ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells

Kenzui Taniue; Akiko Kurimoto; Yasuko Takeda; Takeshi Nagashima; Mariko Okada-Hatakeyama; Yuki Katou; Katsuhiko Shirahige; Tetsu Akiyama

doi:10.1073/pnas.1605938113

New Research In

Edited by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 20, 2016 (received for review April 14, 2016)

Significance

Wnt/β-catenin signaling plays crucial roles in the regulation of proliferation, cell fate, the self-renewal of stem and progenitor cells, and tumorigenesis. Long noncoding RNAs (lncRNAs), non–protein-coding transcripts longer than 200 nt, also play important roles in a number of biological processes and in tumorigenesis. We show that the lncRNA ASBEL [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3) are directly transactivated by β-catenin and form a complex that downregulates the expression of activating transcription factor 3 (ATF3). We further demonstrate that ASBEL–TCF3–mediated downregulation of ATF3 expression is required for the tumorigenicity of colon cancer cells. Our results suggest that the β-catenin–ASBEL–TCF3–ATF3 pathway may be a promising target for colon cancer therapy.

Abstract

Wnt/β-catenin signaling plays a key role in the tumorigenicity of colon cancer. Furthermore, it has been reported that lncRNAs are dysregulated in several steps of cancer development. Here we show that β-catenin directly activates the transcription of the long noncoding RNA (lncRNA) ASBEL [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3), both of which are required for the survival and tumorigenicity of colorectal cancer cells. ASBEL interacts with and recruits TCF3 to the activating transcription factor 3 (ATF3) locus, where it represses the expression of ATF3. Furthermore, we demonstrate that ASBEL–TCF3–mediated down-regulation of ATF3 expression is required for the proliferation and tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of ASBEL. Our results reveal a pathway involving an lncRNA and two transcription factors that plays a key role in Wnt/β–catenin–mediated tumorigenesis. These results may provide insights into the variety of biological and pathological processes regulated by Wnt/β-catenin signaling.

The canonical Wnt signaling pathway plays essential roles in the regulation of proliferation, cell fate, the self-renewal of stem and progenitor cells, and tumorigenesis (1 ⇓⇓–4). In the absence of Wnt signaling, β-catenin is targeted for ubiquitin/proteasome-mediated degradation by a destruction complex composed of the tumor suppressor adenomatous polyposis coli (APC), axin, glycogen synthase kinase-3β, casein kinase I, and TAP/TAZ. Wnt signaling suppresses the function of this complex and induces the association of β-catenin with the TCF/LEF family of transcription factors, thereby activating the transcription of a variety of Wnt target genes. In the majority of colorectal cancers, β-catenin is stabilized by mutations in APC or β-catenin, and thereby Wnt target genes involved in tumorigenesis, including c-Myc and cyclin D1, are constitutively activated.

Mammalian genomes encode numerous long noncoding RNAs (lncRNAs), a class of non–protein-coding transcripts longer than 200 nt (5 ⇓–7). lncRNAs play important roles in a variety of biological processes, including proliferation, differentiation, embryogenesis, neurogenesis, stem cell pluripotency, and tumorigenesis. Accumulating evidence indicates that a number of lncRNAs regulate gene expression by interacting with epigenetic regulators and acting as scaffolds for the assembly of protein complexes (8 ⇓⇓–11). It has also been reported that many lncRNAs regulate transcription by modulating the activity of transcription factors or posttranscriptional processes, including splicing, transport, translation, and degradation of mRNA (12 ⇓⇓⇓⇓–17). Furthermore, it has been shown that lncRNAs are frequently dysregulated in many types of cancer, including colorectal cancer, and can have oncogenic or antioncogenic functions (12, 18, 19).

In the present study, to gain insights into Wnt/β-catenin–mediated tumorigenesis, we attempted to identify lncRNAs that are directly transactivated by β-catenin. We show that β-catenin directly enhances the transcription of the lncRNA ASBEL [antisense ncRNA in the Abundant in neuroepithelium area (ANA)/B-cell translocation gene 3 (BTG3) locus] and transcription factor 3 (TCF3) (20, 21). We previously identified ASBEL as an antisense transcript of the ANA/BTG3 gene, which encodes an antiproliferative protein, and showed that it suppresses the levels of ANA/BTG3 protein and is required for the tumorigenicity of ovarian clear cell carcinoma (13). In this study, we show that ASBEL is required for the tumorigenicity of colon cancer cells and that ASBEL and TCF3 form a complex to suppress the expression of activating transcription factor 3 (ATF3). We further demonstrate that ASBEL–TCF3–mediated down-regulation of ATF3 is critical for the tumorigenicity of colon cancer cells. Finally, we show that ATF3 functions in a negative feedback loop that inhibits ASBEL expression.

Results

The lncRNA ASBEL Is a Target of β-Catenin.

As a first step to identify genes that are the direct targets of β-catenin, we performed RNA-sequencing (RNA-seq) analysis using DLD-1 cells. We found that knockdown of β-catenin led to the up-regulation of 2,072 genes, including 86 genes encoding lncRNAs, and to the down-regulation of 1,512 genes, including 33 genes encoding lncRNAs (Fig. 1A and Fig. S1A and Datasets S1–S4). Functional pathway analyses using the Ingenuity Pathway Analysis (IPA) software revealed that genes involved in cell survival, movement, and proliferation were overrepresented among the affected genes (Fig. S1B and Dataset S5).

Fig. 1.

Transactivation of ASBEL and TCF3 by β-catenin is required for β-catenin–mediated proliferation of colon cancer cells. (A) Depiction of a screen to identify β-catenin target genes. (B) qRT-PCR analysis of the expression of the indicated genes in DLD-1 cells transfected with an siRNA targeting β-catenin or a control siRNA (siCont). Two distinct siRNAs targeting β-catenin (siβ-catenin #1 and #2) were used. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (C) qRT-PCR analysis of the expression of the indicated genes in 293FT cells transfected with an siRNA targeting APC. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D) ChIP assays were performed with DLD-1 cells using anti–β-catenin antibody. The promoter region of AXIN2 was amplified as a positive control. The regions around −3,500 bp of ASBEL and −3,500 bp of TCF3 were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (E) ChIP assays were performed with 293FT cells transfected with an siRNA targeting APC (siAPC) using anti–β-catenin antibody. The promoter region of AXIN2 was amplified as a positive control. The regions around −3,500 bp of ASBEL and −3,500 bp of TCF3 were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05.

Fig. S1.

Transactivation of ASBEL and TCF3 by β-catenin is required for β-catenin–mediated proliferation of colon cancer cells. (A) Venn diagrams showing the overlap of β-catenin–regulated and –occupied genes. DOWN, down-regulated; UP, up-regulated. (B) IPA analysis of molecular and cellular functions of β-catenin–regulated genes. (C and D) GSEA transcription factor (C) and pathway (D) analysis of β-catenin target genes. (E) qRT-PCR analysis of AK092875 expression in DLD-1 cells transfected with an siRNA targeting AK092875. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (F) Cell viability of DLD-1 and HaCaT cells transected with an siRNA targeting AK092875 or ABSEL was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (G) qRT-PCR analysis of the indicated genes in SW480 cells transfected with siRNA targeting β-catenin. Two distinct siRNAs targeting β-catenin (siβ-catenin #1 and #2) were used. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (H) qRT-PCR analysis of Axin2 expression in 293FT cells transfected with an siRNA targeting APC. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (I) ChIP assays were performed with SW480 cells using anti–β-catenin antibody. The promoter region of AXIN2 was amplified as a positive control. The regions around −3,500 bp of ASBEL and −3,500 bp of TCF3 were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (J and K, Left) Schematic representation of the ASBEL promoter luciferase reporter constructs used. Derivatives of the wild-type ASBEL promoter containing mutations in the potential TBEs (TBE1, CTTTGTA → CTTTGGC; TBE2, CTTTGTT → CTTTGGC) were constructed. Luciferase assays were performed with DLD-1 (J) and HeLa (K) cells that had been transfected with a reporter plasmid and the Flag-tagged β-catenin–S33Y or the empty expression plasmid. (Right) Bars represent luciferase activity divided by the activity in cells transfected with the empty expression plasmid and empty reporter plasmid. The pRL-SV40 Renilla luciferase reporter was cotransfected to normalize transfection efficiency. Results are expressed as the mean ± SD (n = 4). *P < 0.05. (L) Expression levels of ASBEL in colon cancer and normal cell lines were quantitated by qRT-PCR as the percentage relative to β-actin mRNA. *P < 0.05. (M) Luciferase assays were performed with HCT116, DLD-1, Caco2, and HaCaT cells that had been transfected with the TOP or FOP reporter plasmid. Bars represent the luciferase activity divided by the activity in cells transfected with the FOP reporter plasmid. The pRL-TK Renilla luciferase reporter was cotransfected to normalize transfection efficiency. Results are expressed as the mean ± SD (n = 4). *P < 0.05.

