p190RhoGAP (p190A) is a negative regulator of RhoA and localizes to membrane protrusions, where its GAP activity is required for directional migration. Here, Binamé et al. identify the protrusion-localization sequence in p190A and show that cancer-associated mutations in this region affect p190A localization and function as well as tumor cell migration.

Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity.

Dopa decarboxylase (DDC) synthesizes serotonin in the developing mouse heart where it is encoded by Ddc_exon1a, a tissue-specific paternally expressed imprinted gene. Ddc_exon1a shares an imprinting control region (ICR) with the imprinted, maternally expressed (outside of the central nervous system) Grb10 gene on mouse chromosome 11, but little else is known about the tissue-specific imprinted expression of Ddc_exon1a. Fluorescent immunostaining localizes DDC to the developing myocardium in the pre-natal mouse heart, in a region susceptible to abnormal development and implicated in congenital heart defects in human. Ddc_exon1a and Grb10 are not co-expressed in heart nor in brain where Grb10 is also paternally expressed, despite sharing an ICR, indicating they are mechanistically linked by their shared ICR but not by Grb10 gene expression. Evidence from a Ddc_exon1a gene knockout mouse model suggests that it mediates the growth of the developing myocardium and a thinning of the myocardium is observed in a small number of mutant mice examined, with changes in gene expression detected by microarray analysis. Comparative studies in the human developing heart reveal a paternal expression bias with polymorphic imprinting patterns between individual human hearts at DDC_EXON1a, a finding consistent with other imprinted genes in human.

Abstract Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.

The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional teneleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.

The mammalian genome is depleted in CG dinucleotides, except at protected regions where they cluster as CpG islands (CGIs). CGIs are gene regulatory hubs and serve as transcription initiation sites and are as expected, associated with gene promoters. Advances in genomic annotations demonstrate that a quarter of CGIs are found within genes. Such intragenic regions are repressive environments, so it is surprising that CGIs reside here and even more surprising that some resist repression and are transcriptionally active within a gene. Hence, intragenic CGI positioning within genes is not arbitrary and is instead, selected for. As a wealth of recent studies demonstrate, intragenic CGIs are embedded within genes and consequently, influence ‘host’ gene mRNA isoform length and expand transcriptome diversity.

Hydroxycarbamide (HC, hydroxyurea) is a cytoreductive drug inducing cell cycle blockade. However, emerging evidence suggests that HC plays a role in the modulation of transcription through the activity of transcription factors and DNA methylation. Examining the global mechanism of action of HC in the context of myeloproliferative neoplasms (MPNs), for which HC is the first-line treatment, will provide a better understanding of its molecular effects. To explore the effects of HC genome-wide, transcriptomic analyses were performed on two clinically relevant cell types at different stages of differentiation treated with HC in a murine MPN model. This study was replicated in MPN patients by profiling genome-wide gene expression and DNA methylation using patient blood samples collected longitudinally, before and following HC exposure. The effects of HC on the transcriptome were not only associated with cell cycle interruption but also with hematopoietic functions. Moreover, a group of genes were restored to normal expression levels in murine hematopoietic stem cells (HSCs) following drug treatment, including the master regulator of hematopoiesis, RUNX1. In humans, HC significantly modifies DNA methylation levels in HSCs at several distal regulatory regions, which we show to be associated with SPI1 binding sites and at the SPI1 locus itself. We have identified novel targets of HC that include pivotal transcription factors involved in hematopoiesis, and for the first time we report abnormal methylation patterns in MPN patients at the master regulator gene SPI1 and its distal binding sites, which HC is able to restore to normal levels.

Glioblastomas represent approximatively half of all gliomas and are the most deadly and aggressive form. Their therapeutic resistance and tumor relapse rely on a subpopulation of cells that are called Glioma Stem Cells (GSCs). Here, we investigated the role of the long non-coding RNA HOXA-AS2 in GSC biology using descriptive and functional analyses of glioma samples classified according to their isocitrate dehydrogenase (IDH) gene mutation status, and of GSC lines. We found that HOXA-AS2 is overexpressed only in aggressive (IDHwt) glioma and GSC lines. ShRNA-based depletion of HOXA-AS2 in GSCs decreased cell proliferation and altered the expression of several hundreds of genes. Integrative analysis revealed that these expression changes were not associated with changes in DNA methylation or chromatin signatures at the promoter of the majority of genes deregulated following HOXA-AS2 silencing in GSCs, suggesting a post-transcriptional regulation. In addition, transcription factor binding motif enrichment and correlation analyses indicated that HOXA-AS2 affects, directly or indirectly, the expression of key transcription factors implicated in GCS biology, including E2F8, E2F1, STAT1, and ATF3, thus contributing to GCS aggressiveness by promoting their proliferation and modulating the inflammation pathway.