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Genome-wide identification of direct HBx genomic targets.

ABSTRACT: The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV.ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication.Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

SUBMITTER: Guerrieri F

PROVIDER: S-EPMC5316204 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Genome-wide identification of direct HBx genomic targets.

Guerrieri Francesca F Belloni Laura L D'Andrea Daniel D Pediconi Natalia N Le Pera Loredana L Testoni Barbara B Scisciani Cecilia C Floriot Oceane O Zoulim Fabien F Tramontano Anna A Levrero Massimo M

BMC genomics 20170217 1

<h4>Background</h4>The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analy ...[more]

PMID: 28212627

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Project description:BackgroundWe have recently identified the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha) as a novel candidate gene for autism spectrum disorder (ASD). Our independent cohort studies have consistently demonstrated the reduction of RORA transcript and/or protein levels in blood-derived lymphoblasts as well as in the postmortem prefrontal cortex and cerebellum of individuals with ASD. Moreover, we have also shown that RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. However, little is known about transcriptional targets of this nuclear receptor, particularly in humans.MethodsHere we identify transcriptional targets of RORA in human neuronal cells on a genome-wide level using chromatin immunoprecipitation (ChIP) with an anti-RORA antibody followed by whole-genome promoter array (chip) analysis. Selected potential targets of RORA were then validated by an independent ChIP followed by quantitative PCR analysis. To further demonstrate that reduced RORA expression results in reduced transcription of RORA targets, we determined the expression levels of the selected transcriptional targets in RORA-deficient human neuronal cells, as well as in postmortem brain tissues from individuals with ASD who exhibit reduced RORA expression.ResultsThe ChIP-on-chip analysis reveals that RORA1, a major isoform of RORA protein in human brain, can be recruited to as many as 2,764 genomic locations corresponding to promoter regions of 2,544 genes across the human genome. Gene ontology analysis of this dataset of genes that are potentially directly regulated by RORA1 reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion and survival, synaptogenesis, synaptic transmission and plasticity, and axonogenesis, as well as higher level functions such as development of the cortex and cerebellum, cognition, memory, and spatial learning. Independent ChIP-quantitative PCR analyses confirm binding of RORA1 to promoter regions of selected ASD-associated genes, including A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2, whose expression levels (in addition to HSD17B10) are also decreased in RORA1-repressed human neuronal cells and in prefrontal cortex tissues from individuals with ASD.ConclusionsFindings from this study indicate that RORA transcriptionally regulates A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2, and strongly suggest that reduction of this sex hormone-sensitive nuclear receptor in the brain causes dysregulated expression of these ASD-relevant genes as well as their associated pathways and functions which, in turn, may contribute to the underlying pathobiology of ASD.

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Genome-wide identification of direct HBx genomic targets.

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Genome-wide identification of direct HBx genomic targets.

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