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DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

Oncogene volume 36, pages 2435–2445 (2017)Cite this article

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Abstract

Sal-like protein 4 (SALL4), an embryonic stem cell transcriptional regulator, is re-expressed by an unknown mechanism in poor prognosis hepatocellular carcinoma (HCC), often associated with chronic hepatitis B virus (HBV) infection. Herein, we investigated the mechanism of SALL4 re-expression in HBV-related HCCs. We performed bisulfite sequencing PCR of genomic DNA isolated from HBV-related HCCs and HBV replicating cells, and examined DNA methylation of a CpG island located downstream from SALL4 transcriptional start site (TSS). HBV-related HCCs expressing increased SALL4 exhibited demethylation of specific CpG sites downstream of SALL4 TSS. Similarly, SALL4 re-expression and demethylation of these CpGs was observed in HBV replicating cells. SALL4 is also re-expressed in poor prognosis HCCs of other etiologies. Indeed, increased SALL4 expression in hepatitis C virus-related HCCs correlated with demethylation of these CpG sites. To understand how CpG demethylation downstream of SALL4 TSS regulates SALL4 transcription, we quantified by chromatin immunoprecipitation (ChIP) assays RNA polymerase II occupancy of SALL4 gene, as a function of HBV replication. In absence of HBV replication, RNA polymerase II associated with SALL4 exon1. By contrast, in HBV replicating cells RNA polymerase II occupancy of all SALL4 exons increased, suggesting CpG demethylation downstream from SALL4 TSS influences SALL4 transcriptional elongation. Intriguingly, demethylated CpGs downstream from SALL4 TSS are within binding sites of octamer-binding transcription factor 4 (OCT4) and signal transducer and activator of transcription3 (STAT3). ChIP assays confirmed occupancy of these sites by OCT4 and STAT3 in HBV replicating cells, and sequential ChIP assays demonstrated co-occupancy with chromatin remodeling BRG1/Brahma-associated factors. BRG1 knockdown reduced SALL4 expression, whereas BRG1 overexpression increased SALL4 transcription in HBV replicating cells. We conclude demethylation of CpGs located within OCT4 and STAT3 cis-acting elements, downstream of SALL4 TSS, enables OCT4 and STAT3 binding, recruitment of BRG1, and enhanced RNA polymerase II elongation and SALL4 transcription.

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Acknowledgements

We thank the French National Biological Resources Centre for frozen human liver tissues, obtained following approved consent from the French Liver Tumor Network Scientific Committee. The French Liver Tumor Network is funded by the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Agence Nationale de la Recherche (ANR). We also thank the Biological Resources Center of Centre Léon Bérard for normal liver tissues obtained following approved consent and ministerial agreement. Financial support: This work was supported by NIH grant DK044533 to OA, and French grants PAIR-CHC 2009 (contract #2009-143, project ENELIVI) from Institute National du Cancer (INCa) to PM. Shared Resources (flow cytometry and DNA sequencing) are supported by NIH grant P30CA023168 to Purdue Center for Cancer Research.

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Authors and Affiliations

  1. Department of Basic Medical Sciences, West Lafayette, IN, USA

    H Fan, Z Cui, H Zhang, S K Mani, A Diab & O Andrisani

  2. Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA

    H Fan, Z Cui, H Zhang, S K Mani, A Diab & O Andrisani

  3. Centre de Recherche en Cancérologie de Lyon, UMR INSERM 1052, Lyon Cedex, France

    L Lefrancois, N Fares & P Merle

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  1. H Fan
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Correspondence to O Andrisani.

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Fan, H., Cui, Z., Zhang, H. et al. DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection. Oncogene 36, 2435–2445 (2017). https://doi.org/10.1038/onc.2016.399

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