We next subjected DLD-1 cells to ChIP-sequencing (ChIP-seq) analysis to examine the genome-wide localization of β-catenin in gene promoter regions, i.e., from approximately −2,000 to approximately +2,000 bp from the transcription start sites (TSSs) (Fig. 1A). We found that β-catenin bound to the promoter regions of 813 genes, including 105 genes encoding lncRNAs (Fig. S1A and Datasets S6 and S7). Comparison of the RNA-seq and ChIP-seq data suggested that β-catenin directly down-regulated 74 genes, including one encoding an lncRNA, and up-regulated 66 genes, including two encoding lncRNAs (Fig. 1A and Fig. S1A and Datasets S8 and S9). Consistent with previous reports (22), Gene Set Enrichment Analysis (GSEA) using the C3 gene set revealed that the β-catenin target genes were enriched for LEF1 motifs in their promoter regions (Fig. S1C and Dataset S10). In addition, GSEA pathway analysis showed that β-catenin regulates the expression of known APC target and ES cell-related genes (Fig. S1D and Datasets S11 and S12).

Among the genes identified in the above experiments, we focused on two lncRNAs that are directly up-regulated by β-catenin, AK092875 and ASBEL (BC028229), the latter of which is known to be required for the tumorigenicity of ovarian cancer (13). We then examined the effects of these lncRNAs on the proliferation of DLD-1 cells and found that knockdown of ASBEL caused stronger growth inhibition than knockdown of AK092875 (Figs. S1 E and F and Fig. S2C). In addition, we found that knockdown of AK092875, but not of ASBEL, inhibited the growth of normal keratinocyte HaCaT cells (Fig. S1F).

Fig. S2.

TCF3 is associated with ASBEL and is required for the proliferation of colon cancer cells. (A) qRT-PCR analysis of ASBEL expression in HCT116, HT29, and DLD-1 cells infected with a lentivirus harboring an shRNA targeting ASBEL. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (B) HT29 and DLD-1 cells infected with a lentivirus expressing an shRNA targeting ASBEL and injected into nude mice. Results are expressed as the mean ± SD (n = 6). *P < 0.05. (C) qRT-PCR analysis of ASBEL expression in HCT116, DLD-1, Caco2, and HaCaT cells transfected with an siRNA targeting ASBEL. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D) Annexin V assays were performed with HCT116 and HaCaT cells that had been transfected with siRNA targeting ASBEL. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (E) qRT-PCR analysis of ASBEL expression in human colon cancerous and noncancerous tissues. ASBEL expression was quantitated as the percentage relative to β-Actin mRNA (N, stage 0, n = 8; I, stage I, n = 5; II, stage II, n = 9; III, stage III, n = 16; IV, stage IV, n = 10). *P < 0.05. (F) qRT-PCR analysis of ANA/BTG3 expression in HCT116 and DLD-1 cells transfected with an siRNA targeting ASBEL. Results are expressed as the mean ± SD (n = 3). (G) Lysates from HCT116 and DLD-1 cells transfected with an siRNA targeting ASBEL were subjected to immunoblotting with anti-ANA/BTG3 and anti–α-tubulin antibodies. α-Tubulin was used as a loading control. (H) qRT-PCR analysis of ANA/BTG3 expression in HCT116 and DLD-1 cells transfected with an siRNA targeting ANA/BTG3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (I) The viability of HCT116 and DLD-1 cells transected with an siRNA targeting ABSEL and/or ANA/BTG3 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 4). *P < 0.05. (J) Schematic representation of IPA analysis. (K) The results of IPA upstream regulator analyses of ASBEL-regulated genes. (L) qRT-PCR analysis of TCF3 expression in HCT116, DLD-1, Caco2, and HaCaT cells transfected with siRNA targeting TCF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (M) Annexin V assays were performed with HCT116, DLD-1, and HaCaT cells transfected with siTCF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (N) qRT-PCR analysis of TCF3 expression in HCT116 cells infected with a lentivirus harboring an shRNA targeting TCF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (O) HCT116 and DLD-1 cells were subjected to subcellular fractionation, and the amount of ASBEL in each fraction was evaluated by qRT-PCR. The amounts of TCF3 and ATF3 were evaluated by immunoblotting analysis. α-Tubulin and GAPDH mRNA were used as markers specific for the cytoplasm. Lamin A/C and U1 snRNA were used as nuclear markers. Results are expressed as the mean ± SD (n = 3). (P) In situ hybridization analysis of ASBEL and immunohistochemical analysis of ATF3 (ab191513) in colon cancer and normal tissues. (Q and R, Left) Schematic representation of the TCF3 promoter luciferase reporter constructs. Derivatives of the wild-type TCF3 promoter containing a mutation in the potential TBE (TTCAAAG → GCCAAAG) were constructed. Luciferase assays were performed with SW480 (Q) and HeLa (R) cells that had been transfected with a reporter plasmid and Flag-tagged β-catenin–S33Y or the empty expression plasmid. (Right) Bars represent the luciferase activity divided by the activity in cells transfected with the empty expression plasmid and empty reporter plasmid. The pRL-SV40 Renilla luciferase reporter was cotransfected to normalize transfection efficiency. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (S) Expression levels of TCF3 in colon cancer and normal cell lines were quantitated by qRT-PCR as the percentage relative to β-actin mRNA. Results are expressed as the mean ± SD (n = 3). *P < 0.05.

We therefore decided to focus our analysis on ASBEL. We first confirmed that knockdown of β-catenin resulted in decreased expression of ASBEL, as well as AXIN2, a known β-catenin target gene, in both DLD-1 and SW480 cells (Fig. 1B and Fig. S1G). We also observed that knockdown of APC caused an increase in the expression of ASBEL and AXIN2 in 293FT cells (Fig. 1C and Fig. S1H). Moreover, ChIP analyses with anti–β-catenin antibody revealed that β-catenin bound to the TSSs but not to the upstream (−3,500 bp) regions of the ASBEL and AXIN2 promoters in DLD-1 and SW480 cells (Fig. 1D and Fig. S1I). In addition, we found that β-catenin is recruited to the TSSs but not to the upstream (−3,500 bp) regions of the ASBEL and AXIN2 promoters in 293FT cells in which APC had been knocked down (Fig. 1E). This recruitment was not observed in control 293FT cells (Fig. 1E). These results suggest that β-catenin transactivates ASBEL directly by binding to its TSS region in colon tumor cells.

We next examined whether β-catenin could up-regulate a region of the ASBEL promoter (from approximately −1,000 to approximately +1 bp) inserted into a reporter construct driving the luciferase gene (WT-ASBEL). We also generated variants of this reporter construct containing a mutated TCF-binding element (TBE) in the ABSEL promoter region (MT1–3-ASBEL; Fig. S1J). When transfected into DLD-1 cells, the activity of WT-ASBEL was significantly higher than in mock or MT1–3-ASBEL reporter plasmids (Fig. S1J). Moreover, we found that cotransfection of WT-ASBEL, but not of mock or MT3-ASBEL, into HeLa cells together with a constitutively active mutant of β-catenin, β-cateninS33Y, resulted in a significant increase in reporter activity (Fig. S1 J and K). Quantitative RT-PC (qRT-PCR) analysis revealed that ASBEL expression was up-regulated in the colon cancer cell lines HCT116, DLD-1, and Caco2, in which Wnt signaling is activated, compared with the normal keratinocyte cell line HaCaT (Fig. S1 L and M). These results suggest that β-catenin up-regulates ASBEL expression by binding to the TBEs in the ASBEL promoter region.

ASBEL Is Required for the Tumorigenicity of Colon Tumor Cells.

To investigate the importance of ASBEL in colorectal tumorigenesis, we infected DLD-1, HCT116, and HT29 cells with a lentivirus expressing an shRNA targeting ASBEL (shASBEL) and examined their tumorigenicity. When these cells were transplanted into nude mice, the cells infected with shASBEL showed significantly retarded cell growth compared with those infected with a control lentivirus (Fig. 2A and Fig. S2 A and B). In addition, Cell Titer-Glo assays revealed that knockdown of ASBEL by siRNA (siASBEL) caused a significant reduction in the growth of DLD-1, HCT116, and Caco2 cells but not of normal keratinocyte HaCaT cells in vitro (Fig. 2B and Fig. S2C). Annexin V assays also showed that knockdown of ASBEL led to a marked increase in apoptotic cell death of HCT116 cells but not of HaCaT cells (Fig. S2 C and D). Moreover, we found that knockdown of ASBEL reduced the invasiveness of DLD-1 and HCT116 cells (Fig. 2C and Fig. S2C).

Fig. 2.

