Alternative splicing decouples local from global PRC2 activity.

The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in virtually all tissues and developmental stages. SUZ12-S drives PRC2.1 formation and favors PRC2 dimerization. While SUZ12-S is necessary and sufficient for the repression of target genes via promoter-proximal H3K27me3 deposition, SUZ12-L maintains global H3K27 methylation levels. Mouse embryonic stem cells (ESCs) lacking either isoform exit pluripotency more slowly and fail to acquire neuronal cell identity. Our findings reveal a physiological mechanism regulating PRC2 assembly and higher-order interactions in eutherians, with impacts on H3K27 methylation and gene repression.

Artificial intelligence in paediatrics: Current events and challenges.

This article examines the use of artificial intelligence (AI) in the field of paediatric care within the framework of the 7P medicine model (Predictive, Preventive, Personalized, Precise, Participatory, Peripheral and Polyprofessional). It highlights various applications of AI in the diagnosis, treatment and management of paediatric diseases as well as the role of AI in prevention and in the efficient management of health care resources and the resulting impact on the sustainability of public health systems. Successful cases of the application of AI in the paediatric care setting are presented, placing emphasis on the need to move towards a 7P health care model. Artificial intelligence is revolutionizing society at large and has a great potential for significantly improving paediatric care.

Inteligencia artificial en pediatría: actualidad y retos / Artificial intelligence in paediatrics: Current events and challenges

Se examina el uso de la inteligencia artificial (IA) en el campo de la atención a la salud pediátrica dentro del marco de la «Medicina de las 7P» (Predictiva, Preventiva, Personalizada, Precisa, Participativa, Periférica y Poliprofesional). Se destacan diversas aplicaciones de la IA en el diagnóstico, el tratamiento y el control de enfermedades pediátricas, así como su papel en la prevención y en la gestión eficiente de los recursos médicos con su repercusión en la sostenibilidad de los sistemas públicos de salud. Se presentan casos de éxito de la aplicación de la IA en el ámbito pediátrico y se hace un gran énfasis en la necesidad de caminar hacia la Medicina de las 7P. La IA está revolucionando la sociedad en general ofreciendo un gran potencial para mejorar significativamente el cuidado de la salud en pediatría.(AU)

This article examines the use of artificial intelligence (AI) in the field of paediatric care within the framework of the 7P medicine model (Predictive, Preventive, Personalized, Precise, Participatory, Peripheral and Polyprofessional). It highlights various applications of AI in the diagnosis, treatment and management of paediatric diseases as well as the role of AI in prevention and in the efficient management of health care resources and the resulting impact on the sustainability of public health systems. Successful cases of the application of AI in the paediatric care setting are presented, placing emphasis on the need to move towards a 7P health care model. Artificial intelligence is revolutionizing society at large and has a great potential for significantly improving paediatric care.(AU)

LOXL2-mediated chromatin compaction is required to maintain the oncogenic properties of triple-negative breast cancer cells.

Oxidation of histone H3 at lysine 4 (H3K4ox) is catalyzed by lysyl oxidase homolog 2 (LOXL2). This histone modification is enriched in heterochromatin in triple-negative breast cancer (TNBC) cells and has been linked to the maintenance of compacted chromatin. However, the molecular mechanism underlying this maintenance is still unknown. Here, we show that LOXL2 interacts with RuvB-Like 1 (RUVBL1), RuvB-Like 2 (RUVBL2), Actin-like protein 6A (ACTL6A), and DNA methyltransferase 1associated protein 1 (DMAP1), a complex involved in the incorporation of the histone variant H2A.Z. Our experiments indicate that this interaction and the active form of RUVBL2 are required to maintain LOXL2-dependent chromatin compaction. Genome-wide experiments showed that H2A.Z, RUVBL2, and H3K4ox colocalize in heterochromatin regions. In the absence of LOXL2 or RUVBL2, global levels of the heterochromatin histone mark H3K9me3 were strongly reduced, and the ATAC-seq signal in the H3K9me3 regions was increased. Finally, we observed that the interplay between these series of events is required to maintain H3K4ox-enriched heterochromatin regions, which in turn is key for maintaining the oncogenic properties of the TNBC cell line tested (MDA-MB-231).

Puckered and JNK signaling in pioneer neurons coordinates the motor activity of the Drosophila embryo.

Central nervous system organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyze the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling in a subset of pioneer neurons, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating JNK signaling activity in pioneer neurons during early embryogenesis directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization.

MAF amplification licenses ERα through epigenetic remodelling to drive breast cancer metastasis.

