A genetic screen reveals Foxa3 and TNFR1 as key regulators of liver repopulation

Abstract

The fundamental question of which genes are most important in controlling liver regeneration remains unanswered. We employed a parallel screen to test the impact of 43 selected genes on liver repopulation in the Fah −/− mouse model of hereditary tyrosinemia. We discovered that the transcription factor Foxa3 was a strong promoter of liver regeneration, while tumor necrosis factor receptor 1 (TNFR1) was the most significant suppressor of repopulation among all of the genes tested. Our approach enabled the identification of these factors as important regulators of liver repopulation and potential drug targets for the promotion of liver repopulation.Supplemental material is available for this article.Received January 19, 2015; revised version accepted April 7, 2015. A better understanding of how the liver regenerates in its attempts to recover from injuries, such as from exposure to drugs or alcohol or from infection by hepatitis viruses, could lead to new treatments with immense impact on the natural history of liver diseases. Previous efforts have relied on the easily accessible partial hepatectomy model in rodents (Fausto and Mead 1989;Fausto 1990Fausto , 2000Fausto , 2004Fausto et al. 1995;Michalopoulos 2010), and many candidate genes were identified by expression profiling after partial hepatectomy (Kelley-Loughnane et al. 2002;Su et al. 2002;Fukuhara et al. 2003;White et al. 2005;Guo and Xu 2008;Cao et al. 2009;Li et al. 2009;Jiang et al. 2011;Schug et al. 2013). These results have implicated numerous pathways as being involved in regeneration, including growth factor pathways, kinases, transcription factors, and nuclear receptors, but did not inform us on the relative impact of the various genes on liver repopulation following toxic injury. Sequencing of multiple hepatocellular carcinomas (HCCs) identified a number of regulators of proliferation as well (Nishida and Kudo 2013;Tornesello et al. 2013). Despite an abundant number of genes that have been identified, no single gene, when mutated in animal models, seems to be absolutely required for the recovery of liver mass following partial hepatectomy, suggesting that genetic redundancy may be an adaptation for such an important and conserved biological process as liver regeneration (Vogel 2006).Genetic screens could be used to sort out the pathways that are key to the genetic control of liver regeneration. Recently, a shRNA screen of genes mutated in HCC was employed in a repopulation model to suggest the map kinase MKK4 as a regulator of liver repopulation (Wuestefeld et al. 2013). The experimental paradigm used was the Fah −/− mouse model of liver repopulation, which has a defect in the last step of tyrosine catabolism, resulting in accumulation of the toxic metabolite fumarylacetoacetate (FAA) and injury to hepatocytes (Grompe 2001). Fah −/− mice can be kept alive by treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC), a drug that inhibits an upstream enzyme in the tyrosine catabolic cascade to prevent formation of FAA. This...

Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform†

“…Nodules of FAH‐positive hepatocytes were found in the livers, consistent with the clonal expansion of stably transduced hepatocytes seen with Sleeping Beauty –mediated gene therapy in Fah –/– mice (Fig. c, middle column) . MYC protein was strongly positive within the nuclei of hepatocytes in all three Fah –/– ; dCas9 + mice injected with TAlib1 but not in Fah –/– control mice.…”

“…Multiple independent gRNAs activated Myc expression to give a robust growth phenotype in a liver repopulation model. The phenotype is analogous to what we previously observed with Myc cDNA overexpression . Indeed, it was shown that liver‐directed Myc cDNA overexpression in transgenic animals resulted in hepatocellular carcinoma by 12‐15 months .…”

“…The results suggest that activation of these loci had a strong epistatic impact on Myc‐dependent tumorigenesis. Previously, we had found that TNFR1 cDNA overexpression with a strong chicken beta‐actin promoter blocked liver repopulation in Fah –/– mice, which was not observed with the CRISPRa platform . It is possible that the level of expression of TNFR1 with the cDNA was greater than that with dCas9 system used here.…”

“…Hydrodynamic tail vein injection of plasmids that contain an Fah cDNA transgene, followed by removal of nitisinone, leads to repopulation of the Fah –/– liver with hepatocytes that stably express FAH . In addition, any “cargo” genes on the plasmids will be expressed in repopulating hepatocytes as well …”

Novel Genetic Activation Screening in Liver Repopulation and Cancer: Now CRISPR Than Ever!

“…The rationale was a previous study by Wangensteen et al demonstrating cMyc up‐regulation and Tnfrsf1a suppression after hepatocyte repopulation in FAH –/– mice. ( ) Further, multiple gRNAs per gene were used as this created a synergistic effect. ( ) In Lib2, the authors maintained gRNAs targeted toward cMyc, Tnfrsf1a, and control and introduced gRNAs specific for solute carrier family 7 member 11 (Slc7a11), another gene up‐regulated during repopulation in FAH –/– mice, and tumor protein P53 (Tp53), a tumor suppressor.…”