ABSTRACT
H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.
Subject(s)
Euchromatin , Heterochromatin , Histone-Lysine N-Methyltransferase , Histones , Methyltransferases , Transcription, Genetic , Animals , Mice , Cell Line , Euchromatin/metabolism , Euchromatin/genetics , Gene Expression Regulation , Heterochromatin/metabolism , Heterochromatin/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Histones/genetics , Methyltransferases/metabolism , Methyltransferases/genetics , Repressor Proteins/metabolism , Repressor Proteins/geneticsABSTRACT
In mammals, the second X chromosome in females is silenced to enable dosage compensation between XX females and XY males. This essential process involves the formation of a dense chromatin state on the inactive X (Xi) chromosome. There is a wealth of information about the hallmarks of Xi chromatin and the contribution each makes to silencing, leaving the tantalising possibility of learning from this knowledge to potentially remove silencing to treat X-linked diseases in females. Here, we discuss the role of each chromatin feature in the establishment and maintenance of the silent state, which is of crucial relevance for such a goal.
Subject(s)
RNA, Long Noncoding , X Chromosome Inactivation , Male , Animals , Female , X Chromosome Inactivation/genetics , RNA, Long Noncoding/genetics , X Chromosome/genetics , Chromatin/genetics , Dosage Compensation, Genetic , Mammals/geneticsABSTRACT
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
Subject(s)
Histone Acetyltransferases/metabolism , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Animals , Cell Line, Tumor , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Structure, TertiaryABSTRACT
Female mouse embryonic stem cells (mESCs) present differently from male mESCs in several fundamental ways; however, complications with their in vitro culture have resulted in an under-representation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female and male mESCs apart. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state that is near indistinguishable from male mESCs. If female pluripotency is to be studied effectively in this system, it is crucial that high-quality cultures of XX mESCs are available. Here, we report a method for better maintaining XX female mESCs in culture that also stabilises the male karyotype and makes study of female-specific pluripotency more feasible.
Subject(s)
Mouse Embryonic Stem Cells , X Chromosome Inactivation , Male , Animals , Female , Mice , Cell Differentiation/physiology , X Chromosome Inactivation/genetics , KaryotypeABSTRACT
Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.
Subject(s)
Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Embryonic Stem Cells/metabolism , Endoderm/metabolism , Lung/embryology , SOX9 Transcription Factor/metabolism , Animals , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Organogenesis , SOX9 Transcription Factor/geneticsABSTRACT
Serum iron has long been thought to exhibit diurnal variation and is subsequently considered an unreliable biomarker of systemic iron status. Circadian regulation (endogenous ~24-h periodic oscillation of a biologic function) governs many critical physiologic processes. It is unknown whether serum iron levels are regulated by circadian machinery; likewise, the circadian nature of key players of iron homeostasis is unstudied. Here we show that serum iron, transferrin saturation (TSAT), hepatic transferrin receptor (TFR1) gene (Tfrc) expression, and erythropoietic activity exhibit circadian rhythms. Daily oscillations of serum iron, TSAT, hepatic Tfrc expression, and erythropoietic activity are maintained in mice housed in constant darkness, where oscillation reflects an endogenous circadian period. Oscillations of serum iron, TSAT, hepatic Tfrc, and erythropoietic activity were ablated when circadian machinery was disrupted in Bmal1 knockout mice. Interestingly, we find that circadian oscillations of erythropoietic activity and hepatic Tfrc expression are maintained in opposing phase, likely allowing for optimized usage and storage of serum iron whilst maintaining adequate serum levels and TSAT. This study provides the first confirmatory evidence that serum iron is circadian regulated, discerns circadian rhythms of TSAT, a widely used clinical marker of iron status, and uncovers liver-specific circadian regulation of TFR1, a major player in cellular iron uptake.
ABSTRACT
BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.
Subject(s)
Necroptosis , Non-alcoholic Fatty Liver Disease , Humans , Female , Male , Mice , Animals , Epigenesis, Genetic , Non-alcoholic Fatty Liver Disease/genetics , Hepatocytes , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Protein Kinases/geneticsABSTRACT
The emergence of large language models (LLMs) and assisted artificial intelligence (AI) technologies have revolutionized the way in which we interact with technology. A recent symposium at the Walter and Eliza Hall Institute explored the current practical applications of LLMs in medical research and canvassed the emerging ethical, legal and social implications for the use of AI-assisted technologies in the sciences. This paper provides an overview of the symposium's key themes and discussions delivered by diverse speakers, including early career researchers, group leaders, educators and policy-makers highlighting the opportunities and challenges that lie ahead for scientific researchers and educators as we continue to explore the potential of this cutting-edge and emerging technology.
Subject(s)
Artificial Intelligence , Biomedical Research , TechnologyABSTRACT
A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. The lack of R/Bioconductor tools for the effective visualization of nanopore methylation profiles between samples from different experimental groups led us to develop the NanoMethViz R package. Our software can handle methylation output generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the sample-level, we use dimensionality reduction to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across individual reads along the genome using the spaghetti plot and heatmaps, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at https://bioconductor.org/packages/NanoMethViz.