MAF amplification increases the risk of breast cancer (BCa) metastasis through mechanisms that are still poorly understood yet have important clinical implications. Oestrogen-receptor-positive (ER+) BCa requires oestrogen for both growth and metastasis, albeit by ill-known mechanisms. Here we integrate proteomics, transcriptomics, epigenomics, chromatin accessibility and functional assays from human and syngeneic mouse BCa models to show that MAF directly interacts with oestrogen receptor alpha (ERα), thereby promoting a unique chromatin landscape that favours metastatic spread. We identify metastasis-promoting genes that are de novo licensed following oestrogen exposure in a MAF-dependent manner. The histone demethylase KDM1A is key to the epigenomic remodelling that facilitates the expression of the pro-metastatic MAF/oestrogen-driven gene expression program, and loss of KDM1A activity prevents this metastasis. We have thus determined that the molecular basis underlying MAF/oestrogen-mediated metastasis requires genetic, epigenetic and hormone signals from the systemic environment, which influence the ability of BCa cells to metastasize.

Thymine DNA glycosylase regulates cell-cycle-driven p53 transcriptional control in pluripotent cells.

Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.

Targeting lymphoid-derived IL-17 signaling to delay skin aging.

Skin aging is characterized by structural and functional changes that contribute to age-associated frailty. This probably depends on synergy between alterations in the local niche and stem cell-intrinsic changes, underscored by proinflammatory microenvironments that drive pleotropic changes. The nature of these age-associated inflammatory cues, or how they affect tissue aging, is unknown. Based on single-cell RNA sequencing of the dermal compartment of mouse skin, we show a skew towards an IL-17-expressing phenotype of T helper cells, γδ T cells and innate lymphoid cells in aged skin. Importantly, in vivo blockade of IL-17 signaling during aging reduces the proinflammatory state of the skin, delaying the appearance of age-related traits. Mechanistically, aberrant IL-17 signals through NF-κB in epidermal cells to impair homeostatic functions while promoting an inflammatory state. Our results indicate that aged skin shows signs of chronic inflammation and that increased IL-17 signaling could be targeted to prevent age-associated skin ailments.

FGF signaling promotes spreading of fat body precursors necessary for adult adipogenesis in Drosophila.

Knowledge of adipogenetic mechanisms is essential to understand and treat conditions affecting organismal metabolism and adipose tissue health. In Drosophila, mature adipose tissue (fat body) exists in larvae and adults. In contrast to the well-known development of the larval fat body from the embryonic mesoderm, adult adipogenesis has remained mysterious. Furthermore, conclusive proof of its physiological significance is lacking. Here, we show that the adult fat body originates from a pool of undifferentiated mesodermal precursors that migrate from the thorax into the abdomen during metamorphosis. Through in vivo imaging, we found that these precursors spread from the ventral midline and cover the inner surface of the abdomen in a process strikingly reminiscent of embryonic mesoderm migration, requiring fibroblast growth factor (FGF) signaling as well. FGF signaling guides migration dorsally and regulates adhesion to the substrate. After spreading is complete, precursor differentiation involves fat accumulation and cell fusion that produces mature binucleate and tetranucleate adipocytes. Finally, we show that flies where adult adipogenesis is impaired by knock down of FGF receptor Heartless or transcription factor Serpent display ectopic fat accumulation in oenocytes and decreased resistance to starvation. Our results reveal that adult adipogenesis occurs de novo during metamorphosis and demonstrate its crucial physiological role.

JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos.

Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), we observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, our data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization.

Quantitative Comparison of Multiple Chromatin Immunoprecipitation-Sequencing (ChIP-seq) Experiments with spikChIP.

The chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) is a powerful technique that enables to characterize the genomic distribution of chromatin-associated proteins, histone posttranslational modifications, and histone variants. However, in the absence of a reference control for monitoring experimental and biological variations, the standard ChIP-seq scheme is unable to accurately assess changes in the abundance of chromatin targets across different experimental samples. To overcome this limitation, the combination of external spike-in material with the experimental chromatin is offered as an effective solution for quantitative comparison of ChIP-seq data across different conditions. Here, we detail (i) the experimental protocol for preparing quality control spike-in chromatin from Drosophila melanogaster cells and (ii) the computational protocol to compare ChIP-seq samples with spike-in based on the use of the spikChIP software.

A computational pipeline to learn gene expression predictive models from epigenetic information at enhancers or promoters.

Here, we present a computational pipeline to obtain quantitative models that characterize the relationship of gene expression with the epigenetic marking at enhancers or promoters in mouse embryonic stem cells. Our protocol consists of (i) generating predictive models of gene expression from epigenetic information (such as histone modification ChIP-seq) at enhancers or promoters and (ii) assessing the performance of these predictive models. This protocol could be applied to other biological scenarios or other types of epigenetic data. For complete details on the use and execution of this protocol, please refer to Gonzalez-Ramirez et al. (2021).1.

JNK signaling and integrins cooperate to maintain cell adhesion during epithelial fusion in Drosophila.

The fusion of epithelial sheets is an essential and conserved morphogenetic event that requires the maintenance of tissue continuity. This is secured by membrane-bound or diffusible signals that instruct the epithelial cells, in a coordinated fashion, to change shapes and adhesive properties and when, how and where to move. Here we show that during Dorsal Closure (DC) in Drosophila, the Jun kinase (JNK) signaling pathway modulates integrins expression and ensures tissue endurance. An excess of JNK activity, as an outcome of a failure in the negative feedback implemented by the dual-specificity phosphatase Puckered (Puc), promotes the loss of integrins [the ß-subunit Myospheroid (Mys)] and amnioserosa detachment. Likewise, integrins signal back to the pathway to regulate the duration and strength of JNK activity. Mys is necessary for the regulation of JNK activity levels and in its absence, puc expression is downregulated and JNK activity increases.

