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.

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.

RNA helicases DDX5 and DDX17 are multitasking proteins that regulate gene expression in different biological contexts through diverse activities. Special attention has long been paid to their function as coregulators of transcription factors, providing insight about their functional association with a number of chromatin modifiers and remodelers. However, to date, the variety of described mechanisms has made it difficult to understand precisely how these proteins work at the molecular level, and the contribution of their ATPase domain to these mechanisms remains unclear as well. In light of their association with long noncoding RNAs that are key epigenetic regulators, an emerging view is that DDX5 and DDX17 may act through modulating the activity of various ribonucleoprotein complexes that could ensure their targeting to specific chromatin loci. This review will comprehensively describe the current knowledge on these different mechanisms. We will also discuss the potential roles of DDX5 and DDX17 on the 3D chromatin organization and how these could impact gene expression at the transcriptional and post-transcriptional levels.

Friend erythroleukemia has been a powerful model for dissection of how multiple oncogenes cooperate to initiate and maintain leukemic transformation. The Friend viral complex contains a replication-defective spleen focus-forming virus (SFFV) and a replication-competent Friend murine leukemia virus (F-MuLV). It induces a multistep erythroleukemic process in susceptible mice (8,33,50). SFFV virus is the pathogenic component responsible for this acute erythroleukemia. During the early stage of the disease, the product of the SFFV env gene, gp55, interacts with the erythropoietin receptor (Epo-R) and constitutively activates signaling pathways allowing the proliferation of proerythroblasts still able to differentiate in the absence of Epo. During this early step, the activation of signaling pathways allowing proerythroblast proliferation is also strictly dependent on the c-Kit receptor and on the small form of the STK receptor tyrosine kinase (20, 57). The second stage of the disease is characterized by a clonal population outgrowth of leukemic proerythroblasts blocked in their differentiation and able to grow as permanent cell lines in vitro. Virtually all tumors display SFFV proviral integration upstream of the Spi-1/PU.1 gene, leading to overexpression of the normal transcription factor Spi-1/PU.1 (hereinafter called Spi-1). It is now well established that the dysregulation of Spi-1 expression is a critical event in the process of SFFV-induced erythroleukemia.Indeed, the terminal erythroid differentiation can be reinitiated in Friend tumor cells by chemical inducers such as hexamethylenebisacetamide (HMBA). This differentiation is associated with a decrease in Spi-1 levels (18,24,27,51), and it can be reversed by Spi-1-enforced expression (43,64). Similarly, enforced expression of Spi-1 together with both gp55 and constitutively activated Epo-R inhibits differentiation of avian erythroid progenitors (42). Furthermore, transgenic mice overexpressing Spi-1 spontaneously develop an erythroleukemia characterized by an Epo-dependent proliferation of proerythroblasts blocked in their differentiation (38). Spi-1 knockdown induced by RNA interference is sufficient to inhibit proliferation and restore terminal erythroid differentiation of erythroleukemic cell lines established from either SFFVinfected (4) or Spi-1 transgenic mice (47). At least one contribution of Spi-1 overexpression to erythroleukemia is through the inhibition of GATA-1 transcriptional activity (39,45,46,67). Indeed, enforced expression of GATA-1 is sufficient to restore the differentiation of SFFV-infected erythroleukemic cells (13,45,46). Recent data have shown that Spi-1 inhibits expression of some GATA-1 target genes by binding to GATA-1 on transcriptional promoters and creating a repressive chromatin structure through the recruitment of pRB, the histone methylase SUV39h, and the heterochromatin protein HP1␣ (55). However, although this mechanism might explain the contribution of Spi-1 to the repression of erythroid-specific GATA-1-dependent gene tr...

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.

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin‐organising transcriptional insulator protein, CCCTC‐binding factor (CTCF), has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knock‐down of CTCF results in a significant increase in pre‐genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre‐genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.

DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3′ end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the ‘tag’ close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.