Epigenetics & Chromatin

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

H1 histones are abundant nuclear proteins that bind to linker DNA at the entry and exit points of nucleosomes. Although individual H1 family members play partially redundant roles in chromatin organization, the discovery of disease-associated mutations in H1 genes has raised interest in their cell-type specific functions. Heterozygous, de novo frameshift mutations in H1-4 cause the neurodevelopmental disorder Rahman Syndrome, which is characterized by mild to severe intellectual disability along with other neurological and morphological features. H1.4 has mostly been studied in dividing cells, and its expression in the brain was poorly understood. Here we characterized the expression of H1f4 mRNA and H1.4 protein in the brains of male and female mice across postnatal development. Using an epitope-tagged H1f4 knockin mouse, we show that this linker histone is robustly expressed throughout the brain including in mature, post-mitotic neurons of adult mice. By chromatin immunoprecipitation, we observe that H1.4 binds broadly across the genome in neural progenitors and accumulates in heterochromatin over the course of neuronal maturation. Finally we show that developmental maturation of chromatin compaction in cerebellar granule neurons is disrupted in H1f2/H1f4 double knockout mice. These data raise the possibility that the neurological changes in Rahman Syndrome may arise from disrupted functions of histone H1.4 in neurons.

Background Complex traits are influenced by numerous variants, most of which have regulatory effects on gene expression that can be mediated by DNA methylation. Molecular QTL mapping is an approach that aims to dissect these effects. However, obtaining molecular phenotypes on a large scale is challenging, particularly in livestock species. In cattle, an epigenotyping array called EpiChip has recently been developed in the European RUMIGEN project. The EpiChip, which contains 43,317 CpG sites distributed all over the bovine genome, enables large-scale measurement of DNA methylation. This study aims to characterize the genetic determinism of blood DNA methylation in cows by estimating heritability and mapping cis- and trans-methylation QTLs (meQTLs). Results Whole blood samples from 4,457 genotyped Holstein cows were epigenotyped. Across all CpG sites, the heritability estimates averaged 24.6%. The local meQTL mapping at sequence-level for variable CpG sites (SD > 2.5%; n = 28,806) detected cis-meQTLs for 80.1% of the CpG sites, with sentinel SNPs located close to their associated CpGs. A two-step analysis was also conducted to identify long-range associations, with a particular focus on trans-meQTL hotspots. First, we identified CpG-SNP trans-associations using medium-density genotypes (50k SNPs) that revealed 31,846 SNPs with significant effects on 1 to 530 trans-CpG sites. Then, regions associated with at least 34 independent trans-CpGs were retained defining 31 hotpots. For each hotspot, a local sequence-level GWAS was conducted using the first principal component derived from the associated trans-CpGs. Out of the 31 detected hotspots, three were located close to transcription factor genes (RUNX1, NFIC and FOXA3) for which the associated trans-CpGs were enriched for the corresponding binding motif. Two other hotspots were located within KDM5A and KDM5B, and their corresponding trans-CpGs were strongly overrepresented in H3K4me3 narrow peaks in blood as well as in other tissues. Conclusions By identifying functional candidate genes associated with blood DNA methylation in cattle, these findings provide new insights into the regulatory architecture of DNA methylation in mammals, highlighting the value of large-scale molecular data from livestock populations.

