Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Sep;20(17):6201-11.
doi: 10.1128/MCB.20.17.6201-6211.2000.

BF-1 interferes with transforming growth factor beta signaling by associating with Smad partners

C Dou  1 , J Lee, B Liu, F Liu, J Massague, S Xuan, E Lai
Affiliations

BF-1 interferes with transforming growth factor beta signaling by associating with Smad partners

C Dou et al. Mol Cell Biol. 2000 Sep.

Abstract

The winged-helix (WH) BF-1 gene, which encodes brain factor 1 (BF-1) (also known as foxg1), is essential for the proliferation of the progenitor cells of the cerebral cortex. Here we show that BF-1-deficient telencephalic progenitor cells are more apt to leave the cell cycle in response to transforming growth factor beta (TGF-beta) and activin. We found that ectopic expression of BF-1 in vitro inhibits TGF-beta mediated growth inhibition and transcriptional activation. Surprisingly, we found that the ability of BF-1 to function as a TGF-beta antagonist does not require its DNA binding activity. Therefore, we investigated whether BF-1 can inhibit Smad-dependent transcriptional responses by interacting with Smads or Smad binding partners. We found that BF-1 does not interact with Smads. Because the identities of the Smad partners mediating growth inhibition by TGF-beta are not clearly established, we examined a model reporter system which is known to be activated by activin and TGF-beta through Smads and the WH factor FAST-2. We demonstrate that BF-1 associates with FAST-2. This interaction is dependent on the same region of protein which mediates its ability to interfere with the antiproliferative activity of TGF-beta and with TGF-beta-dependent transcriptional activation. Furthermore, the interaction of FAST-2 with BF-1 is mediated by the same domain which is required for FAST-2 to interact with Smad2. We propose a model in which BF-1 interferes with transcriptional responses to TGF-beta by interacting with FAST-2 or with other DNA binding proteins which function as Smad2 partners and which have a common mode of interaction with Smad2.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Telencephalic neuroepithelial cells from BF-1 homozygous mutant embryos are more sensitive to growth inhibition by TGF-β. (A) Neuroepithelial cells isolated from E10.75 BF-1+/− heterozygous and BF-1−/− mutant embryos were cultured for 24 h and stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) and nuclear fast red. Cells staining blue are telencephalic neuroepithelial cells with activated BF-1 promoter. Strong and weak staining indicates cells with high and low BF-1 promoter activity, respectively. Bar, 25 μm. (B) RT-PCR for TGF-β receptor type I and II and activin receptor type IIB from RNA isolated from neuroepithelial cells. (C) Neuroepithelial cells isolated from WT, BF-1+/− and BF-1−/− embryos were cultured in the presence or absence of TGF-β or BMP4 (both at 100 pM) for 24 h. Control medium included 1% FBS and 20 ng of FGF-2 per ml. [3H]thymidine was added during the last 6 h, and the amount of [3H]thymidine incorporated was determined as described in Materials and Methods. The [3H]thymidine counts from duplicate wells of a representative experiment were normalized to the cell number. (D) The inhibition of [3H]thymidine incorporation by TGF-β, activin, or BMP4 (mean ± standard error) is reported.
FIG. 2
FIG. 2
(A) BF-1 blunts the growth-inhitory activity of TGF-β. (Inset) Western blot analysis with anti-BF-1 antibody. BF-1 is expressed in clone 8 (lane 2) upon tetracycline withdrawal but not in clone 7 (lane 4). Exponentially growing cells not expressing BF-1 are growth inhibited by TGF-β. Induction of BF-1 (clone 8, −Tet) results in a reduced response to TGF-β. [3H]thymidine incorporation in cells exposed to TGF-β is expressed as a percentage of that in cells not exposed to TGF-β. (B) [3H]thymidine incorporation in cells released from contact inhibition in the presence or absence of TGF-β is shown in the top panel. Cells were cultured for 5 days at contact density to achieve quiescence. BF-1 expression was induced by withdrawal of tetracycline for 48 h prior to replating cells with or without TGF-β (100 pM) for 15 h. When BF-1 is expressed, [3H]thymidine incorporation is increased 10-fold (compare lanes 2 and 4). Hyperphosphorylation of Rb in BF-1-expressing cells treated with TGF-β is shown in the middle