Biology subjects

Stewart, A. F.

Publications and source records attributed to Stewart, A. F..

At least 19 recordsLinked to original sources

bioRxiv2026

A portable C-terminal rheostat calibrates RecE/Redα exonuclease activity for enhanced bacterial recombineering

Recombineering enables precise genome engineering across diverse bacteria, yet efficient tools for large DNA integration remain underdeveloped for Streptomyces and other biotechnologically relevant bacteria. Here we identify a panel of highly active RecET recombinase pairs from actinobacteriophages that mediate efficient homologous recombination in Streptomyces. The lead candidate, Che9cRecET, supports genomic integration of kilobase-scale DNA with only 50bp homology arms and enables accurate insertion of 79kb biosynthetic gene clusters, far outperforming conventional genome editing approaches. Structural and biochemical analyses reveal Che9cRecE as a distinct C3-symmetric toroidal homotrimeric 5'-3' exonuclease, whose extended C-terminal loop acts as a tunable rheostat calibrating exonuclease activity to an optimal window for recombination. We further show this portable C-terminal loop enhances recombineering efficiency across phylogenetically diverse exonucleases including Escherichia coli Redalpha/RecE and Pseudomonas A. Our work uncovers a conserved regulatory mechanism of the RecE/Redalpha exonuclease family and delivers a generalizable strategy for advancing bacterial genome editing.

molecular biology↗

bioRxiv2026

Harmine Plus Exendin-4 Enhances Remission of Recent-Onset Type 1 Diabetes Following Anti-CD3 Therapy

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic {beta}-cells. While anti-CD3 therapy can delay disease progression and preserve residual {beta}-cell function, disease reversal will likely require both immune modulation and {beta}-cell regeneration. We found that the combination of harmine and exendin-4 (H+E) reduced inflammation-induced human {beta}-cell apoptosis, suppressed cytokine signaling and immunogenicity pathways, and improved {beta}-cell function. Although H+E alone did not reverse diabetes in NOD mice, low-dose anti-CD3 followed by H+E normalized blood glucose, increased insulin levels, improved glucose tolerance, expanded {beta}-cell mass, and enhanced diabetes remission. These effects were associated with reduced pro-inflammatory T-cell responses, increased regulatory T cells, and greater expression of exhaustion-related T-cell markers, without broad lymphocyte depletion. Similar immunomodulatory effects were observed in activated human PBMCs. Transcriptomic analyses identified the lncRNA SNHG6 as a key mediator of H+E action; SNHG6 protected {beta}-cells from cytokine-induced stress, apoptosis, and immunogenicity. Together, these findings demonstrate that H+E promotes {beta}-cell recovery and resilience while reducing {beta}-cell immunogenicity, enabling remission of recent-onset T1D when combined with anti-CD3 therapy. SNHG6 emerges as a novel regulator of {beta}-cell protection during inflammation.

cell biology↗

bioRxiv2026

Epigenetic Profiling of Human Insulinomas Reveals AP-1 Family as Critical Regulators of Beta Cell Maturation

Insulinomas are rare and benign human pancreatic adenomas that overproduce insulin and display increased beta cell mass. We and others have shown that transcriptomic and genomic profiling on insulinomas provides a data mine for identifying targets that can be manipulated to induce human beta cell regeneration. Majority of causative genetic variants in insulinomas involve epigenetic regulatory genes. Yet, specifically how these variants lead to human beta cell expansion and increased function is largely unknown. Here, we performed bulk and single-nucleus epigenomic and transcriptomic profiling to define regulatory alterations in human insulinomas. Bulk ATAC-seq and H3K27Ac ChIP-seq revealed significant enrichment of AP-1 transcription factor binding motifs within beta cell-associated open chromatin/enhancer regions in normal islets, accompanied by robust expression of AP-1 family members; in contrast, insulinomas exhibited marked reductions in both AP-1 motif enrichment and AP-1 expression. Our snRNA-seq and snATAC-seq profiling across four independent insulinomas identified a consistent and previously unrecognized signature defined by suppression of AP-1 transcription factors and widespread loss of chromatin accessibility at AP-1 binding sites, particularly at enhancers governing beta cell identity. Collectively, these results establish AP-1-mediated regulatory programs as critical determinants of beta cell maturation and define their disruption as a signature among human insulinomas.

molecular biology↗

bioRxiv2025

Harmine Selectively Drives Human Beta Cell Differentiation and Function Via Protein Kinase A Pathways

