Search

ObjectivesChronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma. A key feature of HBV replication is the synthesis of the covalently close circular (ccc)DNA, not targeted by current treatments and whose elimination would be crucial for viral cure. To date, little is known about cccDNA formation. One major challenge to address this urgent question is the absence of robust models for the study of cccDNA biology.DesignWe established a cell-based HBV cccDNA reporter assay and performed a loss-of-function screen targeting 239 genes encoding the human DNA damage response machinery.ResultsOvercoming the limitations of current models, the reporter assay enables to quantity cccDNA levels using a robust ELISA as a readout. A loss-of-function screen identified 27 candidate cccDNA host factors, including Y box binding protein 1 (YBX1), a DNA binding protein regulating transcription and translation. Validation studies in authentic infection models revealed a robust decrease in HBV cccDNA levels following silencing, providing proof-of-concept for the importance of YBX1 in the early steps of the HBV life cycle. In patients,YBX1expression robustly correlates with both HBV load and liver disease progression.ConclusionOur cell-based reporter assay enables the discovery of HBV cccDNA host factors including YBX1 and is suitable for the characterisation of cccDNA-related host factors, antiviral targets and compounds.

Background Prenatal exposure to androgens during brain development in male individuals may participate to increase their susceptibility to develop neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability. However, little is known about the action of androgens in human neural cells. Methods We used human neural stem cells differentiated from embryonic stem cells to investigate targets of androgens. Results RNA sequencing revealed that treatment with dihydrotestosterone (DHT) leads to subtle but significant changes in the expression of about 200 genes, encoding proteins of extracellular matrix or involved in signal transduction of growth factors (e.g., insulin/insulin growth factor 1). We showed that the most differentially expressed genes (DEGs), RGCC, RNF144B, NRCAM, TRIM22, FAM107A, IGFBP5, and LAMA2, are reproducibly regulated by different androgens in different genetic backgrounds. We showed, by overexpressing the androgen receptor in neuroblastoma cells SH-SY5Y or knocking it down in human neural stem cells, that this regulation involves the androgen receptor. A chromatin immunoprecipitation combined with direct sequencing analysis identified androgen receptor–bound sequences in nearly half of the DHT-DEGs and in numerous other genes. DHT-DEGs appear enriched in genes involved in ASD (ASXL3, NLGN4X, etc.), associated with ASD (NRCAM), or differentially expressed in patients with ASD (FAM107A, IGFBP5). Androgens increase human neural stem cell proliferation and survival in nutrient-deprived culture conditions, with no detectable effect on regulation of neurite outgrowth. Conclusions We characterized androgen action in neural progenitor cells, identifying DHT-DEGs that appear to be enriched in genes related to ASD. We also showed that androgens increase proliferation of neuronal precursors and protect them from death during their differentiation in nutrient-deprived conditions.

International audience; Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position.

The SAGA coactivator complex and the nuclear pore–associated TREX-2 complex couple transcription with mRNA export. Evangelista et al. identify a novel interplay between TREX-2 and the deubiquitination module of SAGA that is necessary to maintain monoubiquitinated H2B levels required for efficient DNA repair through homologous recombination., DNA repair is critical to maintaining genome integrity, and its dysfunction can cause accumulation of unresolved damage that leads to genomic instability. The Spt–Ada–Gcn5 acetyltransferase (SAGA) coactivator complex and the nuclear pore–associated transcription and export complex 2 (TREX-2) couple transcription with mRNA export. In this study, we identify a novel interplay between human TREX-2 and the deubiquitination module (DUBm) of SAGA required for genome stability. We find that the scaffold subunit of TREX-2, GANP, positively regulates DNA repair through homologous recombination (HR). In contrast, DUBm adaptor subunits ENY2 and ATXNL3 are required to limit unscheduled HR. These opposite roles are achieved through monoubiquitinated histone H2B (H2Bub1). Interestingly, the activity of the DUBm of SAGA on H2Bub1 is dependent on the integrity of the TREX-2 complex. Thus, we describe the existence of a functional interaction between human TREX-2 and SAGA DUBm that is key to maintaining the H2B/HB2ub1 balance needed for efficient repair and HR.

Transcription factor IIH (TFIIH) is a eukaryotic multicomponent macromolecular assembly composed of 10 subunits including two deoxyribonucleic acid (DNA)-dependent helicase activities of opposite polarities and a kinase. Mutations in the helicases xeroderma pigmentosum group D protein (XPD) and xeroderma pigmentosum group B protein (XPB) as well as in the p8/TTD-A subunit of human TFIIH are associated with three dramatic genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). Initially recognised as a basal factor responsible for transcription initiation of protein coding genes and transition from initiation to elongation, TFIIH also plays a critical role in nucleotide excision repair via its DNA-opening helicase activity at the site of a lesion. A subcomplex, harbouring its kinase activity, phosphorylates the carboxy-terminal domain (CTD) of ribonucleic acid (RNA) polymerase II as well as a number of transcription regulators including nuclear hormone receptors. Key Concepts: TFIIH is a prime example of multienzyme multitasking macromolecular assembly. Recent data suggest that several components are found in nonTFIIH complexes and that the function of TFIIH is regulated by its subunit composition. The human transcription/DNA repair factor TFIIH is a multi-subunit complex composed two functional and structural entities: core-TFIIH consists of XPB, p62, p52, p44, p34 and p8, whereas the CDK-activating kinase (CAK) subcomplex contains cyclin-dependent kinase 7 (CDK7), cyclin H and MAT1. XPD bridges the core-TFIIH to the CDK activating kinase (CAK) complex, which also exists as a free complex with a distinct function. Although identified as a basal factor responsible for transcription initiation, TFIIH also plays a critical role in nucleotide excision repair and is implicated in the control of cell cycle progression. Key insights into TFIIH result from investigations centred on the analysis of mutations identified in XP, CS and TTD patients. Most mutations identified in patients reside within the noncatalytic regions of the helicases and do not affect the enzymatic activity of TFIIH per se but rather disturb the interactions of the catalytic subunits with their regulatory partners, impairing transcription and DNA repair. Keywords: transcription; DNA repair; helicase; xeroderma pigmentosum; trichothiodystrophy

International audience; The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the “ARCH domain.” By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.