Contrary to other cancer types, stromal antigen 2 (STAG2) expression is associated with shorter survival and an invasive phenotype in muscle invasive bladder cancer (MIBC). As a cohesin complex component, STAG2 regulates genome organization and cell type-specific transcription, yet its mechanistic role in MIBC remains unclear. Here, we uncover mechanisms through which STAG2 coordinates chromatin architecture and gene regulation in MIBC. Modulation of STAG2 rewired chromatin architecture, altering chromatin contacts and compartmentalization, increasing promoter-enhancer interactions, and reducing short-ranged chromatin loops. At specific gene loci, we discovered that STAG2 has context-specific activating and repressive regulatory functions. At the STAG2-activated gene ABCA1, STAG2 maintained A-compartment chromatin and high levels of promoter acetylation, indicators of active transcription. STAG2 KO resulted in loss of promoter acetylation, a shift from A to B compartment chromatin, and increased occupancy of the co-repressor TRIM28, resulting in ABCA1 downregulation and diminished invasive potential. Conversely, at the STAG2-repressed gene SPOCK3, STAG2 KO resulted in B to A compartment switching, aberrant formation of chromatin loops, and SPOCK3 upregulation. Treatment with EZH2 inhibitor tazemetostat augmented STAG2-KO induced SPOCK3 upregulation, suggesting a collaborative role of STAG2 and EZH2 in repressing Polycomb Repressive Complex 2 (PRC2) target genes. Altogether, our results indicate that STAG2 plays a multifaceted role in regulating gene expression in bladder cancer that is dictated by the epigenetic and chromatin landscape of the cells. These findings identify STAG2-dependent vulnerabilities and provide a rationale for therapeutic targeting of chromatin regulators in MIBC.

DNMT3A is a de novo DNA methyltransferase whose recruitment to chromatin regulates its function. Missense mutations within the chromatin-binding PWWP domain are associated with diverse human disorders, yet how mutations in the same domain produce distinct phenotypes remains unclear. Here we systematically characterise 19 clinically reported mutations in the PWWP domain of DNMT3A that are associated with Heyn-Sproul-Jackson syndrome (HESJAS), paraganglioma (PG) and clonal haematopoiesis (CH). We show that all PWWP-domain mutations associated with HESJAS abolished interaction with H3K36me2 modified nucleosomes, defining this as a consistent biochemical feature of HESJAS. In contrast, mutations from all disease classes differentially altered DNA binding of the PWWP domain, driven by alterations in the net charge of the domain. However, these effects are largely overcome by inclusion of the DNNMT3A1 N-terminal region, which is absent from its embryonic isoform, suggesting that PWWP mutations may differentially affect DNMT3A function through development. Changes in the thermal stability of the isolated PWWP domain mutants did not directly translate into altered stability of full-length DNMT3A1 in cells. We show that HESJAS mutations can affect the intramolecular interaction between the PWWP and adjacent ADD domain, an interaction proposed to contribute to the autoinhibitory function of the ADD domain. However, not all mutations behaved in the same way, suggesting that multiple factors govern the intramolecular autoinhibition of DNMT3A. Together, this study advances our understanding of the molecular mechanisms by which DNMT3A PWWP-domain mutations are mechanistically heterogeneous, providing a biochemical framework that contributes to distinct disease phenotypes.

The information content of the genome has been extensively analyzed. However, a comparable quantitative framework for DNA methylation is still lacking. Without such quantification, the magnitude of this regulatory and dynamic epigenetic structure remains conceptually imprecise, even though methylation dysregulation is strongly linked to disease-related phenotypes and altered cellular identity. Here we address this gap by applying Shannon information theory to DNA methylation. We first consider methylation marks as binary or probabilistic regulatory states and estimate the theoretical upper-bound information capacity of the human methylome under simplifying assumptions. We then progressively refine this estimate by incorporating biologically relevant constraints, including methylation bias, bimodal methylation distributions, local CpG correlation, genomic regulatory class, and cell-type-discriminative methylation patterns. This approach allows us to distinguish between theoretical methylation capacity, statistical methylation entropy, and biologically interpretable regulatory information. Finally, we consider methylation information from a discriminative perspective, analyzing its contribution to distinguishing cell types and regulatory cellular states. Within this framework, mutual information between methylation patterns and cell identity provides a biologically constrained estimate of methylations role as an epigenetic identity code. Our layered analysis reconciles megabit-scale methylome capacity with compact, biologically interpretable identity signatures. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/735086v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@f0f0fdorg.highwire.dtl.DTLVardef@5d8a1eorg.highwire.dtl.DTLVardef@116debdorg.highwire.dtl.DTLVardef@79530e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Many human developmental enhancers are characterized by extreme evolutionary constraint in the vertebrate lineage and unique DNA sequence properties, the functional relevance of which is still unknown. Here, we investigate the consequences of their DNA sequence features on three potential aspects important for their function - transcription factor sequence recognition, chromatin accessibility and DNA structure. Using computational predictions in human as well as other vertebrates and invertebrates, we find that conserved non-coding elements (CNEs) are intrinsically nucleosome disfavoring at their core, but favor nucleosome occupancy at their borders driven by distinct nucleotide features conserved over large evolutionary distances. Nevertheless, using genome-wide nucleosome occupancy datasets, we find vertebrate CNEs exhibit higher nucleosome occupancy in comparison to surrounding regions in differentiated cells but a highly accessible conformation in embryonic tissues, suggesting a role for nucleosome positioning in their function. In addition, CNEs are exclusively enriched for homeobox transcription factor motifs, which are found at high density across their sequences. In particular, motifs specifically enriched at the boundary belong to the PBX-HOX, MEIS and POU transcription factor families, known to recognize specific DNA structural features. Consistent with this finding, CNE boundaries are enriched for unusual DNA structural motifs that may constitute a recognition mechanism by transcription factors that bind a narrow minor groove. The finding that extreme nucleotide conservation are likely to be driven by a combination of nucleosome and protein binding constraints provide a potential mechanistic insight into the function of early developmental enhancers.

DNA N6-methyladenine (6mA) has emerged as a significant epigenetic modification across a broad range of eukaryotes, from unicellular protists to metazoa. However, its role in unicellular eukaryotic parasites with highly AT-rich genomes, such as malaria-causing Plasmodium falciparum, remains unclear. Using mass spectrometry, South-western blotting, and Single Molecule Real-Time sequencing (Pacific Biosciences) across four stages of P. falciparum intra-erythrocytic development (IED), we confirmed that 0.02-0.04% of genomic adenines are modified to 6mA, with over 60% of the sites being stably maintained during the IED cycle. Notably, 6mA is enriched at transcription start sites, with genes bearing 6mA marks within their 5 and 3 untranslated regions exhibiting significantly elevated steady-state transcript levels. Consistent with this, 6mA loci show a strong positive correlation with activating histone post-translational modifications, while showing no significant association with repressive histone marks. Furthermore, in contrast to unicellular ciliates such as Oxytricha and Tetrahymena - organisms that share ancestry with Plasmodium - 6mA-marked genomic regions do not occlude nucleosomes. Lastly, we identified a putative 6mA methyltransferase belonging to the METTL4 family in P. falciparum, PfN6AMT encoded by the PF3D7_1303100 gene, and demonstrate that recombinant PfN6AMT exhibits robust methyltransferase activity in vitro, with mutation of its active site residues abolishing catalytic activity. Collectively, our findings demonstrate that 6mA is a low-abundance, yet reproducible, feature of the P. falciparum epigenome that is associated with transcriptionally active chromatin, and that the molecular mechanisms governing DNA adenine methylation may have undergone substantial evolutionary divergence, even among closely related eukaryotic lineages.