In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions.

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

ECM degradation in the Drosophila abdominal epidermis initiates tissue growth that ceases with rapid cell-cycle exit.

During development, multicellular organisms undergo stereotypical patterns of tissue growth in space and time. How developmental growth is orchestrated remains unclear, largely due to the difficulty of observing and quantitating this process in a living organism. Drosophila histoblast nests are small clusters of progenitor epithelial cells that undergo extensive growth to give rise to the adult abdominal epidermis and are amenable to live imaging. Our quantitative analysis of histoblast proliferation and tissue mechanics reveals that tissue growth is driven by cell divisions initiated through basal extracellular matrix degradation by matrix metalloproteases secreted by the neighboring larval epidermal cells. Laser ablations and computational simulations show that tissue mechanical tension does not decrease as the histoblasts fill the abdominal epidermal surface. During tissue growth, the histoblasts display oscillatory cell division rates until growth termination occurs through the rapid emergence of G0/G1 arrested cells, rather than a gradual increase in cell-cycle time as observed in other systems such as the Drosophila wing and mouse postnatal epidermis. Different developing tissues can therefore achieve their final size using distinct growth termination strategies. Thus, adult abdominal epidermal development is characterized by changes in the tissue microenvironment and a rapid exit from the cell cycle.

Highlights regarding prolactin in the dentate gyrus and hippocampus.

Prolactin (PRL) is a pituitary hormone that has been typically related to lactogenesis in mammals. However, it has been described over 300 roles in the organism of vertebrae and its relationship with the central nervous system (CNS) is yet to be clarified. Mainly secreted by the pituitary gland, the source of prolactin in the CNS remains unclear, where some experiments suggest active transport via an unknown carrier or, on the contrary, PRL being synthesized on the brain. So far, it seems to be involved with neurogenesis, neuroprotection, maternal behavior and cognitive processes in the hippocampus and dentate gyrus, among other regions.

Measuring ventral nerve cord stiffness in live flat-dissected Drosophila embryos by atomic force microscopy.

Drosophila is an amenable system for addressing the mechanics of morphogenesis. We describe a workflow for characterizing the mechanical properties of its ventral nerve cord (VNC), at different developmental stages, in live, flat-dissected embryos employing atomic force microscopy (AFM). AFM is performed with spherical probes, and stiffness (Young's modulus) is calculated by fitting force curves with Hertz's contact model. For complete details on the use and execution of this protocol, please refer to Karkali et al. (2022).

The lack of Irs2 induces changes in the immunocytochemical expression of aromatase in the mouse retina.

Insulin receptor substrate (Irs) belongs to a family of proteins that mediate the intracellular signaling of insulin and IGF-1. Insulin receptor substrate 2 (Irs2) is necessary for retinal function, since its failure in Irs2-deficient mice in hyperglycemic situation promotes photoreceptor degeneration and visual dysfunction, like in diabetic retinopathy. The expression of P450 aromatase, which catalyzes androgen aromatization to form 17ß-estradiol, increases in some neurodegenerative diseases thus promoting the local synthesis of neuroestrogens that exert relevant neuroprotective functions. Aromatase is also expressed in neurons and glial cells of the central nervous system (CNS), including the retina. To further understand the role of Irs2 at the retinal level, we performed an immunocytochemical study in adult normoglycemic Irs2-deficient mice. For this aim, the retinal immunoexpression of neuromodulators, such as aromatase, glutamine synthetase (GS), and tyrosine hydroxylase (TH) was analyzed, joint to a morphometric and planimetric study of the retinal layers. Comparing with wild-type (WT) control mice, the Irs2-knockout (Irs2-KO) animals showed a significant increase in the immunopositivity to aromatase in almost all of the retinal layers. Besides, Irs2-KO mice exhibited a decreased immunopositive reaction for GS and TH, in Müller and amacrine cells, respectively; morphological variations were also found in these retinal cell types. Furthermore, the retina of Irs2-KO mice displayed alterations in the structural organization, and a generalized decrease in the retinal thickness was observed in each of the layers, except for the inner nuclear layer. Our findings suggest that the absence of Irs2 induces retinal neurodegenerative changes in Müller and amacrine cells that are unrelated to hyperglycemia. Accordingly, in the Irs2-KO mice, the increased retinal immunocytochemical reactivity of aromatase could be associated with an attempt to repair such neural retina injuries by promoting local neuroprotective mediators.

Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase.

For developmental processes, we know most of the gene networks controlling specific cell responses. We still have to determine how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, we still know little about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. We have performed a promoter analysis, searching for potential regulatory sequences of puckered (puc) and identified different specific enhancers directing gene expression in different tissues and at different developmental times. Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila.