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Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors
Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors
Background: ZEB2 is among transcriptional repressors that regulate epithelial-to-mesenchymal transition through binding to bipartite E-box motifs in gene regulatory regions. Despite the high frequency of E-boxes on the genome and multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets have been identified so far. Hence, we explored genome-wide ZEB2 binding by Chromatin immunoprecipitation-sequencing study under endogenous-ZEB2 expression conditions.

Methods: we designed a ChIP-Seq study by using a homemade anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells with high-endogenous ZEB2 expression. We validated ChIP-Seq targets by ChIP-qPCR assays and explored ZEB2-dependent expression of target genes in ZEB2-repressed and –overexpressed cells by RT-qPCR and Western blotting. Changes in gene expression were assessed in human tumor cDNA arrays by RT-qPCR and, differential expression and correlation analyses were performed in expO and Human Protein Atlas datasets.

Results: more than 500 genes were annotated and intervals related to these genes were found to include 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 GALNT3 gene as a target with the highest but inversely correlated expression. Remarkably, GALNT3 also displayed the highest enrichment in ChIP validation assays. In our analyses of tumor cDNA arrays and expO datasets significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 transcripts were detected in most of the tumors. Moreover, protein expression of ZEB2 and GALNT3 was explored in "Human Protein Atlas" database and a negative correlation was observed in all analyzed tumor types, except malignant melanoma. In contrast to negative or weak expression of ZEB2, tumor tissues displayed strong or moderate GALNT3 staining.

Conclusions: our findings that ZEB2 negatively regulates a GalNAc-transferase involved in O-glycosylation add another level of complexity to the understanding of the role of ZEB2 in EMT and cancer metastasis. This, in turn, implicates that the proteins glycosylated by GALNT3 might be exploited as novel diagnostics and therapeutic targets in human cancers.
ZEB2, GALNT3, antibody validation, ChIP-sequencing, gene regulation, tissue expression
1570-5870
1-15
Balcik-Ercin, Pelin
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Cetin, Metin
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Yalin-Camci, Irem
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Odabas, Gorkem
655b103c-380c-4e8b-95f1-9cb7a0ae1f51
Toktay, Nurettin
964eec0c-03cb-404f-9267-23762eae29e9
Sayan, Abdulkadir
d1dbbcad-9c53-47c1-8b7e-1b45cc56e077
Yagci, Tamer
b2d47a67-27a0-4bbf-a49a-dbc13fd2e387
Balcik-Ercin, Pelin
1bd1b6b3-aad0-4096-8bd4-d4de7a8dd2d1
Cetin, Metin
d23d0fc6-999d-45aa-b66a-4af013d96038
Yalin-Camci, Irem
fa6505c7-cdf4-4f43-ba73-6fc3085f7e5b
Odabas, Gorkem
655b103c-380c-4e8b-95f1-9cb7a0ae1f51
Toktay, Nurettin
964eec0c-03cb-404f-9267-23762eae29e9
Sayan, Abdulkadir
d1dbbcad-9c53-47c1-8b7e-1b45cc56e077
Yagci, Tamer
b2d47a67-27a0-4bbf-a49a-dbc13fd2e387

Balcik-Ercin, Pelin, Cetin, Metin, Yalin-Camci, Irem, Odabas, Gorkem, Toktay, Nurettin, Sayan, Abdulkadir and Yagci, Tamer (2018) Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors. Cellular Oncology, 1-15. (doi:10.1007/s13402-018-0375-7).

Record type: Article

Abstract

Background: ZEB2 is among transcriptional repressors that regulate epithelial-to-mesenchymal transition through binding to bipartite E-box motifs in gene regulatory regions. Despite the high frequency of E-boxes on the genome and multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets have been identified so far. Hence, we explored genome-wide ZEB2 binding by Chromatin immunoprecipitation-sequencing study under endogenous-ZEB2 expression conditions.

Methods: we designed a ChIP-Seq study by using a homemade anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells with high-endogenous ZEB2 expression. We validated ChIP-Seq targets by ChIP-qPCR assays and explored ZEB2-dependent expression of target genes in ZEB2-repressed and –overexpressed cells by RT-qPCR and Western blotting. Changes in gene expression were assessed in human tumor cDNA arrays by RT-qPCR and, differential expression and correlation analyses were performed in expO and Human Protein Atlas datasets.

Results: more than 500 genes were annotated and intervals related to these genes were found to include 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 GALNT3 gene as a target with the highest but inversely correlated expression. Remarkably, GALNT3 also displayed the highest enrichment in ChIP validation assays. In our analyses of tumor cDNA arrays and expO datasets significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 transcripts were detected in most of the tumors. Moreover, protein expression of ZEB2 and GALNT3 was explored in "Human Protein Atlas" database and a negative correlation was observed in all analyzed tumor types, except malignant melanoma. In contrast to negative or weak expression of ZEB2, tumor tissues displayed strong or moderate GALNT3 staining.

Conclusions: our findings that ZEB2 negatively regulates a GalNAc-transferase involved in O-glycosylation add another level of complexity to the understanding of the role of ZEB2 in EMT and cancer metastasis. This, in turn, implicates that the proteins glycosylated by GALNT3 might be exploited as novel diagnostics and therapeutic targets in human cancers.

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sayan cellular oncology - Accepted Manuscript
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Accepted/In Press date: 22 February 2018
e-pub ahead of print date: 7 March 2018
Keywords: ZEB2, GALNT3, antibody validation, ChIP-sequencing, gene regulation, tissue expression

Identifiers

Local EPrints ID: 418576
URI: https://eprints.soton.ac.uk/id/eprint/418576
ISSN: 1570-5870
PURE UUID: 79ad609a-4aed-45b3-b7a2-1c138704f23a

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Date deposited: 12 Mar 2018 17:30
Last modified: 13 Mar 2019 18:49

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Author: Pelin Balcik-Ercin
Author: Metin Cetin
Author: Irem Yalin-Camci
Author: Gorkem Odabas
Author: Nurettin Toktay
Author: Tamer Yagci

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