HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytes.

BACKGROUND: Nuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3. RESULTS: After nuclear transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after nuclear transfer. CONCLUSIONS: This study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration

Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy

AbstractDevelopmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.</jats:p

Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy

Etude des variants de l'histoire H3 : H3.2 et H3.3, au cours du développement embryonnaire d'un vertébré, Xenopus laevis

Proper packaging of eukaryotic genomic DNA into chromatin is important for proper DNA compaction and gene expression regulation. The nucleosome core particle comprises a histone octamer wrapped DNA, and can be modulated by the incoporation of distinct histone variants. Concerning H3, the replicative histone variants H3.1 and H3.2 contribute to the histone supply needed during DNA replication, while the histone variant H3.3 is incorporated throughout the cell cycle. Interestingly, the literature highlights a link between H3.3 and transcription. Moreover, the incorporation of H3.3 depends on a specific assembly pathway that involves the histone chaperone HIRA. The aim of my research project was to determine whether H3.3 and its incorporation via HIRA had specific roles. The context of embryonic development was an ideal situation giving that it requires the fine control of genes expression. The use of the vertebrate Xenopus laevis, which unlike mammals has only one replicative H3 variant: H3.2, allowed me to assess the specific function of H3.2 and H3.3 during development. I showed that despite their similarity these two histone variants are not interchangeable. Remarkably, downregulation of H3.3 expression in embryos or interference with its assembly pathway via HIRA leads to major developmental defects at gastrulation. This phenotype is accompanied by expression defects of mesodermal genes, including the marker Xbra. Moreover, these embryos show overall defects in their chromatin organization. Taken together, these data highlight the importance of H3.3 incorporation into chromatin during a key developmental transition, gastrulationL'organisation en chromatine permet de compacter l'ADN génomique et de réguler finement l'expression du génome. La particule cœur du nucléosome, composée d'un octamère de protéines histones autour desquelles s'enroule l'ADN, peut être modulée par l'incorporation de variants d'histones. Pour l'histone H3, les variants réplicatifs H3.1 et H3.2 permettent une incorporation lors de la réplication de l'ADN, tandis que le variant H3.3 est incorporé tout au long du cycle cellulaire. Les données dans la littérature établissent un lien entre H3.3 et la transcription. L'incorporation d'H3.3 dépend d'une voie d'assemblage faisant intervenir le chaperon HIRA. Mon projet de recherche visait à déterminer si H3.3 et son incorporation via HIRA possédaient un rôle spécifique. Le développement embryonnaire via une régulation fine de l'expression des gènes représentait une situation idéale pour aborder ces questions. L'utilisation du vertébré Xenopus laevis qui ne possède qu'un variant H3 réplicatif : H3.2, m'a permis d'évaluer la fonction de ces variants au cours du développement. J'ai pu montrer que, malgré leur similarité, les variants H3.2 et H3.3 ne sont pas interchangeables. Une altération d'expression d'H3.3 ou l'interférence dans sa voie d'assemblage via son chaperon HIRA conduisent à des défauts majeurs à la gastrulation. Ce phénotype s'accompagne d'un défaut d'expression de gènes mésodermiques, dont le marqueur Xbra. Une désorganisation globale de la chromatine est également observée chez ces embryons. Ces données mettent en lumière l'importance de l'incorporation du variant d'histone H3.3 dans la chromatine au cours d'une étape clé du développement embryonnaire, la gastrulatio

A Developmental Requirement for HIRA-Dependent H3.3 Deposition Revealed at Gastrulation in Xenopus

Discovering how histone variants that mark distinct chromatin regions affect a developmental program is a major challenge in the epigenetics field. To assess the importance of the H3.3 histone variant and its dedicated histone chaperone HIRA, we used an established developmental model, Xenopus laevis. After the early rapid divisions exploiting a large maternal pool of both replicative H3.2 and replacement H3.3, H3.3 transcripts show a distinct peak of expression at gastrulation. Depletion of both H3.2 and H3.3 leads to an early gastrulation arrest. However, with only H3.3 depletion, defects occur at late gastrulation, impairing further development. Providing exogenous H3.3 mRNAs, but not replicative H3.2 mRNAs, rescues these defects. Notably, downregulation of the H3.3 histone chaperone HIRA similarly impairs late gastrulation, and we find a global defect in H3.3 incorporation into chromatin comparable to H3.3 depletion. We discuss how specific HIRA-dependent H3.3 deposition is required for chromatin dynamics during gastrulation

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here we show in mice that the signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/β-catenin signalling and their genetic deletion leads to a rapid decline of nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation

A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI‐NET sequencing

Abstract Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non‐coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA‐seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA‐seq, we delineated a deep intronic TAPT1 mutation (c.1237‐52 G>A) that segregated with the disease. Using SI‐NET‐seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre‐mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein‐null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease‐relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non‐coding variants, as well as in illuminating the molecular mechanisms of human diseases