panel. Western blot analysis with anti-Rb antibody was performed to determine the amount of hyperphosphorylated (Rb-P) and hypophosphorylated (Rb) forms of Rb in these cells. BF-1 overcomes inhibition of Rb phosphorylation by TGF-β (lanes 2 and 4); an RNase protection assay for p15ink4b is shown in the bottom panel. Induction of p15ink4b by TGF-β is reduced in the presence of BF-1 (lane 4). (C) A 2-amino-acid substitution in the WH domain of BF-1 disrupts DNA binding activity. The DNA binding domain (WH domain) of BF-1 or the WH domain with the NH-AA mutation was expressed by translation in reticulocyte lysates. A gel mobility shift assay demonstrates high-affinity binding of the WT BF-1 protein to the S2 double-stranded oligonucleotide (lane 1). This binding activity is abolished by the NH-AA mutation (lane 2). (D) BF-1(NH-AA) antagonizes TGF-β-mediated cell cycle arrest (top panel). Cells cultured with tetracycline to repress BF-1(NH-AA) are inhibited from reentering S phase by TGF-β upon replating at low density (lanes 1 and 2). When BF-1(NH-AA) expression is induced, TGF-β is unable to inhibit [3H]thymidine incorporation. BF-1(NH-AA) inhibits the induction of p15ink4b expression by TGF-β (bottom panel). An RNase protection assay for p15ink4b expression is shown. TGF-β induction of p15ink4b expression is reduced in the presence of BF-1(NH-AA) (compare lanes 2 and 4).
FIG. 3
FIG. 3
Transcriptional activation by TGF-β is inhibited by BF-1. (A) The A3-luc reporter construct, Rous sarcoma virus (RSV)–β-gal, and Myc-FAST-2 were cotransfected into Mv1Lu cells with or without BF-1 expression vector. The cells were treated with 100 pM TGF-β for 24 h before being harvested for luciferase and β-gal assays. Luciferase activity was normalized to cotransfected RSV-β-gal expression. The mean and standard error from duplicate wells is plotted. Very little luciferase activity is detected in the absence of FAST-2 (lane 1). FAST-2 causes a 40-fold transcriptional activation by TGF-β (lane 2). Increasing amounts of cotransfected BF-1 (50 and 100 ng) inhibit FAST-2-mediated A3 luc reporter expression (lanes 3 and 4). In the lower panel, cell lysates from the same experiment were subjected to Western blotting with anti-Myc antibody to monitor the expression levels of FAST-2. (B) BF-1 does not inhibit VD-luc reporter expression. VD-luc reporter, RSV-β-gal, and VD receptor were cotransfected into Mv1Lu cells in the presence or absence of BF-1. The cells were treated with VD (10 nM) or left untreated for 24 h before being harvested. Data were analyzed and plotted in the same way as described for panel A. (C) Inhibition of TGF-β- and FAST-2-dependent activation of A3-luc by levels of BF-1 which do not interfere with cell proliferation (see also Fig. 2B). Clone 8 (Fig. 2A) Mv1Lu cells were cultured with tetracycline (lanes 1, 3, and 4) or without tetracycline to induce BF-1 expression (lane 2). Inhibition by induced BF-1 is comparable to that achieved by cotransfection with an expression plasmid for WT BF-1 (lane 3) or BF-1(NH-AA) (lane 4).
FIG. 4
FIG. 4
BF-1 associates with FAST-2. (A) Comparison of the association between FAST-2 and BF-1 with the association of FAST-2 and Smad-2. Flag-tagged BF-1, Flag-tagged Smad2, and Myc-tagged FAST-2 were transfected into COS cells as indicated. BF-1 and Smad2 coimmunoprecipitate with FAST-2 with similar efficiencies in the absence of TGF-β. TGF-β treatment reduces the association of FAST-2 with BF-1 and enhances its association with Smad2. (Top panel) Cell lysates were immunoprecipitated with anti-Flag antibody and blotted with anti-Myc antibody. (Middle panel) Western blot of cell lysates with anti-Myc antibody. (Bottom panel) Western blot of cell lysates with anti-Flag antibody. (B) Flag-tagged BF-1 was transfected into COS1 cells alone or with several different Myc-tagged FAST-2 constructs. (Top panel) Western blot of cell lysates immunoprecipitated with anti-Myc antibody and probed with anti-FLAG antibody. (Middle panel) Western blot of cell lysates with anti-Flag antibody. (Bottom panel) Western blot of cell lysates with anti-Myc antibody. (C) Requirement of amino acids 314 to 372 in BF-1 for antagonism of TGF-β activity and association with FAST-2. Myc-tagged FAST-2 alone or with different Flag-tagged BF-1 constructs was transfected into COS1 cells. (Top panel) Western blot of cell lysate immunoprecipitated with anti-Flag antibody and probed with anti-Myc antibody. (Middle panel) Western blot of cell lysates with anti-Myc antibody. (Bottom panel) Western blot of cell lysates with anti-Flag antibody.
