Epigenetics & Chromatin

DNA methylation is a key epigenetic mechanism influencing gene regulation and cellular identity. In skeletal muscle, methylation contributes to fiber-type specification, metabolic programming, and satellite cell function, with evidence of sex-specific differences. Here, we investigated whether spatial regionalization of gene expression along the proximal-distal axis of the tibialis anterior (TA) is mirrored by corresponding patterns of DNA methylation. Using MeDseq on TA sections from muscles previously analyzed by spatial transcriptomics, we profiled methylation across transcriptional start sites (TSS), gene bodies, and regulatory elements. Despite robust spatial differences in transcriptomes, methylation patterns were largely uniform along the proximal-distal axis, indicating that DNA methylation does not underlie regional gene expression in adult TA muscle. In contrast, sex emerged as the primary determinant of methylation variation. Male muscles exhibited widespread hypermethylation at TSS, gene-bodies and regulatory regions, corresponding with sex-specific transcriptional programs, including glycolytic fiber enrichment in males and oxidative fiber markers in females. Notably, chromatin- and methylation-associated regulators such as Setd7, Gsk3a, and Bmyc were upregulated in males, suggesting mechanisms linking transcriptional control to epigenetic state. These findings highlight that while spatial gene expression is transcriptionally driven, sex-specific epigenetic programs dominate adult skeletal muscle, underscoring the need to consider sex in multi-omic studies of muscle biology.

BackgroundMaize knobs are regions of constitutive heterochromatin that are readily identified in both meiotic and somatic chromosomes. These structures have been characterized as stable throughout the cell cycle, exhibiting late replication during the S-phase, and are composed of two specific families of highly repetitive DNA sequences: K180 and TR-1. Although widely used as cytogenetic markers due to their variability in number and chromosomal position across inbred lines, hybrids, and landraces, little is known about their chromatin structure and dynamics. In this study, we analyzed chromatin accessibility of knobs using DNS-seq data across four maize tissues representing distinct developmental stages. ResultsOur results reveal that K180 knobs exhibit tissue-specific variation in chromatin accessibility, transitioning between open and closed states during development. In contrast, the TR-1 knob of chromosome 4 remained consistently inaccessible across all tissues analyzed. A knob composed of both K180, and TR-1 further supported this observation, with only the K180 region showing dynamic accessibility. To validate these findings, we also analyzed other repetitive regions such as centromeres, which showed a uniformly closed chromatin structure similar to TR-1. These results suggest a unique developmental modulation of chromatin accessibility associated with K180 repeats. While the chromatin accessibility of knobs does not reach the levels observed at Transcription Start Sites (TSS), the comparison among different classes of repetitive DNA within maize constitutive heterochromatin provides compelling evidence for sequence-specific and tissue-specific chromatin dynamics. ConclusionsOur findings uncover a previously unrecognized property of maize knobs and establish a reference for future studies on chromatin organization and epigenetic regulation of repetitive DNA in plant genomes.

Ehmt2 is a key H3K9 methyltransferase that regulates genome silencing and structural integrity during animal development. In addition to this canonical function, Ehmt2 has also been implicated in neural tissues mediating both direct and indirect transcriptional activation, and exon splicing, to facilitate proper neural cell differentiation and survival. Several germline loss-of-function animal models have been developed showing both conserved and divergent phenotypes that range from embryonic lethality to behavioural deficits in adult, fertile animals. Here, we generated the first maternal-zygotic ehmt2 loss of function mutant in zebrafish using CRISPR-Cas9 mutagenesis. An assessment of the pattern of H3K9 methylation in mutant embryos by ChIP-seq indicates that there are aberrant levels of this repressive mark, including reduction in discrete 5 non-coding regions of genes, but with no significant change in the overall pattern distribution of these marks across the genome. Global transcriptome and morphological analyses demonstrated that mutant embryos displayed greater variation in the timing of developmental progression that is, on average, slower compared to controls. Despite this, mutant embryos ultimately survive and are fertile. Through examination of progenitor cell dynamics and gene expression profiles, we found that the delay in embryonic development was associated with longer rates of S-M phases of the progenitor cell cycle in mutants leading to deficits in tissue growth. Finally, our data suggest a robust network of epigenetic regulators can potentially compensate for Ehmt2 loss of function and permit embryonic development and survival in ehmt2 mutant zebrafish. Our work establishes a zebrafish ehmt2 loss of function model that will facilitate examination of the complex and varied roles of Ehmt2 in vertebrate development.

Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the future egg or sperm cells. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to further mature. The reason for this is unclear. Previous studies in mice revealed that several specific metabolic changes occur during the maturation of these cells, which are essential for their developmental progress. However, very little is known about the metabolic profile of human primordial germ cells. In the severe scarcity of human PGCs, hPGCLCs serve as a research model to study PGC formation. To investigate this, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLC, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the influence of metabolism on human PGC development that could help improve methods for in vitro differentiation and maturation hPGCLCs.

Maternal pancreatic {beta}-cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing {beta}-cell mass, insulin transcription and glucose-stimulated insulin secretion[1-4]. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth[5, 6]. We have previously found that prolactin treatment in islets regulates the expression of epigenetic modifiers[7, 8]. However, whether lactogen signaling in {beta}-cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of {beta}-cells to facilitate transcriptional regulation. Using single-cell ATAC-sequencing, we identified differentially accessible regions (DARs) in {beta}-cells which had 718 overrepresented motifs following prolactin treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within {beta}-cells, including Myc, Mafb and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2 an established regulator of epigenetic landscape within cells. OVOL2 is a transcription factor involved in EMT inhibition and energy homeostasis with unknown roles in pancreatic {beta}-cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on {beta}-cell proliferation, establishing a novel regulator of PRLR signaling.

The actin polymerization machinery, comprising the ARP2/3 complex and its activators, the WASP family proteins, has been implicated in regulating a broad spectrum of nuclear processes, such as transcriptional regulation and nuclear organization. Here, using clustered protocadherin (cPcdh) and {beta}-globin genes as model systems, we showed that WAVE2, a member of the WASP family, regulates chromatin organization by maintaining heterochromatin dynamics. Specifically, by CRISPR DNA-fragment editing, in conjunction with integrated analyses of ChIP-seq, MeDIP-seq, ATAC-seq, 4C-seq, and RNA-seq, we showed that deposition of H3K9me3, a key heterochromatin mark, is significantly decreased at the cPcdh locus upon WAVE2 deletion, concurrent with aberrant accumulation of CTCF/cohesin complex at promoter regions and spatial reorganization of chromatin architecture around nucleolus. In addition, REST/NRSF exerts a similar heterochromatindependent effect on the cPcdh locus. Finally, genetic and genomic data showed that WAVE2 regulates {beta}-globin gene expression by maintaining heterochromatin status. Together our data suggested that WAVE2 and REST/NRSF regulate clustered gene expression in a heterochromatin-dependent manner.

BackgroundGene expression changes in response to developmental and environmental cues rely on cis-regulatory sequence elements (cREs). BRG1/BRM-Associated Factors (BAF) chromatin remodeling complexes maintain chromatin accessibility at many cREs, enabling binding by transcription factors (TFs). However, cREs exhibit a broad range of sensitivity to loss of BAF function, and the basis of this variability remains unknown. ResultsTo identify the characteristics of BAF-dependent cREs, we mapped chromatin accessibility changes following acute pharmacologic BAF inhibition in GM12878 lymphoblastoid cells. We integrated these results with over 100 TF and histone modification ChIP-seq datasets and used machine learning to identify features that predict chromatin accessibility changes. We found that Activator Protein 1 (AP-1) factors and lymphoid lineage-defining TFs including RUNX3 and PU.1 predicted BAF-dependence. Strikingly, we found that cREs bearing the chromatin signature of "primed" enhancers - enriched for H3K4me1 but lacking H3K27ac - were significantly more sensitive to BAF inhibition than typical active enhancers. As primed enhancers are known to facilitate transcriptional responses to stimuli, we tested the requirement of BAF activity in these responses. Acute BAF inhibition was sufficient to prevent both chromatin and transcriptional responses to interferon gamma and dexamethasone. cREs which normally gained accessibility in response to stimulation failed to do so with BAF inhibition, and these cREs were linked to genes with suppressed transcriptional induction. ConclusionsCollectively, our results demonstrate a requirement for continuous BAF activity to enable stimulus response and suggest that defective signal responsiveness may be a pathogenic mechanism in disease states caused by loss-of-function mutations in BAF subunits.

