP-1 and FBI-1, which significantly affects the DNA binding activity of the other protein. Binding by both Sp1 and SREBP-1 of the promoter in a way that favors more Sp1 over SREBP-1 around the GC-box and the SRE/E-box is important in synergistic activation of transcription. Transcription is mainly controlled by the molecular interactions among SRE/E-box, GC-box, FBI-1, Sp1, and SREBP-1. Molecular interaction on the upstream SRE is also expected but has no significant effect on transcription activation by FBI-1. Arrows, DNA-protein interactions. Thickness of the arrow indicated relative binding activity. Tsp (+1), transcription start site.