A functional screen in human cells identifies UBF2 as an RNA polymerase II transcription factor that enhances the beta-catenin signaling pathway

Abstract

beta-Catenin signaling plays an important role in the development of many organisms and has a key part in driving the malignant transformation of epithelial cells comprising a variety of cancers. beta-Catenin can activate gene expression through its association with transcription factors of the lymphoid enhancer factor 1 (LEF-1)/T-cell factor (TCF) family. We designed a screen in human cells to identify novel genes that activate a beta-catenin-LEF/TCF-responsive promoter and isolated the high-mobility group box transcription factor, UBF2. UBF1 and UBF2 are splice variants of a common precursor RNA. Although UBF1 has been shown to activate RNA polymerase I-regulated genes, the function of UBF2 has remained obscure. Here, we show for the first time that both UBF1 and UBF2 activate RNA polymerase II-regulated promoters. UBF2 associates with LEF-1, as shown by coimmunoprecipitation experiments, and potentiates transcriptional activation stimulated by LEF-1/beta-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites. Downregulation of endogenous UBF expression using an RNA interference approach reduces transcriptional activation of a beta-catenin-LEF/TCF-responsive promoter by means of overexpressed beta-catenin, further implicating UBF as a transcriptional enhancer of the beta-catenin pathway.

FIG. 1.

(A) Overview of strategy and process for a mammalian functional screen. Six tandem copies of a LEF/TCF response element (6× CCTTTGATC) were positioned upstream of an IL-2 minimal promoter to drive expression of the reporter GFP gene. The reporter construct was transfected into HT1080 human fibrosarcoma cells, and a stable cell line (REP) was selected. The REP cell lines were further modified by transfection with an expression vector encoding LEF-1 followed by selection of a stable cell line (REP-LEF). The reporter gene is inactive in REP and REP-LEF cells. The REP-LEF cells were used to screen a plasmid-based cDNA library prepared from human tumor cells, GFP-expressing cells were selected by FACS. (B) Reporter gene evaluation by transient transfection with test plasmids. HT1080 cells were cotransfected with the wt reporter gene (wt 6×TCF-IL-2-GFP) (bars 1 to 14) or a reporter gene with mutations in the LEF/TCF binding site (mu 6×TCF-IL-2-GFP) (bars 7a to 10a) and either an empty expression vector (Control Vector) (bars 1, 7, 11, and 7a) or an expression vector encoding β-catenin (wt β-catenin) (bars 2, 8, 12, and 8a), an activated mutant form of β-catenin (ΔN89β-catenin) (bars 3, 9, 13, and 9a), Wnt-1 (bars 4, 10, 14, and 10a), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10 and 7a to 10a, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS. The error bars indicate standard deviations. (C) Confirmation of protein expression. Protein extracts were prepared from the transfected cells evaluated in panel B and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of each protein. LEF-1 comigrates with a background band. (D) Evaluation of the REP cell line, containing a stable integration of the reporter gene, with test vectors. REP cells were transfected with either an empty expression vector (Control Vector) (bars 1, 7, and 11) or expression vectors encoding wt β-catenin (bars 2, 8, and 12), an activated mutant form of β-catenin (ΔN89 β-catenin) (bars 3, 9, and 13), Wnt-1 (bars 4, 10, and 14), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS.

FIG. 1.

(A) Overview of strategy and process for a mammalian functional screen. Six tandem copies of a LEF/TCF response element (6× CCTTTGATC) were positioned upstream of an IL-2 minimal promoter to drive expression of the reporter GFP gene. The reporter construct was transfected into HT1080 human fibrosarcoma cells, and a stable cell line (REP) was selected. The REP cell lines were further modified by transfection with an expression vector encoding LEF-1 followed by selection of a stable cell line (REP-LEF). The reporter gene is inactive in REP and REP-LEF cells. The REP-LEF cells were used to screen a plasmid-based cDNA library prepared from human tumor cells, GFP-expressing cells were selected by FACS. (B) Reporter gene evaluation by transient transfection with test plasmids. HT1080 cells were cotransfected with the wt reporter gene (wt 6×TCF-IL-2-GFP) (bars 1 to 14) or a reporter gene with mutations in the LEF/TCF binding site (mu 6×TCF-IL-2-GFP) (bars 7a to 10a) and either an empty expression vector (Control Vector) (bars 1, 7, 11, and 7a) or an expression vector encoding β-catenin (wt β-catenin) (bars 2, 8, 12, and 8a), an activated mutant form of β-catenin (ΔN89β-catenin) (bars 3, 9, 13, and 9a), Wnt-1 (bars 4, 10, 14, and 10a), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10 and 7a to 10a, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS. The error bars indicate standard deviations. (C) Confirmation of protein expression. Protein extracts were prepared from the transfected cells evaluated in panel B and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of each protein. LEF-1 comigrates with a background band. (D) Evaluation of the REP cell line, containing a stable integration of the reporter gene, with test vectors. REP cells were transfected with either an empty expression vector (Control Vector) (bars 1, 7, and 11) or expression vectors encoding wt β-catenin (bars 2, 8, and 12), an activated mutant form of β-catenin (ΔN89 β-catenin) (bars 3, 9, and 13), Wnt-1 (bars 4, 10, and 14), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS.