Caffeine, the most widely consumed stimulant worldwide and primarily sourced from coffee, is well known for its central nervous system effects. Emerging evidence indicates that caffeine also modulates key cellular processes, including DNA repair. It inhibits the kinase activity of ATM and ATR-essential DNA damage response proteins, and impairs homologous recombination (HR)-mediated repair through multiple mechanisms. However, its effects on nonhomologous end joining (NHEJ), a major double-strand break (DSB) repair pathway, have been underexplored. In a recent study, we reported that caffeine inhibits NHEJ primarily by interfering with Ligase IV/XRCC4 complex, using in vitro and ex vivo model systems. Given coffees role as a primary dietary caffeine source, this study investigates the impact of Coffea arabica decoction on NHEJ-mediated DSB repair. High-performance liquid chromatography (HPLC) quantified caffeine levels in the decoction, followed by in vitro and ex vivo assays to evaluate NHEJ efficiency. Results demonstrate that coffee decoction inhibits end joining of both compatible and noncompatible DNA ends in cell-free systems derived from normal and cancer cells. Extrachromosomal repair assays confirmed impaired intracellular NHEJ, leading to accumulation of unrepaired DSBs in human cells. Kinetic analysis of {gamma}-H2AX foci formation and resolution revealed persistent DNA breaks and reduced repair kinetics. Reconstitution experiments verified that the decoction specifically targets the Ligase IV/XRCC4 complex. These findings, building on our previous work, establish coffee decoction as a potent NHEJ inhibitor, mirroring purified caffeines effects. This underscores caffeines interference with endogenous DNA repair, with profound implications for cancer therapy by sensitizing tumors to genotoxic treatments.

Enhancer RNAs (eRNAs) are non-coding transcripts produced at enhancer regions, which appear to be involved in transcriptional regulation. Up to date, these have been primarily investigated using labor-and cost-intensive genomic techniques. However, the precise mechanisms by which eRNA transcription or the eRNA transcripts themselves mediate transcriptional regulation remain unclear. Here, we present a novel experimental approach that allows us to analyze the characteristics of eRNA transcription in fixed and live whole Drosophila melanogaster embryos. We employ the anterior-posterior patterning genes as a model system to investigate the dynamics of eRNA expression, utilizing an imaging-based approach. We combined high-sensitivity fluorescence in situ hybridization (FISH) chain reaction (HCR) with high-resolution confocal microscopy to detect eRNA and mRNA molecules. Through this experimental assay, we identified foci of elevated transcriptional activity that generate eRNA transcripts correlated with mRNA production at the same gene locus. We could show that this eRNA transcription is independent of promoter activity. Additionally, we demonstrate that insulators can influence eRNA transcription, resulting in loss of eRNA transcription. Moreover, we observe that eRNAs can originate both within classical enhancer regions and outside of them, including from foreign bacterial sequences when these are placed near enhancer sequences, underscoring the strong influence of local regulatory context on eRNA initiation. In live embryos using MS2-MCP live imaging, our analysis of insulators showed a modest reduction in mRNA burst intensity accompanied by a slight increase in burst frequency. Overall, our imaging-based approach offers a novel platform for dissecting enhancer-eRNA interactions and could be adapted for wider applications.

