Evaluating The Transcriptional Regulation of Cdh1 by Grhl3 in Different Cellular Models

Abstract

The calcium-dependent adhesion protein E-cadherin encoded by the Cdh1 gene is a member of a large family conferring proper establishment of adherens junction. The expression of Cdh1 is critical and is observed in epithelial cells. E-cadherin expression is also essential for establishing the pluripotent state in embryonic stem cells. Cdh1 transcriptional regulation has been the focus of research for many years; early reports identified repressors of Cdh1 since its downregulation is essential for the initiation of the epithelial to mesenchymal transition. Restoring the expression of Cdh1 is thought to be simply due to the disengagement of Cdh1 silencers from its promoter. Recent studies supported the presence of dedicated activators of Cdh1 expression, including members of the Grhl and Ets family of transcription factors. Here we evaluated the regulatory potential of Grhl3 on the Cdh1 promoter in different cellular models to understand the extent of the transcriptional relationship with Cdh1. We utilized several approaches, such as the correlation of expression by loss and gain of function, ChIP, and luciferase reporter assays. As a result, we found that Grhl3 is a potent regulator of Cdh1 in cells of epithelial origin. Moreover, Grhl3 was sufficient to re-establish Cdh1 expression in the murine hepatoma cells Hepa1-6. Improved understanding of the regulation of Cdh1 is essential for the perception of how the epithelial to mesenchymal and the mesenchymal to epithelial transitions are regulated, as they play a crucial role in metastasis, which will pave the way for better management of the metastatic disease.

Keywords

• E-cadherin
• transcriptional regulation
• Grhl3

References

1. Almeida, MS and Bray, SJ, 2005. Regulation of post-embryonic neuroblasts by Drosophila Grainyhead. Mech Dev 122 (12): 1282-1293.
2. Alotaibi, H, Basilicata, MF, Shehwana, H, Kosowan, T, Schreck, I, Braeutigam, C, Konu, O, Brabletz, T and Stemmler, MP, 2015. Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition. Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms 1849 (6): 731-742.
3. Auden, A, Caddy, J, Wilanowski, T, Ting, SB, Cunningham, JM and Jane, SM, 2006. Spatial and temporal expression of the Grainyhead-like transcription factor family during murine development. Gene Expr Patterns 6 (8): 964-970.
4. Bedzhov, I, Alotaibi, H, Basilicata, MF, Ahlborn, K, Liszewska, E, Brabletz, T and Stemmler, MP, 2013. Adhesion, but not a specific cadherin code, is indispensable for ES cell and induced pluripotency. Stem Cell Res 11 (3): 1250-1263.
5. Brabletz, T, 2012. To differentiate or not--routes towards metastasis. Nat Rev Cancer 12 (6): 425-436.
6. Brabletz, T, Kalluri, R, Nieto, MA and Weinberg, RA, 2018. EMT in cancer. Nat Rev Cancer 18 (2): 128-134.
7. Fazilaty, H, Rago, L, Kass Youssef, K, Ocana, OH, Garcia-Asencio, F, Arcas, A, Galceran, J and Nieto, MA, 2019. A gene regulatory network to control EMT programs in development and disease. Nat Commun 10 (1): 5115.
8. Frisch, SM, Farris, JC and Pifer, PM, 2017. Roles of Grainyhead-like transcription factors in cancer. Oncogene 36 (44): 6067-6073.

9. Kalluri, R and Weinberg, RA, 2009. The basics of epithelial-mesenchymal transition. J Clin Invest 119 (6): 1420-1428.
10. Lavin, DP and Tiwari, VK, 2020. Unresolved Complexity in the Gene Regulatory Network Underlying EMT. Front Oncol 10: 554.
11. Li, R, Liang, J, Ni, S, Zhou, T, Qing, X, Li, H, He, W, Chen, J, Li, F, Zhuang, Q, Qin, B, Xu, J, Li, W, Yang, J, Gan, Y, Qin, D, Feng, S, Song, H, Yang, D, Zhang, B, Zeng, L, Lai, L, Esteban, MA and Pei, D, 2010. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7 (1): 51-63.
12. Mlacki, M, Kikulska, A, Krzywinska, E, Pawlak, M and Wilanowski, T, 2015. Recent discoveries concerning the involvement of transcription factors from the Grainyhead-like family in cancer. Exp Biol Med (Maywood) 240 (11): 1396-1401.
13. Niwa, H, 2007. How is pluripotency determined and maintained? Development 134 (4): 635-646.
14. Redmer, T, Diecke, S, Grigoryan, T, Quiroga-Negreira, A, Birchmeier, W and Besser, D, 2011. E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming. EMBO Rep 12 (7): 720-726.
15. Sengez, B, Aygun, I, Shehwana, H, Toyran, N, Tercan Avci, S, Konu, O, Stemmler, MP and Alotaibi, H, 2019. The Transcription Factor Elf3 Is Essential for a Successful Mesenchymal to Epithelial Transition. Cells 8 (8).
16. Stemmler, MP, 2008. Cadherins in development and cancer. Mol Biosyst 4 (8): 835-850.
17. Stemmler, MP, Hecht, A and Kemler, R, 2005. E-cadherin intron 2 contains cis-regulatory elements essential for gene expression. Development 132 (5): 965-976.
18. Stemmler, MP, Hecht, A, Kinzel, B and Kemler, R, 2003. Analysis of regulatory elements of E-cadherin with reporter gene constructs in transgenic mouse embryos. Dev Dyn 227 (2): 238-245.
19. Terashima, M, Ishimura, A, Wanna-Udom, S and Suzuki, T, 2018. MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells. J Biol Chem 293 (47): 18016-18030.
20. Thiery, JP, Acloque, H, Huang, RY and Nieto, MA, 2009. Epithelial-mesenchymal transitions in development and disease. Cell 139 (5): 871-890.
21. Voutsadakis, IA, 2015. The network of pluripotency, epithelial-mesenchymal transition, and prognosis of breast cancer. Breast Cancer (Dove Med Press) 7: 303-319.
22. Werth, M, Walentin, K, Aue, A, Schonheit, J, Wuebken, A, Pode-Shakked, N, Vilianovitch, L, Erdmann, B, Dekel, B, Bader, M, Barasch, J, Rosenbauer, F, Luft, FC and Schmidt-Ott, KM, 2010. The transcription factor grainyhead-like 2 regulates the molecular composition of the epithelial apical junctional complex. Development 137 (22): 3835-3845.
23. Xu, H, Liu, C, Zhao, Z, Gao, N, Chen, G, Wang, Y and Cui, J, 2014. Clinical implications of GRHL3 protein expression in breast cancer. Tumour Biol 35 (3): 1827-1831.
24. Zhao, P, Guo, S, Tu, Z, Di, L, Zha, X, Zhou, H and Zhang, X, 2016. Grhl3 induces human epithelial tumor cell migration and invasion via downregulation of E-cadherin. Acta Biochim Biophys Sin (Shanghai) 48 (3): 266-274.