Direct regulation of rRNA transcription by fibroblast growth factor 2

Abstract

Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to approximately 34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may directly regulate ribosome biogenesis, a rate-limiting process in cell growth. Although several growth factors have been shown to accumulate in the nucleolus, their function and mechanism of action remain unclear. Here we show that 18-kDa FGF-2 interacts with upstream binding factor (UBF), an architectural transcription factor essential for rRNA transcription. The maximal activation of rRNA transcription in vitro by 18-kDa FGF-2 requires UBF. The 18-kDa FGF-2 localizes to rRNA genes and is necessary for the full activation of pre-rRNA synthesis in vivo. Our results demonstrate that 18-kDa FGF-2 directly regulates rRNA transcription.

FIG. 1.

Identification of FGF-2 interacting proteins. (a) Proteins in nuclear extracts of HeLa cells that bound to GST (lane 3), GST-FGF-2 (lane 5), or GST-FGF-2 in the presence of a 10-fold excess of hrFGF-2 (lane 6) were separated on a SDS-PAGE gel and stained with Coomassie blue. HeLa cell lysate (lane 1), purified GST (lane 2), and GST-FGF-2 (lane 4) were also separated on the gel. Numbers in lane 5 correspond to proteins bound specifically to FGF-2. (b) Proteins in lanes 1 to 6 of panel a were transferred to a polyvinylidene difluoride membrane and probed with a UBF antibody. Three bands (lane 1) in the HeLa cell lysate likely correspond to UBF1, UBF2, and phosphorylated UBF (35). (c) Purified FLAG-UBF was mixed with either GST (lane 2) or GST-FGF-2 (lane 3). Bound proteins were eluted using glutathione and then detected on immunoblots by a FLAG antibody (anti-FLAG panel) or an FGF-2 antibody (anti-FGF-2 panel). Inputs (10% of total) were loaded in lane 1.

FIG. 2.

FGF-2 interacts with UBF in vivo. (a) Confocal images of HeLa cells expressing GFP (panels a through f) or GFP-FGF-2 (panels g through l) and incubated with actinomycin D (Act D) (panels d through f and j through l) or without actinomycin D (panels a through c and g through i). GFP and GFP-FGF-2 were localized by autofluorescence (panels a, d, g, and j). UBF was detected by using a UBF monoclonal antibody and a Texas Red conjugated secondary antibody (panels b, e, h, and k). Merged images are shown in panels c, f, i, and l. (b) COS-7 cells were cotransfected with p3XFLAG-CMV-14 and pEGFP-N1 (lanes 1 and 2), pFGF-2-3XFLAG-CMV-14 and pEGFP-N1 (lanes 3 and 4), p3XFLAG-CMV-14 and pEGFP-C1-UBF (lanes 5 and 6), or pFGF-2-3XFLAG-CMV-14 and pEGFP-C1-UBF (lanes 7 and 8), and then proteins were immunoprecipitated (IP) with GFP (lanes 1, 3, 5, and 7) or FLAG (lanes 2, 4, 6, and 8) antibodies. Immunoprecipitated proteins were identified using Western blots (WB) probed with FLAG, FGF-2, UBF, or GFP antibodies. The position of GFP-UBF is indicated by arrows. Asterisks in lane 2, 4, 6, and 8 indicate background bands.

FIG. 3.

FGF-2 stimulates rRNA transcription in vitro. (a) The amount of rRNA transcription in the presence of the indicated combinations of purified proteins was measured using S1 nuclease protection. (b) Band intensities in panel a were quantified and normalized to the amount of rRNA transcription in the presence of RNA Pol I and SL1 (lane 1). Bars represent the standard errors of four determinations. Relative to lane 1, P values of lanes 2 to 8 were 0.07, 0.0009, 0.05, 0.0004, 0.005, 0.09, and 0.2, respectively.

FIG. 4.

FGF-2 enhances pre-rRNA synthesis in HEK293 cells. (a) The amount of pre-rRNA in HEK293 cells transfected with pN1 (lane 1), pN1-FGF-2 (lane 2), pN1-FGF-2-K128G (lane 3), pcDNA3-FLAG-UBF (lane 4), pN1-FGF-2 and pcDNA3-FLAG-UBF (lane 5), and pN1-FGF-2-K128G and pcDNA3-FLAG-UBF (lane 6) was determined using S1 nuclease protection and normalized to the protected oligonucleotide in lane 1. The loading controls (18S and 28S RNA) were visualized using ethidium bromide. (b) The protected oligonucleotides in panel c were quantified, and band intensities were normalized to the amount of pre-rRNA in cells transfected with control plasmid (lane 1). Bars represent the standard error of four determinations. Relative to lane 1, P values of lanes 2 to 6 were 0.02, 0.01, 0.07, 0.006, and 0.007, respectively. (c) HEK293 cells were transfected with control plasmid pN1 (lane 1), pN1-FGF-2 (lane 2), or pN1-FGF-2-K128G (lane 3), and then extracts were immunoblotted using antibodies against either FGF-2, phospho-ERK1/ERK2 (T202/Y204), or actin.

FIG. 5.

FGF-2 localizes to rRNA genes. (a) A human rRNA gene repeat contains 18S, 5.8S, and 28S units and the ETS and IGS. The regions targeted in PCR analyses are also indicated. (b) Control PCRs contained the total chromatin (lane 2) or water (lane 1). PCRs of immunoprecipitates using the indicated antibodies are shown in lanes 3 to 7. (c) PCRs of immunoprecipitates from nucleolar chromatin using the indicated antibodies are shown in lanes 1 to 3.