FIG. 1.

(A) Overview of strategy and process for a mammalian functional screen. Six tandem copies of a LEF/TCF response element (6× CCTTTGATC) were positioned upstream of an IL-2 minimal promoter to drive expression of the reporter GFP gene. The reporter construct was transfected into HT1080 human fibrosarcoma cells, and a stable cell line (REP) was selected. The REP cell lines were further modified by transfection with an expression vector encoding LEF-1 followed by selection of a stable cell line (REP-LEF). The reporter gene is inactive in REP and REP-LEF cells. The REP-LEF cells were used to screen a plasmid-based cDNA library prepared from human tumor cells, GFP-expressing cells were selected by FACS. (B) Reporter gene evaluation by transient transfection with test plasmids. HT1080 cells were cotransfected with the wt reporter gene (wt 6×TCF-IL-2-GFP) (bars 1 to 14) or a reporter gene with mutations in the LEF/TCF binding site (mu 6×TCF-IL-2-GFP) (bars 7a to 10a) and either an empty expression vector (Control Vector) (bars 1, 7, 11, and 7a) or an expression vector encoding β-catenin (wt β-catenin) (bars 2, 8, 12, and 8a), an activated mutant form of β-catenin (ΔN89β-catenin) (bars 3, 9, 13, and 9a), Wnt-1 (bars 4, 10, 14, and 10a), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10 and 7a to 10a, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS. The error bars indicate standard deviations. (C) Confirmation of protein expression. Protein extracts were prepared from the transfected cells evaluated in panel B and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of each protein. LEF-1 comigrates with a background band. (D) Evaluation of the REP cell line, containing a stable integration of the reporter gene, with test vectors. REP cells were transfected with either an empty expression vector (Control Vector) (bars 1, 7, and 11) or expression vectors encoding wt β-catenin (bars 2, 8, and 12), an activated mutant form of β-catenin (ΔN89 β-catenin) (bars 3, 9, and 13), Wnt-1 (bars 4, 10, and 14), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS.

FIG. 1.

(A) Overview of strategy and process for a mammalian functional screen. Six tandem copies of a LEF/TCF response element (6× CCTTTGATC) were positioned upstream of an IL-2 minimal promoter to drive expression of the reporter GFP gene. The reporter construct was transfected into HT1080 human fibrosarcoma cells, and a stable cell line (REP) was selected. The REP cell lines were further modified by transfection with an expression vector encoding LEF-1 followed by selection of a stable cell line (REP-LEF). The reporter gene is inactive in REP and REP-LEF cells. The REP-LEF cells were used to screen a plasmid-based cDNA library prepared from human tumor cells, GFP-expressing cells were selected by FACS. (B) Reporter gene evaluation by transient transfection with test plasmids. HT1080 cells were cotransfected with the wt reporter gene (wt 6×TCF-IL-2-GFP) (bars 1 to 14) or a reporter gene with mutations in the LEF/TCF binding site (mu 6×TCF-IL-2-GFP) (bars 7a to 10a) and either an empty expression vector (Control Vector) (bars 1, 7, 11, and 7a) or an expression vector encoding β-catenin (wt β-catenin) (bars 2, 8, 12, and 8a), an activated mutant form of β-catenin (ΔN89β-catenin) (bars 3, 9, 13, and 9a), Wnt-1 (bars 4, 10, 14, and 10a), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10 and 7a to 10a, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS. The error bars indicate standard deviations. (C) Confirmation of protein expression. Protein extracts were prepared from the transfected cells evaluated in panel B and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of each protein. LEF-1 comigrates with a background band. (D) Evaluation of the REP cell line, containing a stable integration of the reporter gene, with test vectors. REP cells were transfected with either an empty expression vector (Control Vector) (bars 1, 7, and 11) or expression vectors encoding wt β-catenin (bars 2, 8, and 12), an activated mutant form of β-catenin (ΔN89 β-catenin) (bars 3, 9, and 13), Wnt-1 (bars 4, 10, and 14), LEF-1 (bar 5), or TCF-4 (bar 6). Bars 1 to 6, cells transfected with the indicated individual expression vectors; bars 7 to 10, cells transfected with the indicated expression vectors along with a LEF-1 expression vector; bars 11 to 14, cells transfected with the indicated expression vectors along with a TCF-4 expression vector. Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS.