Enhancer-regulating epigenetic modifiers play critical roles in normal physiological processes and human pathogenesis. The major enhancer regulator paralogs MLL3 and MLL4 (MLL3/4) belong to the lysine methyltransferase 2 (KMT2) family, which catalyzes the methylation of lysine 4 on histone H3 (H3K4me). MLL3/4 are required for enhancer activation and are essential for mammalian development and stem cell differentiation. Recent studies have linked MLL3/4 with different metabolic pathways in the context of stem cell self-renewal and cancer cell growth; however, the underlying mechanisms remain elusive. Here, we utilize Seahorse extracellular flux analysis, stable isotope tracing, stem cell biology techniques, and transcriptomic analysis to investigate the functional relationship of MLL3/4, cellular respiration, and stem cell differentiation. Our results indicate that the loss of MLL3/4 impairs glycolytic activity and mitochondrial respiration in murine embryonic stem cells by downregulating the rate-limiting glycolytic enzyme Hexokinase 2 (HK2) and impairing the function of the Alpha-ketoglutarate dehydrogenase (OGDH) complex. Furthermore, simultaneously overexpression of HK2 and OGDH rescues defects in both cellular respiration and differentiation caused by MLL3/4 loss. Taken together, our study reveals a novel mechanism by which epigenetic machineries such as MLL3/4 govern the differentiation of pluripotent stem cells and facilitates the understanding of disease pathogenesis driven by enhancer malfunction.

Spatiotemporal organisation of biological molecules is a key driver of cellular processes, including many post-transcriptional epigenetic processes. The germline-specific germ granules are biomolecular condensates that act as hubs for mRNA and small RNA processing and are core regulators of germline gene expression programming. Germ granules have been studied extensively in C. elegans, and recent developments have led to many subdivisions of the germ granule into specialised compartments. Rapid advancements in microscopy and protein-protein interaction (PPI) screening techniques have produced a large amount of data towards characterising the localisation of proteins to specific granules. However, common methods used to probe PPIs are limited in their ability to robustly detect valid interactions, especially the multivalent and sometimes transient ones observed in granule environments. Here we perform a meta-analysis of granule protein interaction screens. While these experiments generally enrich for proteins matching the profile of granule-associated proteins, we find that when considering screens individually, reproducibility is surprisingly low, highlighting not only the variability inherent in these methods but also the dynamic nature of the PPI networks present in granules. We developed an algorithm to provide a measure of each proteins association with specific granules across various experiments. By further clustering and investigation of the resulting score matrix, we demonstrate the power of this holistic approach to provide deeper insights into germ granule organisation and highlight novel can provide a resource to better inform future investigations into granules and their constituent proteins.

Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients eventually develop resistance to TKI therapy. The most common resistance mechanism for the TKI Osimertinib is the overexpression of the MET Proto-Oncogene, Receptor Tyrosine Kinase (MET). We previously demonstrated that miR-411-5p A-to-I edited at position 5 (miR-411ed) can directly target MET in A549 and H1299 cells. MiR-411ed in combination with Osimertinib reduced cell proliferation in two TKI resistant EGFR-mutated cell lines: HCC827R and PC9R. MiR-411ed did not downregulate MET expression in HCC827R, suggesting an alternative mechanism for TKI response. In this study, we aim to identify the mechanism of miR-411ed TKI response using a multi-omics approach of RNAseq and protein mass spectrometry. In our cellular model, we identified miR-411ed affected genes independent of MET activity, resulting in 211 genes (RNAseq) and 36 proteins (proteomics). Pathway analysis identified an increase in interferon signaling for RNAseq and combined omics, and a decrease in ERK/MAPK signaling in proteomics. Using the IsoTar target prediction tool, we identified STAT3 as a key regulator and confirmed STAT3 protein downregulation upon transfection with miR-411ed. We further investigated the effect of miR-411ed in vivo, observing a reduction in tumor size with miR-411ed in combination with Osimertinib but not with miR-411ed or Osimertinib treatment alone, confirming the effectiveness of miR-411ed in TKI response.