TCF3 is associated with ASBEL and is required for the proliferation of colon cancer cells. (A) HCT116 cells infected with a lentivirus expressing an shRNA targeting ASBEL and/or ATF3 were injected into nude mice. Results are expressed as the mean ± SD (n = 6). *P < 0.05. shCont, control. (B) The viability of HCT116, DLD-1, Caco2, and HaCaT cells transfected with an siRNA targeting ASBEL was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (C) Invasion assays were performed with HCT116 and DLD-1 cells transfected with siASBEL followed by Cell Titer-Glo assays to detect invading cells. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D) IPA pathway analysis of ASBEL-regulated genes. (E) qRT-PCR analysis of TCF3 expression in human colon cancerous and noncancerous tissues. TCF3 expression was quantitated as the percentage relative to β-Actin mRNA (N, stage 0, n = 8; I, stage I, n = 5; II, stage II, n = 9; III, stage III, n = 16; IV, stage IV, n = 10). *P < 0.05. (F) Viability of HCT116, DLD-1, Caco2, and HaCaT cells transfected with siTCF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (G) HCT116 cells infected with a lentivirus expressing an shRNA targeting TCF3 and/or ATF3 were injected into nude mice. Results are expressed as the mean ± SD (n = 6). *P < 0.05. (H) Lysates from HCT116 cells were subjected to immunoprecipitation with anti-TCF3 antibody or anti-rabbit IgG antibody followed by qRT-PCR analysis to detect ASBEL. GAPDH, HPRT1, U1, U6, and MALAT were used as negative controls. Results are expressed as the mean ± SD (n = 2). (I) In vitro-transcribed ASBEL interacts with TCF3. Lysates from HCT116 cells were incubated with biotinylated ASBEL or GAPDH generated in vitro, and bound proteins were precipitated with streptavidin beads and subjected to immunoblotting analysis with anti-TCF3 or anti–α-tubulin antibody. α-Tubulin was used as a negative control. (J) The viability of HCT116 cells transfected with an siRNA targeting β-catenin along with the indicated plasmids was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 4). *P < 0.05. HPRT1, hypoxanthine phosphoribosyltransferase 1; MALAT, metastasis associated lung adenocarcinoma transcript; U1, small nuclear RNA U1; U6, small nuclear RNA U6.

We next examined ASBEL expression in human colorectal tumors and adjacent noncancerous tissues by qRT-PCR analysis. We found that ASBEL expression was higher in stage I–III colon cancer than in normal tissues (Fig. S2E). Taken together, these results suggest that ASBEL is required for the proliferation, survival, and tumorigenicity of colon cancer cells.

Knockdown of ASBEL in HCT116 cells resulted in the up-regulation of 375 genes and in the down-regulation of 233 genes, as determined by RNA-seq analysis. Consistent with the above results, this gene set was enriched with genes involved in cell proliferation, death, and movement (Fig. 2D and Datasets S13 and S14).

ASBEL Is Associated with the Transcription Factor TCF3.

We previously identified ASBEL as an antisense transcript of the ANA/BTG3 gene and found that ASBEL promotes the tumorigenicity of ovarian clear cell carcinoma by inhibiting the transportation of ANA/BTG3 mRNA, thereby suppressing its protein levels (13). Consistent with this finding, we found that knockdown of ASBEL resulted in increased levels of ANA/BTG3 protein but had no effect on ANA/BTG3 mRNA in HCT116 and DLD-1 cells (Fig. S2 F and G). However, knockdown of ANA/BTG3 did not restore the viability of HCT116 or DLD-1 cells in which ASBEL had been knocked down (Fig. S2 H and I). Thus, ASBEL-mediated suppression of the ANA/BTG3 protein levels may not be critical for the proliferation of colon cancer cells.

To clarify other potential functions of ASBEL in colon cancer cells, we analyzed ASBEL-regulated genes by RNA-seq analysis and IPA software (Fig. S2J) (23). We found that the gene-expression signature observed in ASBEL-knockdown cells overlapped those regulated by the transcription factor TCF3 (Fig. S2K and Dataset S15). TCF3/E2A belongs to the basic helix–loop–helix (bHLH) family of transcription factors, which bind to E-box (CANNTG) sites present in the promoters or enhancer regions of their target genes (20, 21). The bHLH family member E12/E47 participates in the repression of E-cadherin expression and induction of the epithelial-to-mesenchymal transition, leading to the acquisition of invasive properties (24, 25). Moreover, TCF3/E2A is overexpressed in colon cancer (26, 27), prostate cancer (28, 29), and renal cancer (30) and plays a critical role in the proliferation and survival of tumor cells (29). Consistent with these reports, we found that TCF3 was highly expressed in colon cancer tissues compared with the noncancerous tissues (Fig. 2E) and was required for the viability of DLD-1, HCT116, and Caco2 cells but not of HaCaT cells (Fig. 2F and Fig. S2 L and M). Moreover, when s.c. injected into nude mice, HCT116 cells infected with a lentivirus expressing an shRNA targeting TCF3 showed reduced tumorigenicity compared with control HCT116 cells (Fig. 2G and Fig. S2N).

Based on the above findings, we hypothesized that ASBEL may cooperate with TCF3 and promote its transcriptional activity in colorectal cancer cells. To test this hypothesis, we performed RNA immunoprecipitation (RIP) assays with anti-TCF3 antibody using lysates from HCT116 cells. qRT-PCR analysis of the immunoprecipitates revealed that TCF3 was associated with endogenous ASBEL but not with GAPDH, HPRT1, U1, U6, or MALAT (Fig. 2H). In addition, we carried out RNA pull-down assays and found that TCF3 precipitated with ASBEL generated in vitro but not with GAPDH (Fig. 2I). Consistent with these results, subcellular fractionation followed by qRT-PCR analysis of ASBEL and immunoblotting analysis of TCF3 revealed that ASBEL and TCF3 were localized to the nucleus in colon cancer cells (Fig. S2O). In situ hybridization analysis also showed that ASBEL was localized to the nucleus in colon cancer specimens (Fig. S2P). Anti-TCF3 antibody suitable for immunostaining analysis was not available.

The ASBEL–TCF3 Complex Is Important for Wnt/β–Catenin Signaling.

We noticed that TCF3 is contained in the list of β-catenin target genes in Dataset S8. We confirmed that knockdown of β-catenin reduced the expression of TCF3 in DLD-1 and SW480 cells (Fig. 1B and Fig. S1G), whereas knockdown of APC led to increased expression of TCF3 in 293FT cells (Fig. 1C). We also observed that β-catenin binds to the TSS region but not to the upstream (−3,500 bp) region of the TCF3 promoter in DLD-1, SW480, and 293FT cells in which APC had been knocked down (Fig. 1 D and E and Fig. S1I). Luciferase assays showed that the activity of a reporter containing the promoter and the 5′ UTR region of TCF3 (from approximately −1,000 to approximately +1,000 bp), but not of reporters containing a mutated TBE, was higher than that of the control reporter (Fig. S2Q). Furthermore, this activity was further enhanced by cotransfection of β-cateninS33Y (Fig. S2 Q and R). qRT-PCR analysis revealed that TCF3 expression was up-regulated in the colon cancer cell lines HCT116, DLD-1, and Caco2, in which Wnt signaling is activated, compared with the normal keratinocyte cell line HaCaT (Fig. S1M and Fig. S2S). These results suggest that β-catenin directly enhances the transcription of TCF3.

To investigate the significance of the ASBEL–TCF3 complex in Wnt/β-catenin signaling, we examined the effect of overexpression of ASBEL and TCF3 on the viability of HCT116 cells in which β-catenin had been knocked down by siRNA. Although knockdown of β-catenin caused a reduction in the viability of HCT116 cells, overexpression of ASBEL together with TCF3, but not of ASBEL or TCF3 alone, partially restored their viability (Fig. 2J). These results suggest that the ASBEL–TCF3 complex plays an important role in Wnt/β-catenin–mediated proliferation of colon cancer cells.

ASBEL/TCF3-Mediated Down-Regulation of ATF3 Is Required for the Tumorigenicity of Colon Cancer Cells.

To identify the target genes of the ASBEL–TCF3 complex that are involved in the proliferation of colon cancer cells, we used the IPA software platform to analyze genes whose expression levels are altered by knockdown of ASBEL. We found that the ASBEL–TCF3 complex up-regulated five genes and down-regulated 16 genes (Fig. S2J and Dataset S16). To clarify the significance of the up-regulated genes, we transfected siRNAs targeting these genes into HCT116 cells and examined the viability of these cells. We observed that knockdown of CD79B resulted in decreased cell viability (Fig. S3 A and B). We also examined the viability of cells that had been transfected with siRNAs targeting the down-regulated genes together with siASBEL. We found that knockdown of ATF3, CCNB2, CDH1, or GADD45B partially restored the siASBEL-induced decrease in cell viability (Fig. 3A and Fig. S3 C and D). Moreover, knockdown of ASBEL resulted in the up-regulation of these genes in HCT116 cells but not in HaCaT cells (Fig. S3E). By contrast, AK092875 knockdown did not lead to the up-regulation of these genes in HCT116 cells (Fig. S3F). Of these genes, knockdown of ATF3 had the greatest effect; we hereafter focused our analysis on ATF3.

Fig. 3.