FIG. 2.

Stable integration of a LEF-1 expression plasmid in REP cells to generate REP-LEF cells for a functional screen. (A) Evaluation of the REP-LEF cell line, containing a stable integration of the reporter gene as well as an expression vector encoding LEF-1, with test vectors. REP-LEF cells were transfected with either an empty expression vector (Control Vector) (bar 1) or expression vectors encoding wt β-catenin (bar 2), an activated mutant form of β-catenin (ΔN89 β-catenin) (bar 3), Wnt-1 (bar 4), or LEF-1 (bar 5). Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS. The error bars indicate standard deviations. (B) Confirmation of LEF-1 protein expression in REP-LEF cells. High-salt nuclear extracts were prepared from REP (lanes 1 and 4), REP-LEF (lanes 2 and 5), and COS7 (lane 3 and 6) cells and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of LEF-1 (lanes 1 to 3) or with an antibody directed against LEF-1 (lanes 4 to 6). Markers on the left are in kilodaltons.

FIG. 3.

Selection of genes from a tumor cDNA library using the REP-LEF reporter system. (Top) Outline of protocol for the functional screen with REP-LEF cells. (Middle) GFP-positive REP-LEF cell cultures after initial transfection with the cDNA library (round 1) or after each subsequent round of transfection and enrichment (rounds 2 to 4). The cells were visualized with a fluorescence microscope 48 h after transfection with pools of cDNA expression vectors. (Bottom) FACS analysis of transfected REP-LEF cells after initial transfection (round 1) and after each subsequent round of transfection and enrichment (rounds 2 to 4). The mean GFP fluorescence intensity was measured by FACS in pools of cells 48 h after transfection. For comparison, REP-LEF cells were also transfected with a control vector or expression vectors encoding wt β-catenin or ΔN89 β-catenin.

FIG. 4.

Enrichment and selection of a cDNA encoding a UBF2 fusion protein. (Top) Comparison of amino acid sequences from HMG box 2 of UBF1 and UBF2 isoforms. UBF1 and UBF2 are derived from the same gene by differential splicing; UBF2 contains a 37-amino-acid deletion in HMG box 2. The dashes represent missing amino acids. (Bottom) Amino acid sequence of the carboxyl terminus of fu UBF2, selected in the screen, compared to the carboxyl-terminal sequence of wt UBF1 and wt UBF2. fu UBF2 encodes a fusion between the UBF2 protein, with a carboxyl-terminal truncation of 16 amino acids, and 41 novel carboxyl-terminal amino acids (underlined) derived from out-of-frame translation of ribosomal protein L31. cd UBF2 was constructed to represent the truncated UBF2 protein without the 41-amino-acid carboxyl-terminal extension. The wt UBF1 and wt UBF2 clones were isolated from a human placenta cDNA library.

FIG. 5.

Activities of UBF proteins in the REP-LEF reporter cell line. (A) REP-LEF cells were transfected with a control vector or expression vectors encoding wt β-catenin, wt UBF1, wt UBF2, cd UBF2, or fu UBF2, and GFP induction was analyzed by FACS 48 h after transfection. The error bars indicate standard deviations. (B) Protein extracts were prepared from the cells evaluated in panel A and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of each protein. (C) REP-LEF cells were cotransfected with either a wt luciferase reporter gene (6×TCF-IL-2-LUCIFERASE) or a similar reporter gene with mutated LEF/TCF binding sites (mu) and expression vectors encoding yellow fluorescent protein (control), wt β-catenin, wt UBF1, wtUBF2, cdUBF2, or fuUBF2. The transfections were performed in triplicate, and 48 h after transfection, the induced luciferase activity was measured and normalized to the Renilla activity expressed from a cotransfected internal control vector. The data are presented as n-fold induction, where activated transcription is divided by the basal transcription from the control expression plasmid.

FIG. 6.

UBF2 cooperates with LEF-1 and β-catenin to potentiate a transcriptional response. REP cells were cotransfected with the wt 6×TCF-IL-2-LUCIFERASE reporter gene along with individual expression plasmids encoding the test genes for LEF-1 (bar 1), control Src-SH3 domain (bar 2), wt β-catenin (bar 3), and wt UBF2 (bar 4). LEF-1 was also tested in combination with control Src-SH3 domain (bar 5), wt β-catenin (bar 6), or wt UBF2 (bar 7), and wt UBF2 was tested in combination with wt β-catenin (bar 8), Wnt-1 (bar 9), and TCF-4 (bar 10). The transfections were performed in triplicate, and 48 h after transfection, the induced luciferase activity was measured and normalized to the Renilla activity expressed from a cotransfected internal control vector. The data are presented as n-fold induction where activated transcription is divided by the basal transcription from the control expression plasmid. The error bars indicate standard deviations.