Background: Epigenetic clocks based on DNA methylation (DNAm) have emerged as promising biomarkers of biological aging, yet their associations with cognitive performance remain inconsistent. This study investigates the relationship between epigenetic age acceleration and cognitive performance in older adults using 14 DNAm clocks from five generations of development. Methods: We analyzed data from the Berlin Aging Study II (BASE-II) using genome-wide DNAm profiles and cognitive assessments ascertained at baseline (T0) and two follow-up time points (T1, T2) in up to 1,014 individuals. DNAm-based age and age acceleration estimates were calculated using Biolearn and MethylCIPHER. Analyses focused on cross-sectional and longitudinal associations between DNAm clock estimates and cognitive performance, including sex-specific effects and comparisons with frailty as non-cognitive positive control. Results: Among all tested DNAm clocks, DunedinPACE (a third-generation clock) showed the strongest and most consistent associations with cognitive performance. In addition, the fifth-generation SystemsAge framework also demonstrated robust associations with cross-sectional and longitudinal cognitive outcomes. In contrast, second-generation clocks (GrimAge [v2], PhenoAge) showed occasional nominal associations, while first-generation clocks (Horvath [v1], Hannum) and the causally-informed, fourth-generation clocks (e.g. YingCausAge, YingDamAge) showed no noteworthy signals. Likewise, telomere length estimated from DNAm was not strongly associated with cognitive performance in this dataset. Conclusions: Our findings highlight DunedinPACE as a particularly informative biomarker for various aspects of cognitive aging, while other DNAm aging measures showed no consistent associations. Future work should further refine domain-specific epigenetic biomarkers to improve biological aging assessments and achieve a more reliable early detection of cognitive decline.

Silencing complexes formed by PIWI-clade Argonaute (Ago) proteins and PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, controlling the deleterious activities of transposable elements (TEs) in animal germline. However, our understanding of PIWI-piRNA-directed TE silencing remains incomplete. Here, we systemically characterize the proximity proteome of PIWI members, Piwi, Aubergine (Aub), and Ago3 in the germline of Drosophila ovaries. Functional screening identifies previously uncharacterized factors involved in TE silencing, including H3K4me3 writer and transcriptional coactivator Set1. Transcriptome analysis reveals that Set1 acts as an indispensable repressor for TEs, particularly those forming telomeres. The involvement of Set1 in Piwi pathway is further supported by its critical role in the production of antisense, TE-targeting piRNAs. Notably, catalytic activity of Set1 is dispensable for TE silencing. Genome-wide chromatin binding analysis using CUT&Tag demonstrates that Set1 preferentially associates with TE sequences and localizes to subtelomeric piRNA cluster loci, suggesting a role in promoting piRNA precursor transcription through direct binding. Collectively, these findings uncover a noncanonical function of Set1 in Piwi-mediated TE silencing and telomere control in germline nuclei.

Background/ObjectivesRNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in human is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I could function indirectly derives from studies of its homologs in yeasts called Rpb9. Here, we endogenously humanized the rpb9 gene of the fission yeast Schizosaccharomyces pombe to examine the functional capabilities of POLR2I. MethodsWe edited the genomic rpb9 locus in S. pombe so that it encodes the human POLR2I protein, and investigated functional and structural conservation. ResultsWith our humanized yeast system, we find widespread functional complementation by human POLR2I of S. pombe rpb9 roles in yeast growth, chronological aging, and stress responses. We also find that POLR2I complements novel roles for yeast rpb9 in facultative heterochromatin assembly, resistance against the chemotherapy 5-fluorouracil, and resistance against the fungicide thiabendazole. In contrast, we find that POLR2I cannot complement the role of rpb9 in resistance against the transcription elongation inhibitor 6-azauracil (6-AU) in our system. Interestingly, POLR2I could complement 6-AU resistance if ectopically expressed. Lastly, we observe extensive structural homology between Rpb9 and POLR2I proteins. ConclusionsOur study establishes an endogenous cross-species gene complementation strategy that uncovers both conserved and rewired functions of fission yeast rpb9 and its human homolog, POLR2I. In addition to validating conserved roles, we also identified conservation of previously unrecognized roles of rpb9 in heterochromatin formation and chemoresistance.

The PBAF is one of three biochemically distinct BAF chromatin remodelers in humans. We previously proposed the role of ARID2, a PBAF component, as a bonafide tumor suppressor in colorectal cancer (CRC). Here, we validated loss of tumor suppression under conditions of ARID2 deficiency emanating from a marked reduction in PBAF complex assembly resulting from destabilization of PBAF-specific components BRD7, PHF10, and PBRM1. Transcriptome profiling of ARID2 deficient CRC cells revealed perturbation of disease processes, including CRC and neurodegenerative disorders, as well as CRC relevant pathways including Wnt/{beta}-catenin signalling, but transcript levels of PBAF-specific components remained unchanged, confirmed by RT-qPCR and TCGA data analysis. Our study establishes ARID2 as a critical stabilizer of the PBAF complex of relevance to CRC.

The cohesin complex has conserved roles in sister chromatid cohesion, DNA replication, genome organization, and the DNA damage response. We heterologously expressed the human cohesin complex in yeast to probe the behaviour of human cohesin. Human cohesin was unable to complement loss of function mutations in yeast cohesin, either as single subunits or as complexes, including in the context of co-expressing up to 12 human cohesin-associated genes. Heterologous expression of human cohesin in yeast expressing wildtype yeast cohesin resulted in dominant cohesion dysregulation and DNA damage sensitivity phenotypes. We used co-immunoprecipitation to demonstrate that human SMC proteins interact with endogenous yeast cohesin rings creating dominant-negative hybrid complexes that disrupt endogenous cohesin biology.

Structured AbstractO_ST_ABSINTRODUCTIONC_ST_ABSAdults with Down syndrome (DS) have a higher risk of developing Alzheimers disease (AD). As gut microbiota (GM) alterations have been reported in AD, we investigated their association with cognitive decline and plasma AD biomarkers in DS. METHODSFecal and plasma samples were collected from 58 adults with DS (21-75 years) and 30 euploid controls (CTRL; 25-83 years). GM was profiled using 16S rRNA sequencing. Major Neurocognitive Disorder (NcD) was diagnosed according to DSM-5 criteria. Plasma levels of p-Tau181, NfL, and GFAP were measured using the Simoa platform. RESULTSCompared with CTRL, DS showed significant changes in UBA1819 and Intestinibacter genera, previously reported to be associated with mild cognitive impairment. Furthermore, DS with NcD were characterized by a reduced abundance of Roseburia genus, which was also negatively associated with plasma levels of AD biomarkers. CONCLUSIONAdults with DS display AD-associated changes in GM partially resembling those previously reported in euploid AD patients

Neurofibromin 1 (NF1) is a critical negative regulator of the RAS-RAF-ERK pathway, mutations in which have been clinically implicated in various neurodevelopmental disorders. However, the lack of a high-resolution spatiotemporal map has obscured the understanding of why specific cell populations and developmental processes are uniquely vulnerable to NF1 loss. In this study, we present a comprehensive atlas of NF1 expression in the developing mouse brain. Using in situ hybridization and immunohistochemistry, we characterized NF1 distribution from early embryonic stages through postnatal maturation. We further integrated these findings with single-nuclei RNA-sequencing (snRNA-seq) datasets from adult mouse brain to achieve higher resolution. Our results reveal a previously undocumented graded expression pattern of NF1 across various brain regions and lineages. This comprehensive study will not only help in understanding the fundamental role of NF1 during brain development but will also be pivotal in providing a framework to study NF1-associated brain disorders.

KMT2D, the histone methyltransferase and core component of the COMPASS/MLL4 complex, has been implicated in developmental diseases such as Kabuki Syndrome, interstitial lung disease, and congenital diaphragmatic hernia, with clear links to pediatric pulmonary disorders. Despite this, the mechanism by which KMT2D governs lung development remains unclear. Knock-in mouse models rendering, KMT2D catalytically deactivated (KMT2DKI) and reducing H3K4 methylation, have demonstrated potential in defining KMT2Ds role in pulmonary development. Our examination of the lungs of KMT2DKI mice revealed increased cellular density and impaired sacculation indicated by reduced airspace chord length, thickening of intersaccular septa, and abnormal alveolar cell differentiation. KMT2DKI mice revealed narrowed Sox2+ conducting airways and epithelial differentiation defects characterized by reduced Cc10+ club cells. Accompanying the alveolar and airway hypoplasia, blood vessel luminal area was reduced. Conversely, KMT2DKI lungs had a significantly higher proportion of proliferating cells accompanied by a dramatic expansion in Pdgfr+ mesenchymal progenitor cells. Our findings therefore suggest that KMT2D-mediated H3K4 methylation is vital to normal lung development, and its impairment results in widespread pulmonary hypoplasia and potentially pulmonary hypertension.