The first step in initiating DNA replication is binding of the origin recognition complex (ORC) to chromosomes. Metazoan ORC is recruited to chromatin via the Orc1 intrinsically disordered region (IDR) whose DNA and chromatin binding activity are regulated by Cyclin Dependent Kinase (CDK) phosphorylation. ORC is also enriched in heterochromatin where it is required for the formation and maintenance of a silenced chromatin state. ORCs recruitment to heterochromatin is developmentally and cell cycle regulated but the underlying regulatory mechanism remains unknown. We hypothesized that CDK-dependent phosphorylation of the Orc1 IDR underpins regulated recruitment to heterochromatin. Using bioinformatic analyses, we find that the Drosophila Orc1 IDR (Orc1IDR) contains an exceptionally high density of CDK phospho-sites and, despite considerable sequence variation, the density of sites, but not their position, is conserved. In vitro DNA binding and phase separation experiments reveal that phosphorylation tunes Orc1IDR function in a rheostat-like fashion. Using phospho-mimetic variants, we find that constitutive phosphorylation not only weakens interphase chromatin binding but fully inhibits partitioning of Orc1IDR into heterochromatin. Finally, we use phospho-mimetic variants to probe the importance of site-specific phosphorylation and find that the precise position of sites can be changed provided the new sites are equitably distributed across the sequence. These studies demonstrate that phosphorylation tunes the biochemical properties of the Orc1 IDR to control DNA binding, phase separation, and, consequentially, heterochromatin recruitment. This work suggests that localized dephosphorylation of the DNA binding Orc1 IDR may underlie recruitment of ORC to specific genomic loci.

Induced pluripotent stem cells (iPSCs) are widely used as patient-specific disease models, yet substantial unexplained variability in molecular and functional readouts limits their reliability. Here, we systematically investigated the sources of variation in iPSC-derived neurons for three rare genetic disorders: Myotonic Dystrophy Type 1, chromodomain-DNA-helicase-binding protein 2-related disorder and N-acetylneuraminic acid synthase deficiency. This was performed by profiling multi-omics layers: genomics, epigenomics, transcriptomics, proteomics, metabolomics and lipidomics. Our study found that clonal variability was comparable to inter-patient differences and that neuronal differentiation state and nutrient-driven metabolic activity emerged as dominant contributors to variability observed across omics layers. Clonal differences could partly be attributed to stochastic differences in DNA methylation established during reprogramming. By modeling and correcting the observed variation, we improved the detection of disease-associated molecular signatures. Our study provides guidelines for improved study design and data analysis to minimize variability, enabling robust biomarker discovery and reliable iPSC-based disease modeling.

Polycomb group (PcG) proteins are evolutionarily conserved epigenetic regulators that maintain transcriptional states during development and are frequently misregulated in disease. Here, we generated a comprehensive collection of endogenously HA-tagged alleles for the core components of Polycomb Repressive Complex 1 (PRC1) and additional chromatin regulators in Drosophila melanogaster using CRISPR/Cas9-mediated genome engineering. We show that endogenous HA tagging preserves protein expression, chromatin localization, and genome-wide binding profiles, enabling direct comparison using a common antibody to analyze distinct PcG subunits. Proteomic analyses recovered known components of canonical and non-canonical PRC1 and PRC2 complexes and identified additional PcG-associated factors. Notably, multiple components of the nuclear pore complex (NPC) were detected as PcG interactors, and genetic analyses confirmed a functional interaction between them. This resource provides a platform for standardized analysis of Polycomb function and chromatin regulation that can easily be extended to other chromatin regulatory complexes and applied across different developmental stages. Article SummaryPolycomb group (PcG) proteins are essential regulators of development and genome function. Comparative studies of individual PcG components are often limited by the availability and quality of specific antibodies. Here, we generated a collection of Drosophila melanogaster lines carrying endogenous HA epitope tags in core components of the canonical Polycomb Repressive Complex 1 (PRC1), along with additional chromatin regulators. These tagged proteins faithfully reproduce native chromatin binding and can be used for chromatin mapping and protein interaction studies using a single, highly specific antibody. This standardized resource provides a practical tool for investigating PcG function and chromatin regulation in vivo.

Limited oxygen supply or hypoxia can impair fetal development and lead to developmental disorders, but the molecular mechanism underlying this phenomenon remains poorly understood. It is also well known that hypoxia results in transcriptomic alterations and epigenetic reprogramming. Drosophila melanogaster (fruit fly) has been used for decades as a powerful model to dissect the molecular mechanisms regulating development. To better understand the role of early hypoxic stress on development, we performed single-cell joint analysis of chromatin accessibility and transcriptome to characterize the influence of hypoxia on Drosophila embryonic development. We identified hypoxia-induced alterations in both gene expression and chromatin accessibility across 22 cell groups, especially in the genes regulating organogenesis and development of neuronal, tracheal, and muscular systems, including a reduction of germ cells, suggesting a long-lasting influence of hypoxic stress at an early embryonic stage on development and reproduction. In summary, this study demonstrates that early embryonic hypoxia induces cell type- and dose-dependent changes in chromatin accessibility and gene expression, leading to distinct developmental phenotypic responses, such as reduced number of germ cells under both 3% and 5% O2. We further conclude that the tramtrack (ttk) gene is critical in germ cell development and reproduction in Drosophila melanogaster.

Developmental genes are frequently regulated by multiple enhancers distributed across large cis-regulatory regions. How these enhancers communicate with their target promoter and how their interactions are shaped by distinct developmental transcriptional environments remain incompletely understood. Here, we investigate the chromatin organization of the Drosophila shavenbaby locus, a developmental gene controlled by seven distal enhancers. Tissue-specific UMI-4C revealed extensive enhancer-promoter and enhancer-enhancer interactions, including in cell populations where individual enhancers are inactive. Quantitative three-dimensional DNA-FISH revealed compact enhancer-promoter hubs enriched in shavenbaby-expressing cells, yet also present in non-expressing cells and prior to expression. Perturbation of shavenbaby regulators, transcription factors, and architectural proteins revealed that multiple factors contribute to hub organization. Their relative contributions differed between epidermal populations, indicating that similar hubs can be supported by different combinations of regulators. Perturbations that reduced hub organization were frequently associated with reduced shavenbaby-dependent trichome formation. Together, our results identify a robust, multi-factorial enhancer-promoter hub that is shaped by distinct regulatory inputs across developmental contexts.

Serous tubal intraepithelial carcinoma (STIC) is a known precursor of high-grade serous ovarian cancer (HGSOC). Yet, molecular events driving progression from STIC to HGSOC remain poorly defined. Aberrant DNA methylation is a hallmark of cancer, yet its role in early HGSOC remains unclear. We performed a comprehensive meta-analysis of publicly available Illumina Infinium DNA methylation EPIC array datasets assessing 255 samples comprising STIC, HGSOC, and histologically normal fallopian tube tissues. We mapped DNA methylation alterations during early tumorigenesis, identified conserved methylation patterns across STIC and HGSOC, and assessed RNA-sequencing data to define transcriptional consequences within genomic and epigenomic landscapes. STIC and HGSOC exhibited widespread DNA hypomethylation relative to normal tissue, accompanied by focal hypermethylation in CpG islands and 5' regulatory regions. DNA Hypomethylation intensifies during progression from STIC to HGSOC, particularly in cis-regulatory enhancer domains and intergenic regions. We identified 11,660 CpG sites and 447 genomic regions with conserved DNA methylation patterns across STIC and HGSOC. Within these, 70 genes showed coordinated DNA methylation and expression changes, including TRIM15, NKAPL, and RIPPLY3. These findings reveal that epigenetic remodelling occurs in STIC lesions, prior to malignant transformation. DNA methylation alterations at regulatory regions may drive invasion and offer novel avenues for early detection and targeted intervention.

Epigenetic mechanisms provide sophisticated regulatory layers that modulate gene expression across diverse organisms, yet their organization and crosstalk remain poorly understood in non-dikarya fungi (NDF). Here, we characterize the genome-wide landscape of chromatin organization in the fungus Rhizopus microsporus, revealing a compartmentalized architecture where active histone modifications (H3K4me1, H3K4me3, H3K27ac) define transcriptionally active euchromatin distinct from H3K9me3-marked constitutive heterochromatin. Through comprehensive ChIP-seq analysis, we demonstrate that these modifications exhibit distinct distribution patterns over gene bodies and co-localize with 6-methyladenine (6mA) clusters (MACs), an essential epigenetic mark that is associated with transcription in this fungus. We identified functional specialization among H3K4 methyltransferase Set1 paralogs, where Set1a primarily deposits H3K4me3 and Set1b deposits H3K4me1. In contrast, both Gcn5 paralogs function redundantly in H3K27 acetylation. Knockout analysis reveals that these enzymes are critical for sporulation, stress resistance, and pathogenesis. Importantly, we uncover an epigenetic crosstalk in which active histone modifications restrict off-target 6mA deposition, regulate methylation cluster stability, and buffer transcriptional variation. Our findings reveal conserved principles of epigenetic crosstalk between active histone modifications and the essential DNA modification 6mA that may represent a fundamental mechanism of chromatin regulation in eukaryotes. SIGNIFICANCEEpigenetic mechanisms regulate gene activity without altering the DNA sequence, yet how different epigenetic marks interact remains poorly understood. Here, we characterize the genome-wide distribution of active histone modifications and DNA N6-methyladenine (6mA) in the fungus Rhizopus microsporus, revealing that they define distinct active and inactive chromatin domains. While 6mA plays a central role in transcriptional regulation, active histone modifications direct its accurate deposition and maintenance, thereby reducing transcriptional variability. These findings uncover conserved crosstalk between histone modifications and 6mA that may represent a fundamental principle of chromatin regulation across eukaryotes.

Cohesin is a large multisubunit protein complex that plays essential roles in cell proliferation, genome organisation, and gene regulation in metazoans. Germline mutations in cohesin subunits or regulators cause a group of human developmental disorders collectively known as cohesinopathies. Increasing evidence indicates that individual cohesin subunits can confer distinct molecular functions to the complex; for example, STAG1 and STAG2 have both overlapping and non-overlapping roles in genome organisation. The zebrafish tailbud provides an excellent developmental model for investigating the coordination of cell proliferation and differentiation, processes in which cohesin has crucial functions. We previously demonstrated that loss of Stag2 disrupts Wnt signalling and mesoderm patterning in the zebrafish tailbud. Here, we show that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood. In contrast to Stag2 deficiency, loss of Stag1 impairs cell cycle progression, activates p53 signalling, and induces a metabolic shift towards catabolism. BMP signalling is reduced in Stag1-deficient embryos and is accompanied by expansion of BMP antagonist chordin expression. Stag1 loss also alters chromatin accessibility at the chordin locus and affects accessibility at chromatin domain boundaries. We propose that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly. Together, these findings reveal distinct contributions of Stag1 and Stag2 to cell-cycle regulation, chromatin architecture, and developmental signalling during vertebrate embryogenesis.

Nucleosomes are fundamental units of DNA packaging and gene regulation in eukaryotes. In mammalian sperm, most nucleosomes are replaced by protamines causing extreme chromatin compaction. Various epigenomic studies reported conflicting results on the distribution of residual nucleosomes in mammalian sperm, questioning their potential role in mediating intergenerational inheritance of paternal epigenetic information. Here we performed single-molecule footprinting through Nucleosome Occupancy and Methylome (NOMe) sequencing and applied the Bayesian statistical model nomeR to determine frequencies of nucleosome removal and retention at 103 specific genomic regions in thousands of developing haploid spermatids and mature spermatozoa of mice. While we readily detected footprints of nucleosomes and the transcription factor CTCF in round spermatids, chromatin became transiently highly accessible in elongating spermatids with loss of such footprints, indicating extensive chromatin reprogramming during spermiogenesis. In mature sperm, following nuclear decondensation with recombinant nucleoplasmin, we measured nucleosome occupancy frequencies ranging ~1.2 to 1.7% at mouse loci. In human sperm, nucleosome occupancy varied between ~2.3 to 4.5% at 163 genomic loci profiled. Contrasting mice, chromatin in ~25% of human sperm was accessible upon reducing disulfide bonds between protamines arguing for species specific protamine packaging. Our findings support a stochastic rather than programmed potential role of residual nucleosomes in mammalian sperm in regulating paternal gene expression during ensuing embryonic development.

Mutations in components of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodelling complex are among the most common genetic alterations in human cancers, yet their functional consequences are difficult to predict as they are highly context-dependent. ARID1A, a core subunit of the canonical BAF complex, is the most frequently mutated SWI/SNF gene and is recurrently altered in breast cancer, with a notable enrichment in the lobular carcinoma subtype. While previous studies in ductal breast cancer have linked ARID1A loss to deregulated oestrogen receptor (ER) signalling and endocrine resistance through its association with the pioneer factor FOXA1, the role of ARID1A in lobular cancer remains poorly understood. Here, we define the genomic, transcriptomic, and chromatin accessibility landscapes governed by ARID1A in lobular breast cancer cells. We show that ARID1A and its catalytic partner SMARCA4 co-occupy distal regulatory regions enriched for forkhead and AP-2 transcription factor motifs. ARID1A depletion leads predominantly to loss of chromatin accessibility at these putative enhancer regions and downregulation of associated genes, indicating a primary role in maintaining a permissive regulatory landscape. Extensive co-binding and reciprocal dependencies between ARID1A, FOXA1, and AP-2 transcription factors reveal a coordinated regulatory network distinct from that observed in ductal breast cancers. ARID1A loss does not impair ER-mediated transcriptional responses in the lobular subtype but instead alters basal expression of a subset of oestrogen-responsive genes. Importantly, ARID1A, FOXA1, and AP-2 jointly regulate genes implicated in skeletal system development and bone metastasis, mirroring mutational patterns observed in lobular breast cancer patient datasets. These findings highlight a unique ARID1A-centred transcriptional programme in lobular breast cancer with potential implications for metastatic behaviour and therapeutic vulnerability.

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Orc6 is the smallest and least evolutionarily conserved among all ORC subunits. In Drosophila, Orc6 binds tightly with the core ORC(1-5) and is required for DNA binding and replication initiation, whereas in Xenopus and human systems Orc6 loosely associates with the rest of the complex resulting in some differences for replication-associated activities. Despite these variations, Orc6 remains essential for viability in all species. In current study we analyzed specific residues within the C-terminal 11 helix that is critical for stable association of Orc6 with the ORC complex in Drosophila. Human Orc6 lacks these residues, however it possesses a strong nuclear localization signal (NLS) that is absent in Drosophilidae. We propose that this NLS drives human protein to the nucleus and compensates for weaker Orc6-ORC(1-5) interactions by increasing the nuclear concentration of Orc6 and shifting the equilibrium toward formation of the fully assembled ORC complex at the DNA.

IDH1 R132 mutations are among the most frequent hotspot mutations in cancer, but their mutational origins have remained unclear. Here, we provide evidence that IDH1 R132C, the predominant IDH1 mutation in cholangiocarcinoma, acute myeloid leukemia, and melanoma, likely arises through APOBEC3-mediated mutagenesis. IDH1 R132C is a TpC>TpT substitution on the lagging-strand DNA template within a hairpin-forming sequence context, consistent with APOBEC3 susceptibility. In vitro assays showed that APOBEC3A can deaminate the relevant cytosine, and APOBEC3A and APOBEC3B were relatively highly expressed in tumor types with recurrent IDH1 R132C mutations. IDH1 R132G, a TpC>TpG substitution at the same site, may similarly result from APOBEC3 activity. By contrast, IDH1 R132H, the predominant IDH1 mutation in lower grade glioma and glioblastoma, is a CpG>TpG substitution at a methylated cytosine on the leading-strand DNA template, a pattern more consistent with DNA polymerase epsilon replication error. Concordantly, tumor types enriched for IDH1 R132H showed relatively low POLE expression. Together, these in vitro and bioinformatic analyses provide insight into the distinct mutational mechanisms that likely underlie recurrent IDH1 hotspot mutations in cancer.