Subject(s)
DNA Methylation/genetics , Genomics/methods , Nanopore Sequencing/methods , Sequence Analysis, DNA/methods , Software , Animals , Humans , MiceABSTRACT
Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains - the N-terminal ATPase and C-terminal SMC hinge - suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing.
Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Protein Multimerization , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Binding Sites/genetics , Chromatin/genetics , Chromosomal Proteins, Non-Histone/genetics , HEK293 Cells , Humans , Immunoblotting , Microscopy, Fluorescence , Mutation , Protein Binding , Protein Domains , Scattering, Small Angle , Substrate Specificity , Ubiquitin/chemistry , Ubiquitin/metabolism , X-Ray DiffractionABSTRACT
Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of â¼10, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterize, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions (ICRs) as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new ICRs.
Subject(s)
Genome , Genomic Imprinting , Genotyping Techniques , Haplotypes , Sequence Analysis, DNA/statistics & numerical data , Alleles , Animals , Chromosome Mapping , CpG Islands , DNA Methylation , Embryo, Mammalian/chemistry , Embryo, Mammalian/metabolism , Female , High-Throughput Nucleotide Sequencing , Male , Mice , Placenta/chemistry , Placenta/metabolism , PregnancyABSTRACT
The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.
Subject(s)
Chromosomal Proteins, Non-Histone/physiology , DNA Methylation , Homeodomain Proteins/genetics , Microsatellite Repeats/genetics , Cells, Cultured , Cellular Reprogramming/genetics , Choanal Atresia/genetics , Choanal Atresia/metabolism , DNA Methylation/genetics , Epigenesis, Genetic/genetics , Gene Expression Regulation , HCT116 Cells , HEK293 Cells , Homeodomain Proteins/metabolism , Humans , Male , Microphthalmos/genetics , Microphthalmos/metabolism , Muscular Dystrophy, Facioscapulohumeral/genetics , Muscular Dystrophy, Facioscapulohumeral/metabolism , Muscular Dystrophy, Facioscapulohumeral/pathology , Nose/abnormalitiesABSTRACT
In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Haploinsufficiency/physiology , Animals , Blotting, Western , Cells, Cultured , Chromosomal Proteins, Non-Histone/genetics , DNA Methylation/genetics , DNA Methylation/physiology , Fibroblasts/metabolism , Flow Cytometry , Haploinsufficiency/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Keratinocytes/metabolism , Mice , Mice, Transgenic , Muscle, Skeletal/metabolism , Muscular Dystrophy, Facioscapulohumeral/genetics , Muscular Dystrophy, Facioscapulohumeral/metabolism , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Skin , ThymocytesABSTRACT
It has very recently become clear that the epigenetic modifier SMCHD1 has a role in two distinct disorders: facioscapulohumoral muscular dystrophy (FSHD) and Bosma arhinia and micropthalmia (BAMS). In the former there are heterozygous loss-of-function mutations, while both gain- and loss-of-function mutations have been proposed to underlie the latter. These findings have led to much interest in SMCHD1 and how it works at the molecular level. We summarise here current understanding of the mechanism of action of SMCHD1, its role in these diseases, and what has been learnt from study of mouse models null for Smchd1 in the decade since the discovery of SMCHD1.
Subject(s)
Choanal Atresia/genetics , Chromosomal Proteins, Non-Histone/genetics , Epigenesis, Genetic , Microphthalmos/genetics , Muscular Dystrophy, Facioscapulohumeral/genetics , Nose/abnormalities , Animals , DNA Methylation/genetics , Heterozygote , Humans , Mice , MutationABSTRACT
Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.
