Sirt6-dependent gene regulation of oncofetal gene loci in hepatocytes
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Abstract
Hepatocellular carcinoma (HCC), the primary malignant tumor of liver, represents the third leading cause of cancer-related deaths globally due to the lack of effective treatment options. Accumulating studies have revealed that SIRT6, a NAD+-dependent histone deacetylase, plays pivotal roles in liver-related disease and liver tumor formation. Genetic deletion of Sirt6 in mice develops a pro-oncogenic phenotype in the liver with dramatic elevation of oncofetal genes typical for HCC, such as Igf2, H19 and α-Fetoprotein (Afp). However, the underlying mechanisms regulating these genes remain unknown. For delineating the role of Sirt6 in the tumor-related phenotype, Sirt6 knockout mice model together with primary human hepatocytes and human hepatoma cells (Hep3B) were used in this study.
The present study shows that Sirt6 suppresses the expression of these oncofetal genes. Functional analysis of the Igf2/H19 gene locus as a model showed that loss of Sirt6 causes nucleosome remodeling, altered chromatin loop, enhancer switch from the poised (enriched with H3K27me3 and H3K4me1) to active (enriched with H3K27ac and H3K4me1) by recruiting pioneer factors Foxa1 and Foxa2. Interestingly, these alterations establish a super-enhancer to drive H19 and Igf2 transcription. Moreover, Sirt6 deletion causes cytoplasmic translocation of Suz12, a core component of the Polycomb repressive complex 2 (PRC2), and weakens PRC2-mediated repression at the Igf2/H19 gene locus. Further analysis of Hep3B cells in relation to primary human hepatocytes indicated that this regulatory mechanism is relevant for human cancer development.
Besides, this study also suggests the role of Sirt6 in hepatic differentiation. Sirt6 knockout leads to the increased expression of embryonic hepatoblast markers and the reduced transcription of adult hepatocytes markers. Correspondingly, this trend reversed when SIRT6 was overexpressed in Hep3B cells. Functional analysis of this regulatory mechanism at the Alb/Afp gene locus as a model showed that Sirt6 deletion causes epigenetic and genetic alterations, which induces Foxa2 switch from enhancer of Alb (hepatocytes marker) to Afp (hepatoblast marker). However, PRC2-mediated repression is not evident at these temporal enhancers.
Altogether, in hepatocytes, loss of Sirt6 induces disruption of enhancer activity through genetic and epigenetic alterations, resulting in activation of a tumorigenic phenotype.