The ASBEL–TCF3 complex represses the expression of ATF3 in colon cancer cells. (A) The viability of HCT116 cells transfected with an siRNA targeting ASBEL along with a control siRNA (siCont) or siATF3 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (B) qRT-PCR analysis of ATF3 expression in human colon cancerous and noncancerous tissues. ATF3 expression was quantitated as the percentage relative to β-Actin mRNA (N, stage 0, n = 8; I, stage I, n = 5; II, stage II, n = 9; III, stage III, n = 16; IV, stage IV, n = 10). *P < 0.05. (C and D, Left) qRT-PCR analysis of ATF3 mRNA in HCT116 cells transfected with an siRNA targeting ASBEL (C) or TCF3 (D). Results are expressed as the mean ± SD (n = 3). *P < 0.05. (Right) Cell lysates were subjected to immunoblotting analysis with anti-ATF3 or anti–α-tubulin antibody. α-Tubulin was used as a loading control. The asterisk indicates an irrelevant background band.

Fig. S3.

The ASBEL–TCF3 complex represses the expression of ATF3 in colon cancer cells. (A) HCT116 cells were transfected with siRNAs targeting the indicated genes and were subjected to qRT-PCR analysis. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (B) The viability of HCT116 cells transfected with siRNAs targeting the indicated genes. Results are expressed as the mean ± SD (n = 4). *P < 0.05. (C) HCT116 cells were transfected with siRNAs targeting the indicated genes and were subjected to qRT-PCR analysis. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D) The viability of HCT116 cells transfected with siRNAs targeting the indicated genes. Results are expressed as the mean ± SD (n = 4). *P < 0.01, **P < 0.001. (E) qRT-PCR analysis of the indicated mRNAs in HCT116 and HaCaT cells transfected with an siRNA targeting ASBEL. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (F) qRT-PCR analysis of the indicated mRNAs in HCT116 cells transfected with an siRNA targeting AK092875. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (G and H) qRT-PCR analysis of ATF3 mRNA in DLD-1 and Caco2 cells transfected with an siRNA targeting ASBEL (G) or TCF3 (H). Results are expressed as the mean ± SD (n = 3). *P < 0.05. (I) 293FT cells were transfected with siRNAs targeting the indicated genes and were subjected to qRT-PCR analysis. Results are expressed as the mean ± SD (n = 3). *P < 0.05.

ATF3 is a member of the ATF/CREB family of transcription factors whose expression is induced rapidly by a wide range of cellular stresses, including DNA damage, cellular injury, and oxidative stress (31). ATF3 has been shown to suppress tumor growth and metastasis in many cancer types, including glioblastoma, colon, bladder, and lung cancer (32 ⇓⇓–35). We examined the expression of ATF3 in human colorectal tumor tissues and found that ATF3 was localized to the nucleus (Fig. S2P). Subcellular fractionation followed by immunoblotting analysis also revealed that ATF3, as well as ASBEL and TCF3, was localized to the nucleus of HCT116 and DLD-1 cells (Fig. S2O). qRT-PCR analysis revealed that ATF3 mRNA was expressed at lower levels in colorectal tumors than in noncancerous tissues (Fig. 3B). Moreover, we found that knockdown of ASBEL or TCF3 resulted in increased expression of the ATF3 mRNA and protein (Fig. 3 C and D and Fig. S3 G and H). Furthermore, we found that knockdown of either ASBEL or TCF3 restored the expression of ATF3 in 293FT cells in which APC had been knocked down (Fig. S3I). Consistent with these results, in situ hybridization analysis of ASBEL and immunohistochemical analysis of ATF3 revealed that an inverse correlation between ASBEL and ATF3 is also found in colorectal tissues (Fig. S3J). These results raised the possibility that the growth inhibition caused by siASBEL or siTCF3 may be an indirect consequence of increased ATF3 protein expression. In line with this notion, knockdown of ATF3 could partially rescue HCT116 cells from the reduction in cell viability caused by either ASBEL knockdown or TCF3 knockdown (Figs. 3A and 4A). In addition, knockdown of ATF3 had similar effects on the growth of cells in which β-catenin had been knocked down (Fig. 4B). Furthermore, we found that ATF3 knockdown could restore the tumorigenicity of HCT116 cells infected with a lentivirus targeting ASBEL or TCF3 (Fig. 2A and G and Figs. S2 A and N and S4A).

Fig. 4.

Down-regulation of ATF3 is required for the tumorigenicity of colon cancer cells. (A) The viability of HCT116 cells transfected with an siRNA targeting TCF3 along with siCont or siATF3 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (B) The viability of HCT116 cells transfected with an siRNA targeting β-catenin and/or ATF3 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (C) Schematic representation of the human ATF3 promoter region. Amplified regions are indicated by black bars. The regions around −5,500 bp (UP) and +4,000 bp (DO) were amplified as negative controls. (D) ChIP assays were performed with HCT116 cells that had been transfected with siASBEL using anti-TCF3 antibody. The regions around UP (−5,500) and DO (+4,000) were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (E) RNA ChIP assays were performed with HCT116 cells that had been transfected with biotinylated sense or antisense ASBEL RNA and subjected to precipitation using streptavidin beads. The regions around UP (−5,500 bp) and DO (+4,000 bp) were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (F) Schematic representation of the Luc-Mock and Luc-ATF3-Enh (Intron 1; ATF3 enhancer) reporter constructs for enhancer assays. (G and H) HCT116 cells that had been transfected with an siRNA targeting ASBEL (G) or TCF3 (H) or control siRNA were transfected with the ATF3 enhancer reporter construct and subjected to luciferase assays. pRL-SV40 was used as an internal control. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (I) HeLa cells were transfected with a mock, ASBEL, and/or TCF3 expression vector along with an enhancer reporter construct containing ATF3 intron 1 sequence (Luc-ATF3-Enh) and were subjected to luciferase assays. pRL-TK was used as an internal control. Results are expressed as the mean ± SD (n = 4). *P < 0.05. (J) The ASBEL–TCF3 complex is required for the tumorigenicity of colon cancer cells. β-Catenin directly transactivates ASBEL and TCF3 in colon cancer. ASBEL is associated with TCF3 and represses the expression of ATF3. The ASBEL–TCF3–mediated down-regulation of ATF3 is required for the tumorigenicity of colon cancer cells. DO, downstream; UP, upstream.

Fig. S4.

ATF3 expression is required for the tumorigenicity of colon cancer cells. (A) qRT-PCR analysis of ATF3 expression in HCT116 cells infected with a lentivirus harboring an shRNA targeting ATF3. Results are expressed as the mean ± SD (n = 2). *P < 0.05. (B) Data from the ENCODE project showing an association of TCF3 with a region downstream of the ATF3 gene TSS. (C, Left) qRT-PCR analysis of TCF3 expression in HCT116 cells transfected with an siRNA targeting ASBEL. Results are expressed as the mean ± SD (n = 3). (Right) Cell lysates were subjected to immunoblotting analysis with anti-TCF3, anti-ATF3, or anti–α-tubulin. α-Tubulin was used as a loading control. The asterisk indicates a background signal. (D) Lysates from 293FT cells transfected with the indicated expression constructs were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblotting with anti-Flag or anti-HA antibody. (E) Lysates from HCT116 cells transfected with the indicated expression constructs and/or an siRNA targeting ASBEL were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblotting with anti-Flag or anti-HA antibody.

As a possible mechanism underlying ASBEL-mediated down-regulation of ATF3 expression, we searched the ATF3 locus for sites that may bind TCF3. Data from the ENCODE project (https://genome.ucsc.edu/ENCODE/) indicated an association of TCF3 with a region 1,641–2,220 bp downstream (the TCF-binding site, TBS) of the ATF3 gene TSS, as determined in the lymphoblastoid cell line GM12878 (Fig. S4B). Consistent with these data, our ChIP analysis with anti-TCF3 antibody confirmed that TCF3 was indeed associated with a TBS ∼2,000 bp downstream of the ATF3 gene but not with two other regions located upstream and downstream of the gene (Fig. 4 C and D). Furthermore, knockdown of ASBEL resulted in a significant decrease in TCF3 binding to the TBS in the ATF3 gene (Fig. 4 C and D). In addition, we found by qRT-PCR and immunoblotting analyses that ASBEL did not affect the expression levels of TCF3 in HCT116 cells (Fig. S4C), nor did it affect TCF3 dimer formation, which is critical for the binding of TCF3 to TBSs (Fig. S4 D and E) (20, 21). We next examined whether ASBEL interacts physically with the TBS at the ATF3 locus. We transfected HCT116 cells with biotin-labeled ASBEL RNA and precipitated ASBEL-associated DNA using streptavidin-coated beads. We found that ASBEL, but not antisense ASBEL, precipitated ATF3 sequences containing the TBS but did not precipitate the upstream or downstream region (Fig. 4 C and E).

We next investigated the cis-activating potential of the DNA region containing this TBS. The ATF3 TBS region was inserted downstream of a reporter cassette consisting of the SV40 promoter and luciferase gene (Fig. 4F). We found that knockdown of ASBEL or TCF3 resulted in a significant increase in luciferase reporter activity (Fig. 4 G and H). By contrast, overexpression of both ASBEL and TCF3, but not of either alone, resulted in the inhibition of reporter activity (Fig. 4I). These results raise the possibility that ASBEL recruits TCF3 to the ATF3 locus and thereby represses ATF3 expression in colon cancer cells.