Subject(s)
Cellular Reprogramming , Pluripotent Stem Cells/cytology , Cell Differentiation , Fibroblasts/cytology , Genomic Instability , Humans , Kruppel-Like Factor 4ABSTRACT
The structural maintenance of chromosomes hinge domain containing protein 1 (SMCHD1) is a large multidomain protein involved in epigenetic gene silencing. Variations in the SMCHD1 gene are associated with two debilitating human disorders, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). Failure of SMCHD1 to silence the D4Z4 macro-repeat array causes FSHD, yet the consequences on gene silencing of SMCHD1 variations associated with BAMS are currently unknown. Despite the interest due to these roles, our understanding of the SMCHD1 protein is in its infancy. Most knowledge of SMCHD1 function is based on its similarity to the structural maintenance of chromosomes (SMC) proteins, such as cohesin and condensin. SMC proteins and SMCHD1 share similar domain organisation and affect chromatin conformation. However, there are important differences between the domain architectures of SMC proteins and SMCHD1, which distinguish SMCHD1 as a non-canonical member of the family. In the last year, the crystal structures of the two key domains crucial to SMCHD1 function, the ATPase and hinge domains, have emerged. These structures reveal new insights into how SMCHD1 may bind and regulate chromatin structure, and address how amino acid variations in SMCHD1 may contribute to BAMS and FSHD. Here, we contrast SMCHD1 with canonical SMC proteins, and relate the ATPase and hinge domain structures to their roles in SMCHD1-mediated epigenetic silencing and disease.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Epigenesis, Genetic , Adenosine Triphosphatases/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Gene Silencing , Humans , Polymorphism, Single Nucleotide , Protein Domains , Structure-Activity RelationshipABSTRACT
The tendency of 5-methylcytosine (5mC) to undergo spontaneous deamination has had a major role in shaping the human genome, and this methylation damage remains the primary source of somatic mutations that accumulate with age. How 5mC deamination contributes to cancer risk in different tissues remains unclear. Genomic profiling of 3 early-onset acute myeloid leukemias (AMLs) identified germ line loss of MBD4 as an initiator of 5mC-dependent hypermutation. MBD4-deficient AMLs display a 33-fold higher mutation burden than AML generally, with >95% being C>T in the context of a CG dinucleotide. This distinctive signature was also observed in sporadic cancers that acquired biallelic mutations in MBD4 and in Mbd4 knockout mice. Sequential sampling of germ line cases demonstrated repeated expansion of blood cell progenitors with pathogenic mutations in DNMT3A, a key driver gene for both clonal hematopoiesis and AML. Our findings reveal genetic and epigenetic factors that shape the mutagenic influence of 5mC. Within blood cells, this links methylation damage to the driver landscape of clonal hematopoiesis and reveals a conserved path to leukemia. Germ line MBD4 deficiency enhances cancer susceptibility and predisposes to AML.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , Endodeoxyribonucleases/genetics , Gene Expression Regulation, Leukemic , Hematopoiesis , Leukemia, Myeloid, Acute/genetics , Adult , DNA Methyltransferase 3A , Female , Gene Deletion , Germ Cells/metabolism , Germ Cells/pathology , Humans , Leukemia, Myeloid, Acute/pathology , Male , Mutation , Mutation AccumulationABSTRACT
Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1) plays important roles in epigenetic silencing and normal mammalian development. Recently, heterozygous mutations in SMCHD1 have been reported in two disparate disorders: facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS). FSHD2-associated mutations lead to loss of function; however, whether BAMS is associated with loss- or gain-of-function mutations in SMCHD1 is unclear. Here, we have assessed the effect of SMCHD1 missense mutations from FSHD2 and BAMS patients on ATP hydrolysis activity and protein conformation and the effect of BAMS mutations on craniofacial development in a Xenopus model. These data demonstrated that FSHD2 mutations only result in decreased ATP hydrolysis, whereas many BAMS mutations can result in elevated ATPase activity and decreased eye size in Xenopus Interestingly, a mutation reported in both an FSHD2 patient and a BAMS patient results in increased ATPase activity and a smaller Xenopus eye size. Mutations in the extended ATPase domain increased catalytic activity, suggesting critical regulatory intramolecular interactions and the possibility of targeting this region therapeutically to boost SMCHD1's activity to counter FSHD.
Subject(s)
Adenosine Triphosphate/metabolism , Choanal Atresia/genetics , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Eye Diseases/pathology , Microphthalmos/genetics , Muscular Dystrophy, Facioscapulohumeral/genetics , Mutation, Missense , Nose/abnormalities , Adenosine Triphosphatases , Amino Acid Sequence , Animals , Choanal Atresia/pathology , Chromosomal Proteins, Non-Histone/genetics , Crystallography, X-Ray , Eye Diseases/genetics , Eye Diseases/metabolism , Humans , Mice , Microphthalmos/pathology , Muscular Dystrophy, Facioscapulohumeral/pathology , Nose/pathology , Protein Conformation , Protein Domains , Sequence Homology , Xenopus laevisABSTRACT
Epigenetic mechanisms involved in the establishment of lung epithelial cell lineage identities during development are largely unknown. Here, we explored the role of the histone methyltransferase Ezh2 during lung lineage determination. Loss of Ezh2 in the lung epithelium leads to defective lung formation and perinatal mortality. We show that Ezh2 is crucial for airway lineage specification and alveolarization. Using optical projection tomography imaging, we found that branching morphogenesis is affected in Ezh2 conditional knockout mice and the remaining bronchioles are abnormal, lacking terminally differentiated secretory club cells. Remarkably, RNA-seq analysis revealed the upregulation of basal genes in Ezh2-deficient epithelium. Three-dimensional imaging for keratin 5 further showed the unexpected presence of a layer of basal cells from the proximal airways to the distal bronchioles in E16.5 embryos. ChIP-seq analysis indicated the presence of Ezh2-mediated repressive marks on the genomic loci of some but not all basal genes, suggesting an indirect mechanism of action of Ezh2. We found that loss of Ezh2 de-represses insulin-like growth factor 1 (Igf1) expression and that modulation of IGF1 signaling ex vivo in wild-type lungs could induce basal cell differentiation. Altogether, our work reveals an unexpected role for Ezh2 in controlling basal cell fate determination in the embryonic lung endoderm, mediated in part by repression of Igf1 expression.