Harmine and other small molecule inhibitors of the kinase DYRK1A induce human beta cells to replicate and regenerate in vitro and in vivo, and are effective at reversing diabetes in animal models. In addition to its beta cell proliferative and regenerative effects, harmine also induces expression of transcription factors and other genes involved in beta cell differentiation and function, exemplified by PDX1, MAFA, NKX6.1, GLP1R, PCSK1 and many others. Harmine also rapidly enhances glucose-stimulated insulin secretion in vitro and in vivo, reversing diabetes within days in diabetic mice transplanted with a marginal mass of human islets. We had assumed that this pro-differentiation or pro-function effect was a common feature of all DYRK1A inhibitors, and was mediated by DYRK1A inhibition. Thus, DYRK1A is a primary target (we refer to it as Target 1) for harmine. Here, to our surprise, we report that the pro-differentiation effect is not a generalized action for all small molecule DYRK1A inhibitors and does not result from DYRK1A interference or genetic silencing. Instead, the prodifferentiation effect is restricted to a small select subset of DYRK1A inhibitors (harmine, 2-2c and 5-IT). Remarkably, the pro-differentiation effect results from the unique ability of these drugs to activate protein kinase A (PKA), a dual mechanism that distinguishes this class from other DYRK1A inhibitors. The beneficial effects of harmine on PKA appear to be indirect, driven by an as yet unidentified, Target 2 in the PKA pathway. These findings make it clear that all DYRK1A inhibitors are not interchangeable, and that those that drive both proliferation and differentiation/function will likely be preferable in human clinical therapeutic settings. They also provide a novel target or roadmap for enhancing human beta cell differentiation in Type 1 and Type 2 diabetes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/692818v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@bd015corg.highwire.dtl.DTLVardef@a46f9borg.highwire.dtl.DTLVardef@19b4f65org.highwire.dtl.DTLVardef@179aa28_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Nutrient excess remodels islet autonomic innervation via pancreatic schwann cells

Nutrient excess results in short- and long-term adaptations in the structure and function of metabolic organs, such as pancreatic islets. Pancreatic innervation contributes to islet architecture and function in many species, including humans, but little is known about its adaptation to over-nutrition. Here, we use 3D imaging to show that short-term high fat diet rapidly remodels islet sympathetic innervation while long-term high fat diet results in islet-specific cholinergic neuropathy. Using highly targeted, organ- and circuit-specific approaches, we demonstrate that remodeling of pancreatic innervation contributes to impaired glycemic control with short- and long-term nutrient excess. Counteracting the nutrient-induced changes in sympathetic and parasympathetic inputs to the pancreas using neuromodulation and repurposed FDA-approved approaches, we can significantly improve glycemic control even with long-term high fat diet. Finally, we identify islet Schwann cells as a source of the neurotrophic factor, S100b, contributing to rapid sympathetic hyperinnervation with short-term overnutrition. Our findings reveal novel adaptations in islet innervation contributing to nutrition-mediated metabolic dysfunction that can be ameliorated by targeting pancreatic neural circuits.

physiology↗

bioRxiv2025

A non-catalytic role for MLL2 in controlling chromatin organisation and mobility during the priming of pluripotent cells for differentiation

How the chromatin regulator MLL2 (KMT2B) influences cell differentiation remains poorly understood. MLL2 is the main histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for ESCs to differentiate into neuroectoderm. We show here that this requirement occurs during exit from naive pluripotency, days before neuroectoderm differentiation is impaired. Although MLL2 knockout has only a subtle effect on transcription during exit, reducing the expression of a few important neuroectodermal transcription factors, it substantially remodels chromatin architecture, disrupting 3D chromatin loops associated with bivalent promoters. The enzymatic activity of MLL2 is not needed for stabilising these loops or for neuroectoderm differentiation. This non-catalytic function of MLL2 in stabilising 3D chromatin loops has implications for lineage specification. Because MLL2 shares features with all four MLLs, chromatin tethering, rather than H3K4 methylation, may represent the primary function of MLL proteins during lineage commitment.

cell biology↗

bioRxiv2024

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including {gamma}CaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that {gamma}CaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

neuroscience↗

bioRxiv2024

The Rad52 superfamily as seen by AlphaFold

1Rad52, a highly conserved eukaryotic protein, plays a crucial role in DNA repair, especially in double-strand break repair. Recent findings reveal that its distinct structural features, including a characteristic {beta}-sheet and {beta}-hairpin motif, are shared with the lambda phage single-strand annealing proteins, Red{beta}, indicating a common superfamily. Our analysis of over 10,000 single-strand annealing proteins (SSAPs) across all kingdoms of life supports this hypothesis, confirming their possession of the characteristic motif despite variations in size and composition. We found that archaea, representing only 1% of the studied proteins, exhibit most of these variations. Through the examination of four representative archaeal SSAPs, we elucidate the structural relationship between eukaryotic and bacterial SSAPs, highlighting differences in {beta}-sheet size and {beta}-hairpin complexity. Furthermore, we identify an archaeal SSAP with a structure nearly identical to the human variant and screen over 100 million unannotated proteins for potential SSAP candidates. Our computational analysis complements existing sequence with structural evidence supporting the suggested orthology among five SSAP families across all kingdoms: Rad52, Red{beta}, RecT, Erf, and Sak3.

