Guillaume Giraud, Sophie Terrone and Cyril F. Bourgeois. BMB Reports 2018;51:613-22. https://doi.org/10.5483/BMBRep.2018.51.12.234

Chromatin domains and loops are important elements of chromatin structure and dynamics, but much remains to be learned about their exact biological role and nature. Topological associated domains and functional loops are key to gene expression and hold the answer to many questions regarding developmental decisions and diseases. Here, we discuss new findings, which have linked chromatin conformation with development, differentiation and diseases and hypothesized on various models while integrating all recent findings on how chromatin architecture affects gene expression during development, evolution and disease.

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

Spi-1 and Fli-1 are ETS transcription factors recurrently deregulated in mouse erythroleukemia induced by Friend viruses. Since they share the same core DNA binding site, we investigated whether they may contribute to erythroleukemia by common mechanisms. Using inducible knockdown, we demonstrated that Fli-1 contributes to proliferation, survival, and differentiation arrest of erythroleukemic cells harboring an activated fli-1 locus. Similarly, we used inducible Fli-1 knockdown and either hexamethylenebisacetamide (HMBA)- or small interfering RNA-mediated Spi-1 knockdown to investigate their respective contributions in erythroleukemic cells harboring an activated spi-1 locus. In these cells, simple or double knockdown of both Spi-1 and Fli-1 additively contributed to induce proliferation arrest and differentiation. Transcriptome profiling revealed that virtually all transcripts affected by both Fli-1 knockdown and HMBA are affected in an additive manner. Among these additively downregulated transcripts, more than 20% encode proteins involved in ribosome biogenesis, and conserved ETS binding sites are present in their gene promoters. Through chromatin immunoprecipitation, we demonstrated the association of Spi-1 and Fli-1 on these promoters in Friend erythroleukemic cells. These data lead us to propose that the oncogenicity of Spi-1, Fli-1, and possibly other ETS transcription factors may involve their ability to stimulate ribosome biogenesis.

Clonal erythroleukemia developing in susceptible mice infected by Friend virus complex are associated with highly recurrent proviral insertions at one of three loci called Spi-1, Fli-1 or Fli-3, leading to deregulated expression of oncogenic Spi-1 or Fli-1 transcription factors or miR-17-92 miRNA cluster, respectively. Deregulated expression of each of these three oncogenes has been independently shown to contribute to cell proliferation of erythroleukemic clones. Previous studies showed a close relationship between Spi-1 and Fli-1, which belong to the same ETS family, Spi-1 activating fli-1 gene, and both Spi-1 and Fli-1 activating multiple common target genes involved in ribosome biogenesis. In this study, we demonstrated that Spi-1 and Fli-1 are also involved in direct miR-17-92 transcriptional activation through their binding to a conserved ETS binding site in its promoter. Moreover, we demonstrated that physiological re-expression of exogenous miR-17 and miR-20a are able to partially rescue the proliferation loss induced by Fli-1 knock-down and identified HBP1 as a target of these miRNA in erythroleukemic cells. These results establish that three of the most recurrently activated oncogenes in Friend erythroleukemia are actually involved in a same oncogenic network controlling cell proliferation. The putative contribution of a similar ETS-miR-17-92 network module in other normal or pathological proliferative contexts is discussed.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.