FIG. 5
FIG. 5
Four Mv1Lu cell lines with inducible expression of different Flag-tagged BF-1 constructs (A) were used in growth inhibition (B) and A3-luc reporter (C) assays. (A) Expression of Flag-tagged BF-1 (F-BF-1) (lanes 1 and 2) and three deletion mutants of BF-1 (F-Δ373–480 [lanes 3 and 4], F-Δ276–372 [lanes 5 and 6], and F-Δ1–149 [lanes 7 and 8]) was induced by tetracycline withdrawal and monitored by Western blotting. (B) Inhibition of [3H]thymidine incorporation by TGF-β is antagonized by Flag-tagged BF-1 and F-Δ1–149 (lanes 2 and 8) and partially antagonized by F-Δ373–480 (lane 4). F-Δ276–372 is inactive in this assay (lane 6). (C) Activation of the A3-luc reporter by TGF-β in Mv1Lu cells cotransfected with FAST-2 is inhibited by Flag-tagged BF-1 and F-Δ1–149 (lanes 2 and 8) and partially inhibited by F-Δ373–480 (lane 4). F-Δ276–372 is inactive in this assay (lane 6).
FIG. 6
FIG. 6
Schematic diagram of BF-1 constructs used in the experiments in Fig. 4 and 5B and C. The relative efficiency of each construct in coimmunoprecipitating FAST-2 is compared with their ability to inhibit TGF-β-stimulated A3 luc reporter expression and to antagonize the growth-inhibitory activity of TGF-β. ++, strong activity; +, low to moderate activity; −, no activity; nd, not determined. The asterisk indicates that the BF-1(NH-AA) protein in this stable cell line is not Flag tagged.
FIG. 7
FIG. 7
BF-1 interferes with the formation of the FAST-2–Smad2 complex. (A) Interference by BF-1Δ1–119. (Top panel) Flag blot of proteins immunoprecipitated with Myc antibody. Flag-tagged Smad2 (F-Smad2) coimmunoprecipitates with Myc-lagged FAST-2 when cotransfected into COS cells. Formation of this complex is enhanced by TGF-β. Coexpression of Myc-tagged BF-1Δ1–119 reduces the amount of Flag-tagged Smad2 which is coimmunoprecipitated with Myc-tagged FAST-2. (Middle and bottom panels) Western blots of cell lysates indicate the relative amounts of Flag-tagged Smad2 (middle) and Myc-tagged FAST-2 or Myc-tagged BF-1Δ1–119 (bottom). (B) Interference by BF-1. (Top panel) Myc blot of proteins immunoprecipitated with Flag antibody. Coexpression of Myc-tagged BF-1 reduces the amount of Flag-tagged Smad2 which is coimmunoprecipitated with Myc-tagged FAST-2. (Middle and bottom panels) Western blots of cell lysates indicate the relative amounts of Myc-tagged FAST-2 or Myc-tagged BF-1 (middle) and Flag-tagged Smad2 (bottom).
FIG. 8
FIG. 8
BF-1 interferes with multiple TGF-β responses by interference with FAST-2 and other Smad2 partners. (A) In this model, we propose that BF-1 associates with FAST-2 through a specific motif within the Smad interaction domain of the FAST-2 protein to inhibit TGF-β- and FAST-2-dependent transcriptional activation from the A3-luc reporter gene. ARE, activin response element. (B) BF-1 also interferes with other TGF-β responses such as the transcriptional activation of the p15 gene. The specific Smad2 partner for this and other responses remains to be established. We postulate that BF-1 will interact with a subset of these DNA binding partners, i.e., those that share with FAST-2 a mode of interaction with Smad2. (C) This model also predicts that other DNA binding partners which interact with Smad2 through a distinct mechanism will not be susceptible to interference by BF-1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Catron K, Zhang H, Marshall S, Inostroza J, Wilson J, Abate C. Transcriptional repression by msx-1 does not require homeodomain DNA-binding sites. Mol Cell Biol. 1995;15:861–871. - PMC - PubMed
    1. Caviness V J, Takahashi T, Nowakowski R. Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model. Trends Neurosci. 1995;18:379–383. - PubMed
    1. Chen X, Rubock M, Whitman M. A transcriptional partner for MAD proteins in TGF-B signalling. Nature. 1996;383:691–696. - PubMed
    1. Chen X, Weisberg E, Fridmacher V, Watanabe M, Naco G, Whitman M. Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature. 1997;389:85–89. - PubMed
    1. Clark K, Halay E, Lai E, Burley S. Cocrystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature. 1993;364:412–420. - PubMed

Publication types

MeSH terms

LinkOut - more resources