FIG. 7.

Coimmunoprecipitation of UBF proteins with LEF-1. (A) COS7 cells were cotransfected with an expression vector encoding LEF-1 and expression vectors encoding either wt β-catenin as a positive control (lanes 1), wt UBF2 (lanes 2), fu UBF2 (lanes 3), cd UBF2 (lanes 4), Mad4 as a negative control (lanes 5), or Src-SH3 domain as an additional negative control (lanes 6). HA-tagged LEF-1 (lanes 7) was transfected alone as a positive control for LEF-1 expression and HA epitope tag detection. Extracts from transfected cells were immunoprecipitated with an antibody directed against LEF-1, and immunoprecipitates (IP) (top), along with aliquots of total cell extract (Total) (bottom), were analyzed by Western immunoblotting with an antibody against the HA epitope tag encoded at the amino terminus of each protein. The top asterisk indicates the position of the coimmunoprecipitated proteins, the middle asterisk indicates the HA-tagged LEF-1 protein, and the bottom asterisk indicates the HA-tagged Src-SHC domain. (B) COS7 cells were cotransfected with an expression vector encoding β-catenin and expression vectors encoding either wt UBF2 (lanes 1), cd UBF2 (lanes 2), fu UBF2 (lanes 3), LEF-1 as a positive control (lanes 4), or Raf as a negative control (lanes 5). Extracts from transfected cells were immunoprecipitated with an antibody directed against β-catenin, and immunoprecipitates (top), along with aliquots of total cell extract (bottom), were analyzed by Western immunoblotting with an antibody against the HA epitope tag encoded at the amino terminus of each protein. M, markers.

FIG. 8.

UBF2 and LEF-1 colocalize in the nuclei of human cells. HT1080 cells were transfected (tx) with either an empty expression vector or individual vectors encoding LEF-1, HA-tagged Src-SH3 domain, HA-tagged wt UBF2 protein, or a combination of expression vectors encoding LEF-1 plus HA-tagged wt UBF2. The Src-SH3 domain- and wt UBF2-expressing cells were stained with a polyclonal antibody directed against the HA tag, followed by a green fluorescent secondary antibody. Cells transfected with the empty control vector and the expression vector encoding LEF-1 were stained with a monoclonal antibody directed against LEF-1, followed by a red fluorescent secondary antibody.

FIG. 9.

UBF1 and UBF2 proteins activate a cyclin D1 promoter-reporter gene by cooperative interactions with LEF-1 protein. (A) REP-LEF cells were cotransfected with either a promoter-luciferase reporter construct consisting of the wt cyclin D1 promoter, including a LEF/TCF binding site (bars 1 to 4), or a similar reporter gene with a mutation in the LEF/TCF binding site (mu) (bars 5 to 8), along with individual expression vectors encoding a control yellow fluorescent protein gene (bars 1 and 5), wt β-catenin (bars 2 and 6), wt UBF1 (bars 3 and 7), or wt UBF2 (bars 4 and 8). The wt cyclin D1 reporter was also cotransfected into the REP cell line, along with the same test vectors (bars 9 to 12). The transfections were performed in triplicate, and the induced luciferase activity was measured and normalized to the Renilla activity from a constitutive expression vector cotransfected as an internal control. The final numbers represent n-fold induction, where activated transcription is divided by the basal transcription from the control expression plasmid. The error bars indicate standard deviations. (B) Protein extracts were prepared from the cells evaluated in panel A and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of each protein.

FIG. 10.

Downregulation of endogenous UBF by shRNA reduces the ability of ΔN89 β-catenin to activate a LEF/TCF-responsive reporter. (A) REP-LEF cells were cotransfected with an expression vector encoding ΔN89 β-catenin along with either MSCV-U6 empty vector (bar 2) or MSCV-U6-UBF (bar 3). Forty-eight hours after transfection, the fluorescence level of GFP-expressing cells was determined by FACS. Untx, untransfected. (B) REP-LEF cells were cotransfected with an expression vector encoding HA-UBF2, along with either MSCV-U6 empty vector (lane 3) or MSCV-U6-UBF (lane 4). High-salt nuclear extracts were prepared from the transfected cells and analyzed by Western immunoblotting with an antibody directed against the HA epitope tag encoded at the amino terminus of UBF2. −, absent.