Negative Feedback Regulation of the Expression of ASBEL by ATF3 in Colon Cancer Cells.

The GSEA transcription factor analysis in Fig. 1A revealed that ATF/CREB (TGACGTCA) motifs are enriched in genes targeted by β-catenin (Fig. S1C and Dataset S10). We therefore examined whether ATF3 regulates the expression of ASBEL in colon cancer cells. We found that knockdown of ATF3 indeed resulted in increased expression of ASBEL in HCT116 cells (Fig. S5 A and B). ChIP analyses with anti-ATF3 antibody revealed that ATF3 was associated with the TSS but not with the upstream (−3,500 bp) region of the ASBEL promoter or the CCND1 promoter (Fig. S5C). Furthermore, we found that knockdown of ATF3 resulted in increased activity of a reporter plasmid containing the ASBEL promoter (Fig. S5D). Taken together, these results suggest that ATF3 functions as a component in the negative feedback loop that inhibits ASBEL expression.

Fig. S5.

Negative feedback regulation of the expression of ASBEL by ATF3 in colon cancer cells. (A) qRT-PCR analysis of ASBEL expression in HCT116 cells transfected with siRNA targeting ATF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (B) qRT-PCR analysis of ATF3 expression in HCT116 cells transfected with an siRNA targeting ATF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (C) ChIP assays were performed with HCT116 cells using anti-ATF3 antibody. The promoter region of CCND1 was amplified as a positive control. The region around −3,500 bp of ASBEL was amplified as a negative control. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D) HCT116 cells that had been transfected with siRNA targeting ATF3 or control siRNA were transfected with the ASBEL promoter reporter constructs and were subjected to luciferase assays. The pRL-TK vector was used as an internal control. Results are expressed as the mean ± SD (n = 3). *P < 0.05.

Discussion

In this study, we attempted to identify lncRNAs that are critical for Wnt/β-catenin–mediated tumorigenesis. We performed RNA-seq and ChIP-seq analyses of colorectal cancer cells and found that β-catenin directly activates the transcription of the lncRNA ASBEL. We observed that ASBEL is required for the proliferation and tumorigenicity of colon cancer cells. Furthermore, analysis of ASBEL-regulated genes using IPA software revealed that ASBEL cooperates with the transcription factor TCF3, which is also directly transactivated by β-catenin. ASBEL forms a complex with TCF3, and this complex, but not either individual molecule, plays an important role in Wnt/β-catenin signaling in colon cancer cells. Interestingly, overexpression of ASBEL together with TCF3 restores the viability of colon cancer cells in which β-catenin has been knocked down, whereas overexpression of ASBEL or TCF3 alone has no effect.

In agreement with previous reports (26, 27), we found that TCF3 is overexpressed in colon cancer tissues compared with adjacent noncancerous tissues. It has also been reported that TCF3 is highly expressed in various cancers, including prostate, gastric, and renal cancers (28 ⇓–30). Furthermore, it has been reported recently that TCF3 is up-regulated by hypomethylation of its promoter and that this up-regulation is correlated with recurrence in stage II and III colorectal cancers (27). Thus, TCF3 is up-regulated by multiple mechanisms in colon cancer, including aberrant activation of Wnt/β-catenin signaling and hypomethylation of its promoter.

We previously identified ASBEL as an antisense transcript of the antiproliferative ANA/BTG3 gene and found that it is required for the proliferation and tumorigenicity of ovarian clear cell carcinoma (13). We found that ASBEL forms complexes with ANA/BTG3 mRNA in the nucleus and suppresses its cytoplasmic transportation, thereby suppressing the levels of ANA/BTG3 protein. Furthermore, we reported that knockdown of ANA/BTG3 rescues the growth inhibition caused by ASBEL knockdown. Thus, ASBEL may promote the tumorigenicity of ovarian clear cell carcinoma by inhibiting its cytoplasmic transportation of the sense gene, thereby suppressing its translation. Similarly, we observed that ASBEL suppresses the expression of ANA/BTG3 protein in colon cancer cells. However, we found that knockdown of ANA/BTG3 does not restore the reduced viability of HCT116 or DLD-1 cells caused by ASBEL knockdown. Thus, ASBEL-mediated suppression of the levels of ANA/BTG3 protein may not be critical for the growth of colon cancer cells. Our results suggest that ASBEL is a multifunctional lncRNA and that the significance of each function varies depending on cell type.

We identified ATF3 as a target of the ASBEL–TCF3 complex and showed that ASBEL- and TCF3-mediated repression of ATF3 expression is required for the tumorigenicity of colon cancer cells. Our results suggest that repression of ATF3 is critical for Wnt/β-catenin–mediated tumorigenesis. We also found that ATF3 expression is down-regulated in colon cancer cells compared with noncancerous tissues. These results appear to be consistent with a previous report showing that ATF3 suppresses tumor growth in colon cancer and many other types of cancer, including glioblastoma and bladder and lung cancer (32 ⇓⇓–35).

Our ChIP analysis with anti-TCF3 antibody showed that TCF3 is associated with the TBS at the ATF locus in an ASBEL-dependent manner. Consistent with this result, we confirmed that ASBEL interacts physically with the ATF3 locus TBS. Furthermore, we showed that both ASBEL and TCF3 are required for the repression of ATF3 expression. These results suggest that the ASBEL–TCF3 complex binds to the ATF3 locus and thereby represses ATF3 expression in colon cancer cells. The function of TCF3 is known to be regulated at the level of dimer formation. TCF3 needs to form a homodimer or heterodimer via its HLH domain to bind to the TBS in the promoter or enhancer regions of its target genes (20, 21). In addition, the DNA-binding activity of TCF3 is inhibited by heterodimer formation with ID1–3 (inhibitor of DNA binding 1–3, dominant-negative helix–loop–helix proteins) (20, 21, 36). However, we found that ASBEL does not affect TCF3 homodimer formation. We also observed that ASBEL does not affect the levels of TCF3 protein or mRNA. We therefore speculate that ASBEL may facilitate the recruitment of TCF3 to the ATF3 locus. Interestingly, it has been reported recently that the lncRNA MEG3 regulates TGF-β pathway genes by binding to chromatin through formation of RNA–DNA triplex structures (37). We also speculate that the ASBEL–TCF3 complex may recruit corepressor factors that participate in repressing ATF3 expression.

It has been reported that AP-1 motifs (TGANTCA) are enriched in genes targeted by β-catenin/TCF/LEF (38 ⇓–40). We found that in addition to AP-1 motifs, ATF/CREB motifs (TGAYRTCA) are also enriched in genes targeted by β-catenin. Furthermore, we observed that ATF3 binds to the ATF motif in the promoter region of ASBEL and represses its expression in colon cancer cells. Therefore it is possible that ATF3 also represses β-catenin target genes other than ASBEL. These negative feedback loops may play important roles in the regulation of Wnt/β-catenin signaling in both tumor and normal cells.

In conclusion, we found that ASBEL and TCF3 are directly transactivated by β-catenin and form a complex that down-regulates the expression of ATF3. We further showed that the ASBEL–TCF3 complex–mediated down-regulation of ATF3 is required for the tumorigenicity of colon cancer cells (Fig. 4J). The pathway identified in this study may play important roles in Wnt/β-catenin–mediated tumorigenesis as well as in various other biological processes. These findings may provide insights into the development of cancer treatments.

Materials and Methods

Further details are provided in SI Materials and Methods. All animal experimental protocols were performed in accordance with the guidelines of the Animal Ethics Committee of the University of Tokyo. Primer sequences and shRNA sequences are listed in Dataset S17. Cell lines are described in Dataset S18. Cell viability was determined by measuring the intracellular levels of ATP using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega). Luminescence was measured using a Mithras LB 940 (Berthold).

SI Materials and Methods

qRT-PCR Analysis.

Total RNA was isolated using the Total RNA Isolation kit (MACHEREY-NAGEL) and treated with DNase I (Takara). One microgram of RNA was reverse transcribed using PrimeScript RT Master Mix (RR036A; Takara). qRT-PCR analysis of cDNA was performed on a LightCycler 480 (Roche Applied Science) using SYBR Green PCR Master Mix (Applied Biosystems). TissueScan Cancer and Normal Tissue cDNA Arrays (Colon Cancer cDNA Array IV) were obtained from OriGene Technologies. Primer sequences are listed in Dataset S17.

Cell Culture.

HeLa cells (ATCC) were cultured in minimum essential medium (MEM) supplemented with 10% (vol/vol) bovine serum, MEM non-essential amino acids solution, and sodium pyruvate (Gibco). DLD-1 cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) bovine serum. Caco2, HaCaT, and 293FT cells (ATCC) were maintained in DMEM supplemented with 10% (vol/vol) bovine serum. HCT116 and HT29 cells (ATCC) were grown in McCoy’s 5A medium supplemented with 10% (vol/vol) bovine serum. SW480 cells (ATCC) were cultured in L-15 medium supplemented with 10% (vol/vol) bovine serum. A description of the cell lines is given in Dataset S18.

