HELLS regulates chromatin remodeling and its overexpression induces epigenetic silencing of multiple tumor suppressors in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer and a leading death causing cancer. Both genetic and epigenetic alterations are accumulated during the long-term HCC development. In our study, we identified a deregulation of a novel epigenetic regulator, HELicase, Lymphoid-Specific (HELLS) in HCC and unearthed its molecular mechanism in carcinogenesis. Previous studies have shown that HELLS involved in DNA methylation during development, while its chromatin remodelling capacity was largely elusive and remained to be explored. Clinicopathologically, our research showed the overexpression of HELLS was associated with aggressive tumor features, including advanced tumor stages, early onset age and present of direct liver invasion etc. Molecularly, we discovered a new HELLS upstream regulator, a transcription factor called SP1 whose hyperactivation caused HELLS deregulation in HCC. Functionally, activating HELLS expression in HCC cell lines by CRISPR SAM system augmented cell proliferation, colony formation ability and migration rate. Consistently, depleting HELLS in HCC cell lines by CRISPR/Cas9 genome editing system and lentiviral shRNA infection resulted in remarkably cell growth inhibition, cell migration suppression, apoptosis induction and reverse of cancer metabolic features in HCC cell line model. In the animal models, the subcutaneous and orthotopic tumor implantation models showed that ablation of HELLS also mitigated tumorigenicity and lung metastasis in vivo. Mechanistically, our RNA-seq revealed that knockout of HELLS leads to the re-expression of multiple tumor suppressor genes, including CDH1, XAF1, and CREB3L3 etc. These re-expressed genes were significantly enriched in the EMT and migration pathway. Moreover, the underlying mechanism was uncovered by Reduced representation bisulfite sequencing (RRBS), MNase-seq and ChIP-qPCR. Consistent with previous studies, HELLS play an important role in maintaining DNA methylation. Knockout of HELLS induced genome-wide hypomethylation, especially in intragenic regions and activated silenced genes in HCC cells. Importantly, MNase-Seq results revealed that HELLS controls the nucleosome-depletion region (NDR) in the transcription start site (TSS). HELLS binds to the TSS and enhancer regions and control the nucleosome occupancy around these areas. The shift of nucleosome can block the accession of transcription factors to gene promoters and enhancers. This may subsequently silence the expression of HELLS regulated tumor suppressor genes and then promote cancer progression. We also developed a CRISPR-based HELLS-depended epigenetic editing system to further validate that tethering HELLS to nucleosome-free region was sufficient to induce epigenetic silencing of CDH1. Collectively, our study confirmed that HELLS overexpressed in HCC and was contributed to hyperactivation of SP1 transcription factor. By using various techniques, we demonstrated HELLS regulates DNA methylation and chromatin remodelling in HCC. Up-regulation of HELLS silenced multiple important tumor suppressors involving in multiple cancer hallmarks to promote HCC progression.