Abstract Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines. The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. Furthermore, we demonstrated that TCF-4J conferred high tumorigenic potential to HCC cells in contrast to TCF-4K (PLoS ONE 2012). However, the anti-apoptotic protein Bcl-xL was much expressed in TCF-4K-overexpressing HCC cells than the level in TCF-4J-overexpressing cells, suggesting that the SxxSS was involved in Bcl-xL expression. Indeed, Wnt/β-catenin signaling needs to control cell apoptosis during embryogenesis and carcinogenesis, possible direct interaction between the TCF-4 isoforms and the bcl-xL promoter region was suggested. Thus, the AIM of this study was to assess the protein-DNA interaction and its functional consequences by using ChIP assay and luciferase-reporter assay, respectively. Methods: The human HCC cell lines HAK-1A and HAK-1B were used. HAK-1B was an aggressive sister cell line derived from HAK-1A. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J (J cells), K (K cells), and K-mutants (269A, 272A, and 273A cells, respectively) were established. Western blot analysis and real-time PCR were employed to evaluate protein and mRNA expression levels, respectively. ChIP assay was performed by using SimpleChIP assay kit (Cell Signaling Technology). Two primer pairs for bcl-xL promoter region (BCL2L1(-)01Kb and BCL2L1(-)02Kb) were obtained from QIAGEN. The promoter assay was done by using LightSwitch Luciferase Assay System (SWITCHGEAR GENOMICS). Results: Robust expression of Bcl-xL protein was found in HAK-1B cells, in contrast to its low expression in HAK-1A cells. Consistently, the mRNA level in HAK-1B was 2-fold of that in HAK-1A. In ChIP assay, clear binding of TCF-4 with bcl-xL promoter regions was confirmed, encouraging us to compare the binding affinity in J cells, K cells, 269 cells, and control cells. As a result, significant TCF-4-DNA interaction was found in K cells, and, of note, the interaction was abolished in 269A cells. In the promoter assay, K cells showed the highest luciferase activity. Conclusions: The findings suggest that TCF-4 directly regulates the Bcl-xL expression. We suggest that phosphorylation at serine 269 in the TCF-4K isoform is critical to fine-tune anti-apoptotic potential through increasing the Bcl-xL expression in HCC. Citation Format: Hironori Koga, Miran Kim, Anna Nakamura, Hirohisa Yano, Mitsuhiko Abe, Yu Ikezono, Toru Nakamura, Takuji Torimura, Jack R. Wands, Michio Sata. The Wnt signal transcription factor TCF-4 directly regulates Bcl-xL expression in human hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 330. doi:10.1158/1538-7445.AM2014-330