High-Resolution Genomic Profiling of Liver Cancer Links Etiology With Mutation and Epigenetic Signatures.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization. METHODS: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection. RESULTS: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway. CONCLUSIONS: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic "scarring" of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

Quantitative effect of target translation on small RNA efficacy reveals a novel mode of interaction.

Small regulatory RNAs (sRNAs) in bacteria regulate many important cellular activities under normal conditions and in response to stress. Many sRNAs bind to the mRNA targets at or near the 5' untranslated region (UTR) resulting in translation inhibition and accelerated degradation. Often the sRNA-binding site is adjacent to or overlapping with the ribosomal binding site (RBS), suggesting a possible interplay between sRNA and ribosome binding. Here we combine quantitative experiments with mathematical modeling to reveal novel features of the interaction between small RNAs and the translation machinery at the 5'UTR of a target mRNA. By measuring the response of a library of reporter targets with varied RBSs, we find that increasing translation rate can lead to increased repression. Quantitative analysis of these data suggests a recruitment model, where bound ribosomes facilitate binding of the sRNA. We experimentally verified predictions of this model for the cell-to-cell variability of target expression. Our findings offer a framework for understanding sRNA silencing in the context of bacterial physiology.

Large-scale mapping of sequence-function relations in small regulatory RNAs reveals plasticity and modularity.

Two decades into the genomics era the question of mapping sequence to function has evolved from identifying functional elements to characterizing their quantitative properties including, in particular, their specificity and efficiency. Here, we use a large-scale approach to establish a quantitative map between the sequence of a bacterial regulatory RNA and its efficiency in modulating the expression of its targets. Our approach generalizes the sort-seq method, introduced recently to analyze promoter sequences, in order to accurately quantify the efficiency of a large library of sequence variants. We focus on two small RNAs (sRNAs) in E. coli, DsrA and RyhB, and their regulation of both repressed and activated targets. In addition to precisely identifying functional elements in the sRNAs, our data establish quantitative relationships between structural and energetic features of the sRNAs and their regulatory activity, and characterize a large set of direct and indirect interactions between nucleotides. A core of these interactions supports a model where specificity can be enhanced by a rigid molecular structure. Both sRNAs exhibit a modular design with limited cross-interactions, dividing the requirements for structural stability and target binding among modules.

Functional consequences of necdin nucleocytoplasmic localization.

BACKGROUND: Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin ß1. Finally, exclusion of necdin from the nucleus triggered extensive cell death. CONCLUSIONS/SIGNIFICANCE: These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization.