Abstract
Background
ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions.
Methods
For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets.
Results
Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression.
Conclusions
Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.
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Abbreviations
- EMT:
-
Epithelial-to-mesenchymal transition
- ChIP:
-
Chromatin immunoprecipitation
- ChIP-Seq:
-
Chromatin immunoprecipitation-sequencing
- ChIP-qPCR:
-
Chromatin immunoprecipitation-quantitative polymerase chain reaction
- HCC:
-
Hepatocellular carcinoma
- RT-qPCR:
-
Real time- quantitative polymerase chain reaction
- ZEB2:
-
Zinc finger E-box binding homeobox 2
- GALNT3:
-
Polypeptide N-acetylgalactosaminyltransferase 3
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Acknowledgements
The study was funded by the Scientific and Technical Research Council of Turkey (TUBITAK) grant 111S484 (to T.Y.).
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T.Y., A.E.S. and P.B-E. designed the study. P.B-E., M.C., I.Y-C. and G.O. performed the experiments. P.B-E., N.T. and T.Y. analyzed and interpreted the data. T.Y., A.E.S. and P.B-E. wrote the manuscript. All authors read and approved the final manuscript.
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Balcik-Ercin, P., Cetin, M., Yalim-Camci, I. et al. Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors. Cell Oncol. 41, 379–393 (2018). https://doi.org/10.1007/s13402-018-0375-7
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DOI: https://doi.org/10.1007/s13402-018-0375-7