Antibodies.

Antibodies against TCF3 (sc349), ATF3 (sc-188), and β-catenin (sc7199) were obtained from Santa Cruz Biotechnology. Anti-ATF3 (ab191513) and anti–α-tubulin antibody (CP-06) were obtained from Abcam and Calbiochem, respectively. Secondary antibodies and ECL-plus were purchased from GE Healthcare.

Lentivirus Production.

A lentiviral vector (CS-Rfa-CG) harboring an shRNA driven by the H1 promoter was transfected with the packaging vectors pCAG-HIV-gp and pCMV-VSV-G-RSV-Rev into 293FT cells using polyethylenimine MAX (catalog no. 24765; Polysciences, Inc.). All plasmids were kindly provided by H. Miyoshi, RIKEN BioResource Center, Tsukuba-shi, Ibaraki, Japan. Virus supernatants were purified by ultracentrifugation at 25,000 rpm for 90 min (Optima XE-90 Ultracentrifuge, SW28 rotor, Beckman Coulter). Infection efficiency was monitored by GFP expression because it was driven by the CMV promoter. The sequences of shRNAs are shown in Dataset S17.

Tumorigenesis Assay.

HCT116, HT29, and DLD1 cells infected with a lentivirus expressing an shRNA targeting ASBEL, TCF3, or ATF3 were injected s.c. into 6-wk-old nude mice (BALB/cAJcl-nu/nu; CLEA Japan, Inc.). All animal experimental protocols were performed in accordance with the guidelines of the Animal Ethics Committee of the University of Tokyo.

RNAi.

siRNA duplexes targeting β-catenin, APC, TCF3, ATF3, MSMO1, SMARCC1, LMO2, GM2A, CD79B, TOP2A, SLC7A5, CCNB2, CCNA2, CDH1, JUN, KIF11, ECT2, GPAM, NFIL3, LMO4, KLF4, H2AFX, GADD45B, or ANLN were purchased from Ambion. siRNA duplexes targeting ASBEL or AK092875 were purchased from Cosmo Bio. Cells were transfected with RNA duplexes using Lipofectamine RNAiMAX (Invitrogen). Sequences of siRNAs are shown in Dataset S17. Silencer Select negative control siRNA #2 (Ambion) or Cosmo Bio negative control (S5C-0600) was used as a control.

ChIP Assay.

ChIP assays were performed according to the manufacturer’s instructions (Upstate) with some modifications as previously described (41, 42). Cells from approximately one confluent 10-cm plate were used. For ChIP analyses with anti–β-catenin antibody shown in Fig. 1 and Fig. S1, nuclear fractions from DLD-1, SW480, and 293FT cells were prepared as follows. Cells were transfected with siRNA, cultured for 72 h, suspended in a 5× volume of cell lysis buffer [10 mM Hepes-KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40], and then resuspended in a 3× volume of cell lysis buffer using a 21-gauge syringe. The nuclei were fixed initially with 1 mM dithiobis (succinimidyl propionate (DSP; Pierce) and rotated for 30 min at 25 °C. The nuclei then were fixed with 1% formaldehyde for 10 min at 25 °C. The reaction was stopped by adding 0.5 mL of 2.5 M glycine and rotating for 5 min. Cross-linked chromatin was resuspended in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS] containing proteinase inhibitors and sonicated to yield 300- to 1,000-bp DNA fragments. Immunoprecipitation was performed using anti–β-catenin antibody and Protein G Dynabeads (Invitrogen). qRT-PCR was performed as described above. For the RNAi-coupled ChIP analyses shown in Fig. 4, HCT116 cells were treated with siRNA duplexes for 72 h and then were fixed with 1% formaldehyde for 10 min at 25 °C. Immunoprecipitation was performed using anti-TCF3 antibody. Primer sequences for qRT-PCR are shown in Dataset S17.

Constructs and Transfection.

ASBEL and TCF3 were amplified by PCR using the corresponding primers and were cloned into pcDNA3.1(+). For pull-down assays, ASBEL and GAPDH were cloned into pBlueScript II SK+. A fragment containing the promoter region of ASBEL (−1,000 bp to +1 bp from the TSS) or TCF3 (−1,000 bp to +1,000 bp from the TSS) was amplified by PCR from HCT116 genome and was subcloned into the pGL3-basic luciferase vector (Promega). Reporter constructs containing a mutated TCF-binding element were created by PCR using KOD -Plus- (Toyobo). Luc-ATF3-Enh was constructed by cloning the region containing the first intron of the human ATF3 gene (+1,641 bp to +2,240 bp from the TSS) into the pGL3-Promoter Vector (Promega). Primer sequences are listed in Dataset S17. Plasmids were transfected into cells using polyethylenimine MAX (catalog no. 24765; Polysciences, Inc.).

Subcellular Fractionation.

Cell pellets were resuspended in one packed cell volume of Hypotonic buffer A [10 mM Hepes (pH 7.5), 0.5 mM DTT, 10 mM KCl, 1.5 mM MgCl₂] containing protease inhibitors and RNase Inhibitor (Promega). After incubation on ice for 10 min, cells were disrupted by 10 passages through a 25-gauge needle and were centrifuged for 10 min at 1,000 × g at 4 °C. The supernatant containing the cytoplasmic fraction was collected by further centrifugation at 15,000 × g for 15 min. The remaining pellets were washed twice with hypotonic buffer, resuspended in hypotonic buffer B [10 mM Hepes (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 0.5% Nonidet P-40] containing protease inhibitors and RNase inhibitor (Promega), and incubated at 4 °C for 30 min with gentle rotation. After centrifugation for 10 min at 6,000 × g at 4 °C, the pellets were washed once with hypotonic buffer and were resuspended in RIPA buffer [50 mM Tris⋅HCl (pH 7.5), 1,500 mM KCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA pH 8.0] containing protease inhibitors and RNase inhibitor (Promega) followed by incubation at 4 °C for 30 min with gentle rotation. The supernatant containing the nuclear fraction was collected by centrifugation at 15,000 × g for 20 min.

In Situ Hybridization.

In situ hybridization was carried out with the ISH Reagent Kit (Genostaff) according to the manufacturer’s instructions (43). Paraffin-embedded sections of human colon adenocarcinoma tissues [PA12F44F77 (patient 1), PA0000B56E (patient 2)] were obtained from OriGene Technologies. A 643-bp DNA fragment corresponding to the nucleotide position 25 bp–667 bp of ASBEL was subcloned into the pGEM-T vector (Promega) and was used to generate the antisense RNA probe. A 570-bp plant DNA fragment (guanine-cytosine content: 57.9%) was used as a negative control (Genostaff). Digoxigenin (DIG)-labeled RNA probes were prepared with DIG RNA Labeling Mix (Roche). Paraffin-embedded sections were deparaffinized, treated with Proteinase K (Wako Pure Chemical Industries), and hybridized with a probe at concentration of 300 ng/mL in G-Hybo-L (Genostaff) for 16 h at 60 °C. The sections then were incubated with anti-DIG AP conjugate (Roche Diagnostics) diluted 1:2,000 with 50× G-Block (Genostaff) in Tris-buffered saline and Tween 20 for 1 h at 25 °C. Coloring reactions were performed with NBT/BCIP solution (Sigma-Aldrich) overnight followed by washing with PBS. The sections were counterstained with Kernechtrot stain solution (Muto Pure Chemicals) and were mounted with G-Mount (Genostaff).

Immunohistochemical Analysis.

Tissue sections were deparaffinated with xylene and were rehydrated through an ethanol series and PBS. Antigen retrieval was performed by microwaving the sections for 20 min with citrate buffer, pH 6.0. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min followed by incubation with Protein Block (Genostaff). The sections were incubated with anti-ATF3 mouse monoclonal antibody (ab191513; Abcam) at 4 °C overnight and then were incubated with Histofine Simple Stain MAX PO (MULTI) (Nichirei) for 30 min at room temperature. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer’s hematoxylin (Muto), dehydrated, and then mounted with malinol (Muto).

RNA Pull-Down Assay.

Biotinylated ASBEL or GAPDH was incubated with nuclear extracts (200 μg) from HCT116 cells and then was mixed with streptavidin beads, washed, and boiled in SDS buffer as described previously (12). The associated proteins were analyzed by gel electrophoresis followed by immunoblotting analysis.

RIP Assay.

Cells growing in six-well dishes were lysed in 0.5 mL of lysis buffer [0 mM Hepes (pH 7.5), 150 mM KCl, 0.5% Nonidet P-40, 2 mM EDTA, 1 mM NaF] containing protease inhibitors and RNase inhibitor (Promega) and were centrifuged at 16,400 × g for 10 min. The supernatants were incubated with anti-TCF3 or anti-rabbit IgG antibody for 3 h at 4 °C with gentle rotation. Thirty microliters of Protein G Dynabeads (Invitrogen) were added and incubated for 3 h at 4 °C with gentle rotation. The beads were washed three times with RIP wash buffer [50 mM Hepes (pH 7.5), 150 mM KCl, 0.05% Nonidet P-40] containing RNase inhibitor (Promega) and then were washed twice with PBS containing RNase inhibitor (Promega). RNA was extracted using the Total RNA Isolation kit (MACHEREY-NAGEL). qRT-PCR was performed as described above. Primer sequences for qRT-PCR are shown in Dataset S17.

