Focus on the 9th Annual Seminar organized by the Canceropôle Provence-Alpes-Côte-d'Azur: Highlights of the event.

The 9th Annual Seminar of the Canceropôle Provence-Alpes-Côte-d'Azur took place on July, 5th-6th 2022 in Saint-Raphaël, south of France. Annual meeting of the regional scientific community working in the field of cancer research, this seminar brings together a large and diverse audience, with 285 people attending in 2022: PhD students, postdocs, PIs (Principal Investigators) and senior researchers, clinicians, patient associations, funding partners of the Canceropôle. This document reviews the major scientific results presented and key moments of the event.

Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse.

The Myc transcriptional regulators are implicated in a range of cellular functions, including proliferation, cell cycle progression, metabolism and pluripotency maintenance. Here, we investigated the expression, regulation and function of the Myc family during mouse embryonic axis elongation and segmentation. Expression of both cMyc (Myc - Mouse Genome Informatics) and MycN in the domains in which neuromesodermal progenitors (NMPs) and underlying caudal pre-somitic mesoderm (cPSM) cells reside is coincident with WNT and FGF signals, factors known to maintain progenitors in an undifferentiated state. Pharmacological inhibition of Myc activity downregulates expression of WNT/FGF components. In turn, we find that cMyc expression is WNT, FGF and Notch protein regulated, placing it centrally in the signalling circuit that operates in the tail end that both sustains progenitors and drives maturation of the PSM into somites. Interfering with Myc function in the PSM, where it displays oscillatory expression, delays the timing of segmentation clock oscillations and thus of somite formation. In summary, we identify Myc as a component that links NMP maintenance and PSM maturation during the body axis elongation stages of mouse embryogenesis.

Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro.

Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene Nkx1.2 we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.

The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability.

The interplay between methylation and demethylation of histone lysine residues is an essential component of gene expression regulation and there is considerable interest in elucidating the roles of proteins involved. Here we report that histone demethylase KDM4A/JMJD2A, which is involved in the regulation of cell proliferation and is overexpressed in some cancers, interacts with RNA Polymerase I, associates with active ribosomal RNA genes and is required for serum-induced activation of rDNA transcription. We propose that KDM4A controls the initial stages of transition from 'poised', non-transcribed rDNA chromatin into its active form. We show that PI3K, a major signalling transducer central for cell proliferation and survival, controls cellular localization of KDM4A and consequently its association with ribosomal DNA through the SGK1 downstream kinase. We propose that the interplay between PI3K/SGK1 signalling cascade and KDM4A constitutes a mechanism by which cells adapt ribosome biogenesis level to the availability of growth factors and nutrients.

Neuromesodermal progenitors and the making of the spinal cord.

Neuromesodermal progenitors (NMps) contribute to both the elongating spinal cord and the adjacent paraxial mesoderm. It has been assumed that these cells arise as a result of patterning of the anterior neural plate. However, as the molecular mechanisms that specify NMps in vivo are uncovered, and as protocols for generating these bipotent cells from mouse and human pluripotent stem cells in vitro are established, the emerging data suggest that this view needs to be revised. Here, we review the characteristics, regulation, in vitro derivation and in vivo induction of NMps. We propose that these cells arise within primitive streak-associated epiblast via a mechanism that is separable from that which establishes neural fate in the anterior epiblast. We thus argue for the existence of two distinct routes for making central nervous system progenitors.

Global regulation of heterochromatin spreading by Leo1.

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution.

Histone demethylases in chromatin cross-talks.

The 'histone code' hypothesis states that chromatin-based regulation of nuclear processes such as transcription is brought about by the combination of distinct modifications (histone marks) at specific loci. Its correct establishment involves chromatin cross-talks, ensuring an ordered and concerted deposition/removal of a particular set of modifications that act together to give the correct transcriptional outcome. Histone methylation on lysine residues can negatively or positively impact on gene transcription, depending on the residue and on its degree of methylation. Thanks to this complexity and given the number of chromatin 'readers' that can recognize methylated lysine residues, histone methylation plays a very special role in specifying the various chromatin states. The recent discovery of histone demethylases, which represent a large family of enzymes often containing histone modification binding modules, sheds new light on cross-talk mechanisms involving methylated residues. In the present review, after a brief overview of the various families of histone demethylases, we describe the different mechanisms by which they participate in chromatin cross-talks and how these mechanisms are integrated to achieve the mutual exclusion or the link between chromatin marks, leading to the establishment of the correct histone code.

A new isoform of the histone demethylase JMJD2A/KDM4A is required for skeletal muscle differentiation.

In proliferating myoblasts, muscle specific genes are silenced by epigenetic modifications at their promoters, including histone H3K9 methylation. Derepression of the promoter of the gene encoding the myogenic factor myogenin (Myog) is key for initiation of muscle differentiation. The mechanism of H3K9 demethylation at the Myog promoter is unclear, however. Here, we identify an isoform of the histone demethylase JMJD2A/KDM4A that lacks the N-terminal demethylase domain (ΔN-JMJD2A). The amount of ΔN-JMJD2A increases during differentiation of C2C12 myoblasts into myotubes. Genome-wide expression profiling and exon-specific siRNA knockdown indicate that, in contrast to the full-length protein, ΔN-JMJD2A is necessary for myotube formation and muscle-specific gene expression. Moreover, ΔN-JMJD2A promotes MyoD-induced conversion of NIH3T3 cells into muscle cells. ChIP-on-chip analysis indicates that ΔN-JMJD2A binds to genes mainly involved in transcriptional control and that this binding is linked to gene activation. ΔN-JMJD2A is recruited to the Myog promoter at the onset of differentiation. This binding is essential to promote the demethylation of H3K9me2 and H3K9me3. We conclude that induction of the ΔN-JMJD2A isoform is crucial for muscle differentiation: by directing the removal of repressive chromatin marks at the Myog promoter, it promotes transcriptional activation of the Myog gene and thus contributes to initiation of muscle-specific gene expression.