Immunoblotting Analysis.

Cells (1 × 10⁶) were lysed for 20 min with lysis buffer [50 mM Hepes (pH 7.5), 150 mM KCl, 0.5% Nonidet P-40, 2 mM EDTA, 1 mM NaF) containing protease inhibitors. After centrifugation at 16,400 × g for 15 min at 4 °C, samples were resolved by SDS/PAGE followed by immunoblotting analysis using HRP-conjugated secondary antibodies. Visualization was performed using the Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare) and an LAS-4000EPUVmini Luminescent Image Analyzer (GE Healthcare).

Apoptosis.

Phosphatidylserine exposure at the cell surface was detected using the Annexin V-Biotin Apoptosis Detection Kit (MBL) and streptavidin–APC conjugates (S888; Invitrogen).

Luciferase Assay.

Dual luciferase assays were performed according to the manufacturer’s protocol (Promega). In brief, cells were seeded in a 96-well plate 24 h before transfection. siRNA transfection was performed 48 h before the assays, as described above. Cells were cotransfected with the pGL3 reporter vector, the pTOP- or pFOP-tk-luciferase vector, the Renilla luciferase vector, and/or the β-catenin S33Y, TCF3, or ASBEL expression vector. At 24 h after transfection, cells were lysed with passive lysis buffer, and lysates were subjected to dual luciferase assays with a Mithras LB 940 microplate reader (Berthold).

RNA Transfection and RNA ChIP Assay.

Biotin-labeled RNA was synthesized using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Ambion), with template plasmid (1 μg) and biotin-CTP (Invitrogen). HCT116 cells were transfected with biotin-labeled ASBEL or anti-ASBEL using the TransIT transfection reagent (Mirus). After 24 h, cells were washed twice with ice-cold PBS containing proteinase inhibitor and RNase inhibitor (Promega), fixed with 1% formaldehyde for 10 min at 25 °C, and then lysed in nuclear lysis buffer [10 mM Tris⋅HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 1% SDS) containing proteinase inhibitor and RNase inhibitor (Promega). Chromatin was sheered by sonication and centrifuged, and the supernatant was incubated for 1 h at 4 °C with streptavidin beads. The beads were washed twice with ChIP dilution buffer, twice with RIP wash buffer, and then twice with Tris-EDTA (TE) buffer containing proteinase inhibitor and RNase inhibitor (Promega). Subsequent elution, proteinase K treatment, and de–cross-linking procedures were performed as described previously (42). DNA was extracted using the QIAquick PCR purification kit (Qiagen), and qRT-PCR was performed as described above. Primer sequences for qRT-PCR are shown in Dataset S17.

Sequence Data Analysis.

ChIP-seq samples prepared from DLD-1 cells using anti–β-catenin antibody were sequenced using the Illumina HiSeq 2500 system, and raw reads were mapped to the human reference genome (hg18) using Bowtie2 2.1.0. ChIP-seq peaks were called using MACS 2.0.9 (https://github.com/taoliu/MACS/) with default parameters. Additional information such as gene symbols and mRNA names were annotated according to the University of California. Santa Cruz (UCSC) hg18 and fRNADB databases (44). Distal promoter regions of the genes were defined as sequences that mapped ±2 kb upstream from the TSS.

RNA-seq samples from DLD-1 cells transfected with an siRNA targeting β-catenin were also sequenced using the Illumina HiSeq. 2500 system, and raw reads were mapped to the human reference genome (hg18) using TopHat 2.0.8 (ccb.jhu.edu/software/tophat/index.shtml). Gene-expression levels were calculated by Cuffdiff 2 (cole-trapnell-lab.github.io/cufflinks/). Additional information such as gene symbols and mRNA names were annotated according to the iGenome UCSC hg18 and fRNADB databases. Genes with Q-value <0.05 were considered to be differentially expressed.

RNA-seq samples from HCT116 cells transfected with an siRNA targeting ASBEL were also sequenced using the Illumina HiSeq. 2000 system, and raw reads were mapped to the human reference genome (hg19) using TopHat 2.0.8. Gene-expression levels were calculated by Cuffdiff 2. Genes with fewer than one fragment per kilobase of exon per million reads mapped (FPMK) in either ASBEL knockdown or control samples were removed. Genes with P value < 0.05 and FPKM >1 were considered to be differentially expressed.

Functional characterization of these genes was performed using the Molecular Signatures Database (MSigDB) Investigate Gene Sets (41). Analyses of upstream regulator and molecular and cellular functions were performed using the IPA software tool (Ingenuity Systems).

Statistical Analysis.

Statistical analysis was performed using a Mann–Whitney u test or an unpaired two-tailed Student’s t test. A P value < 0.05 was considered statistically significant.

Acknowledgments

This work was supported by the Innovative Technology Research Program of Innovative Cell Biology (Integrated Systems Analysis of Cellular Oncogenic Signaling Networks), Grants-in-Aid for Scientific Research on Innovative Areas “Integrative Research on Cancer Microenvironment Network” and “Non-Coding RNA Neo-Taxonomy,” Grants-in-Aid for Scientific Research (C), and the Project for the Development of Innovative Research on Cancer Therapeutics, Ministry of Education, Culture, Sports, Science, and Technology, Japan, and Takeda Science Foundation.

Footnotes

↵¹K.T. and A.K. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: akiyama{at}iam.u-tokyo.ac.jp.

Author contributions: K.T., A.K., and T.A. designed research; K.T., A.K., Y.T., Y.K., and K.S. performed research; K.T., T.N., and M.O.-H. analyzed data; Y.K. and K.S. performed sequence analysis; and K.T. and T.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the DNA Data Bank of Japan Sequence Read Archive (DRA) database, trace.ddbj.nig.ac.jp/dra/index.html (accession no. DRA004515).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1605938113/-/DCSupplemental.

View Abstract

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Proceedings of the National Academy of Sciences: 118 (3)

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[1] ↵
Clevers H,
Nusse R
(2012) Wnt/β-catenin signaling and disease. Cell 149(6):1192–1205.
.
OpenUrl CrossRef PubMed

[2] Clevers H,

[3] Nusse R

[4] ↵
Lien W-H,
Fuchs E
(2014) Wnt some lose some: Transcriptional governance of stem cells by Wnt/β-catenin signaling. Genes Dev 28(14):1517–1532.
.
OpenUrl Abstract/FREE Full Text

[5] Lien W-H,

[6] Fuchs E

[7] ↵
Niehrs C
(2012) The complex world of WNT receptor signalling. Nat Rev Mol Cell Biol 13(12):767–779.
.
OpenUrl CrossRef PubMed

[8] Niehrs C

[9] ↵
Holland JD,
Klaus A,
Garratt AN,
Birchmeier W
(2013) Wnt signaling in stem and cancer stem cells. Curr Opin Cell Biol 25(2):254–264.
.
OpenUrl CrossRef PubMed

[10] Holland JD,

[11] Klaus A,

[12] Garratt AN,

[13] Birchmeier W

[14] ↵
Rinn JL,
Chang HY
(2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166.
.
OpenUrl CrossRef PubMed

[15] Rinn JL,

[16] Chang HY

[17] ↵
Wang KC,
Chang HY
(2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43(6):904–914.
.
OpenUrl CrossRef PubMed

[18] Wang KC,

[19] Chang HY

[20] ↵
Ulitsky I,
Bartel DP
(2013) lincRNAs: Genomics, evolution, and mechanisms. Cell 154(1):26–46.
.
OpenUrl CrossRef PubMed

[21] Ulitsky I,

[22] Bartel DP

[23] ↵
Rinn JL, et al.
(2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129(7):1311–1323.
.
OpenUrl CrossRef PubMed

[24] Rinn JL, et al.

[25] ↵
Zhao J,
Sun BK,
Erwin JA,
Song J-J,
Lee JT
(2008) Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322(5902):750–756.
.
OpenUrl Abstract/FREE Full Text

[26] Zhao J,

[27] Sun BK,

[28] Erwin JA,

[29] Song J-J,

[30] Lee JT

[31] ↵
Nagano T, et al.
(2008) The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322(5908):1717–1720.
.
OpenUrl Abstract/FREE Full Text

[32] Nagano T, et al.

[33] ↵
Wang X, et al.
(2008) Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 454(7200):126–130.
.
OpenUrl CrossRef PubMed

[34] Wang X, et al.

[35] ↵
Taniue K, et al.
(2016) Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1. Proc Natl Acad Sci USA 113(5):1273–1278.
.
OpenUrl Abstract/FREE Full Text

[36] Taniue K, et al.

[37] ↵
Yanagida S, et al.
(2013) ASBEL, an ANA/BTG3 antisense transcript required for tumorigenicity of ovarian carcinoma. Sci Rep 3:1305.
.
OpenUrl PubMed

[38] Yanagida S, et al.

[39] ↵
Tripathi V, et al.
(2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39(6):925–938.
.
OpenUrl CrossRef PubMed

[40] Tripathi V, et al.

[41] ↵
Yoon J-H, et al.
(2012) LincRNA-p21 suppresses target mRNA translation. Mol Cell 47(4):648–655.
.
OpenUrl CrossRef PubMed

[42] Yoon J-H, et al.

[43] ↵
Carrieri C, et al.
(2012) Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491(7424):454–457.
.
OpenUrl CrossRef PubMed

[44] Carrieri C, et al.

[45] ↵
Gong C,
Maquat LE
(2011) lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements. Nature 470(7333):284–288.
.
OpenUrl CrossRef PubMed

[46] Gong C,

[47] Maquat LE

[48] ↵
Huarte M
(2015) The emerging role of lncRNAs in cancer. Nat Med 21(11):1253–1261.
.
OpenUrl CrossRef PubMed

[49] Huarte M

[50] ↵
Gupta RA, et al.
(2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464(7291):1071–1076.
.
OpenUrl CrossRef PubMed

[51] Gupta RA, et al.

[52] ↵
Slattery C,
Ryan MP,
McMorrow T
(2008) E2A proteins: Regulators of cell phenotype in normal physiology and disease. Int J Biochem Cell Biol 40(8):1431–1436.
.
OpenUrl CrossRef PubMed

[53] Slattery C,

[54] Ryan MP,

[55] McMorrow T

[56] ↵
Kee BL
(2009) E and ID proteins branch out. Nat Rev Immunol 9(3):175–184.
.
OpenUrl CrossRef PubMed

[57] Kee BL

[58] ↵
Watanabe K, et al.
(2014) Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer. PLoS One 9(3):e92317.
.
OpenUrl CrossRef PubMed

[59] Watanabe K, et al.

[60] ↵
Krämer A,
Green J,
Pollard J Jr,
Tugendreich S
(2014) Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30(4):523–530.
.
OpenUrl Abstract/FREE Full Text

[61] Krämer A,

[62] Green J,

[63] Pollard J Jr,

[64] Tugendreich S

[65] ↵
Perez-Moreno MA, et al.
(2001) A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions. J Biol Chem 276(29):27424–27431.
.
OpenUrl Abstract/FREE Full Text

[66] Perez-Moreno MA, et al.

[67] ↵
Cubillo E, et al.
(2013) E47 and Id1 interplay in epithelial-mesenchymal transition. PLoS One 8(3):e59948.
.
OpenUrl CrossRef PubMed

[68] Cubillo E, et al.

[69] ↵
Kijanka G, et al.
(2010) Human IgG antibody profiles differentiate between symptomatic patients with and without colorectal cancer. Gut 59(1):69–78.
.
OpenUrl Abstract/FREE Full Text

[70] Kijanka G, et al.

[71] ↵
Li C,
Cai S,
Wang X,
Jiang Z
(2014) Hypomethylation-associated up-regulation of TCF3 expression and recurrence in stage II and III colorectal cancer. PLoS One 9(11):e112005.
.
OpenUrl CrossRef PubMed

[72] Li C,

[73] Cai S,

[74] Wang X,

[75] Jiang Z

[76] ↵
Asirvatham AJ,
Carey JPW,
Chaudhary J
(2007) ID1-, ID2-, and ID3-regulated gene expression in E2A positive or negative prostate cancer cells. Prostate 67(13):1411–1420.
.
OpenUrl CrossRef PubMed

[77] Asirvatham AJ,

[78] Carey JPW,

[79] Chaudhary J

[80] ↵
Patel D,
Chaudhary J
(2012) Increased expression of bHLH transcription factor E2A (TCF3) in prostate cancer promotes proliferation and confers resistance to doxorubicin induced apoptosis. Biochem Biophys Res Commun 422(1):146–151.
.
OpenUrl CrossRef PubMed

[81] Patel D,

[82] Chaudhary J

[83] ↵
Krishnamachary B, et al.
(2006) Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B. Cancer Res 66(5):2725–2731.
.
OpenUrl Abstract/FREE Full Text

[84] Krishnamachary B, et al.

[85] ↵
Thompson MR,
Xu D,
Williams BRG
(2009) ATF3 transcription factor and its emerging roles in immunity and cancer. J Mol Med (Berl) 87(11):1053–1060.
.
OpenUrl CrossRef PubMed

[86] Thompson MR,

[87] Xu D,

[88] Williams BRG

[89] ↵
Gargiulo G, et al.
(2013) In vivo RNAi screen for BMI1 targets identifies TGF-β/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis. Cancer Cell 23(5):660–676.
.
OpenUrl CrossRef PubMed

[90] Gargiulo G, et al.

[91] ↵
Hackl C, et al.
(2010) Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition. BMC Cancer 10:668.
.
OpenUrl CrossRef PubMed

[92] Hackl C, et al.

[93] ↵
Yuan X, et al.
(2013) ATF3 suppresses metastasis of bladder cancer by regulating gelsolin-mediated remodeling of the actin cytoskeleton. Cancer Res 73(12):3625–3637.
.
OpenUrl Abstract/FREE Full Text

[94] Yuan X, et al.

[95] ↵
Jan Y-H, et al.
(2012) Adenylate kinase-4 is a marker of poor clinical outcomes that promotes metastasis of lung cancer by downregulating the transcription factor ATF3. Cancer Res 72(19):5119–5129.
.
OpenUrl CrossRef PubMed

[96] Jan Y-H, et al.

[97] ↵
Engel I,
Murre C
(2001) The function of E- and Id proteins in lymphocyte development. Nat Rev Immunol 1(3):193–199.
.
OpenUrl CrossRef PubMed

[98] Engel I,

[99] Murre C

[100] ↵
Mondal T, et al.
(2015) MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures. Nat Commun 6:7743.
.
OpenUrl CrossRef PubMed

[101] Mondal T, et al.

[102] ↵
Bottomly D,
Kyler SL,
McWeeney SK,
Yochum GS
(2010) Identification of β-catenin binding regions in colon cancer cells using ChIP-Seq. Nucleic Acids Res 38(17):5735–5745.
.
OpenUrl Abstract/FREE Full Text

[103] Bottomly D,

[104] Kyler SL,

[105] McWeeney SK,

[106] Yochum GS

[107] ↵
Hatzis P, et al.
(2008) Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells. Mol Cell Biol 28(8):2732–2744.
.
OpenUrl Abstract/FREE Full Text

[108] Hatzis P, et al.

[109] ↵
Frietze S, et al.
(2012) Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3. Genome Biol 13(9):R52.
.
OpenUrl CrossRef PubMed

[110] Frietze S, et al.

[111] ↵
Matsuura K, et al.
(2011) Identification of a link between Wnt/β-catenin signalling and the cell fusion pathway. Nat Commun 2:548.
.
OpenUrl CrossRef PubMed

[112] Matsuura K, et al.

[113] ↵
Taniue K,
Oda T,
Hayashi T,
Okuno M,
Akiyama T
(2011) A member of the ETS family, EHF, and the ATPase RUVBL1 inhibit p53-mediated apoptosis. EMBO Rep 12(7):682–689.
.
OpenUrl Abstract/FREE Full Text

[114] Taniue K,

[115] Oda T,

[116] Hayashi T,

[117] Okuno M,

[118] Akiyama T

[119] ↵
Hoshino M, et al.
(1999) Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1. J Biol Chem 274(25):17837–17844.
.
OpenUrl Abstract/FREE Full Text

[120] Hoshino M, et al.

[121] ↵
Kin T, et al.
(2007) fRNAdb: A platform for mining/annotating functional RNA candidates from non-coding RNA sequences. Nucleic Acids Res 35(Database issue):D145–D148.
.
OpenUrl Abstract/FREE Full Text

[122] Kin T, et al.

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ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells

Significance

Abstract

Results

The lncRNA ASBEL Is a Target of β-Catenin.

ASBEL Is Required for the Tumorigenicity of Colon Tumor Cells.

ASBEL Is Associated with the Transcription Factor TCF3.

The ASBEL–TCF3 Complex Is Important for Wnt/β–Catenin Signaling.

ASBEL/TCF3-Mediated Down-Regulation of ATF3 Is Required for the Tumorigenicity of Colon Cancer Cells.

Negative Feedback Regulation of the Expression of ASBEL by ATF3 in Colon Cancer Cells.

Discussion

Materials and Methods

SI Materials and Methods

qRT-PCR Analysis.

Cell Culture.

Antibodies.

Lentivirus Production.

Tumorigenesis Assay.

RNAi.

ChIP Assay.

Constructs and Transfection.

Subcellular Fractionation.

In Situ Hybridization.

Immunohistochemical Analysis.

RNA Pull-Down Assay.

RIP Assay.

Immunoblotting Analysis.

Apoptosis.

Luciferase Assay.

RNA Transfection and RNA ChIP Assay.

Sequence Data Analysis.

Statistical Analysis.

Acknowledgments

Footnotes

References

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