bioinformatics↗

bioRxiv2024

Select DYRK1A Inhibitors Enhance Both Proliferation and Differentiation in Human Pancreatic Beta Cells

The small molecule DYRK1A inhibitor, harmine, induces human beta cell proliferation, expands beta cell mass, enhances expression of beta cell phenotypic genes, and improves human beta cell function in vitro and in vivo. It is unknown whether the "pro-differentiation effect" is a DYRK1A inhibitor class-wide effect. Here we compare multiple commonly studied DYRK1A inhibitors. Harmine, 2-2c and 5-IT increase expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC2A2, SLC30A8, ENTPD3 in normal and T2D human islets. Unexpectedly, GNF4877, CC-401, INDY, CC-401 and Leucettine fail to induce expression of these essential beta cell molecules. Remarkably, the pro-differentiation effect is independent of DYRK1A inhibition: although silencing DYRK1A induces human beta cell proliferation, it has no effect on differentiation; conversely, harmine treatment enhances beta cell differentiation in DYRK1A-silenced islets. A careful screen of multiple DYRK1A inhibitor kinase candidate targets was unable to identify pro-differentiation pathways. Overall, harmine, 2-2c and 5-IT are unique among DYRK1A inhibitors in their ability to enhance both beta cell proliferation and differentiation. While beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor pharmaceutical development.

molecular biology↗

bioRxiv2023

Human Pancreatic α-Cell Heterogeneity and Trajectory Inference Analysis Using Integrated Single Cell- and Single Nucleus-RNA Sequencing Platforms

Prior studies have shown that pancreatic -cells can transdifferentiate into {beta}-cells, and that {beta}-cells de-differentiate and are prone to acquire an -cell phenotype in type 2 diabetes (T2D). However, the specific human -cell and {beta}-cell subtypes that are involved in -to-{beta}-cell and {beta}-to--cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into -{beta} cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG-expressing human -cell subclusters [1, 2, -{beta}-transition 1 (AB-Tr1), -{beta}-transition 2 (AB-Tr2), and -{beta} (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The 1, 2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for -cell function while AB cells are enriched in genes related to pancreatic progenitor and {beta}-cell pathways; 4) Trajectory inference analysis of extracted - and {beta}-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both - and {beta}-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards {beta}-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards -cells. 6) Remarkably, in contrast to the events in vitro, the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from -to-{beta}-cells in vivo. 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from {beta}-to--cells compatible with dedifferentiation or conversion into -cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro, in vivo, in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between - and {beta}-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo. Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.

cell biology↗

bioRxiv2023

Single Cell RNA-Seq Analysis of Regenerative Drug-Treated Human Pancreatic Islets Identifies A Cycling Alpha Cell Population As Key Beta Cell Progenitors

Diabetes ultimately results from an inadequate number of functional, insulin-producing human beta cells. Although current attempts to replenish the remaining beta cell pool in people with diabetes are encouraging, scalability and cost limit access for the millions of people with diabetes. The small molecule DYRK1A inhibitor class of beta cell regenerative drugs, either alone or in combination with GLP1 receptor agonists or TGF{beta} superfamily inhibitors, are capable of inducing beta cell replication in vitro and increasing beta cell mass in vivo. Despite these advances, the precise mechanisms of action of DYRK1A inhibitors remain incompletely understood. To address the mechanisms more deeply, we performed single cell RNA sequencing on human pancreatic islets treated with a DYRK1A inhibitor, either alone, or in combination with a GLP1 receptor agonist or a TGF{beta} superfamily inhibitor. We identify a cluster of Cycling Alpha Cells as the cells most responsive to DYRK1A inhibition. Velocity and pseudotime lineage trajectory analyses suggest that Cycling Alpha Cells serve as the primary target cell type for of DYRK1A inhibitors, and may serve as precursor cells that transdifferentiate into functional human beta cells in response to the DYRK1A inhibition. In addition to providing a novel mechanism of action for DYRK1A inhibitors, our findings suggest that efforts to target regenerative drugs to human beta cells may be mis-directed: the proper target may be Cycling Alpha Cells.

cell biology↗

bioRxiv2023

Short oligomers rather than rings of human RAD52 promote single-strand annealing

Genome maintenance and stability rely on the repair of DNA double-strand breaks. The break repair can be mediated by the single-strand annealing protein RAD52. RAD52 forms rings that are thought to promote annealing. However, RAD52s annealing activity decreases with increasing concentrations that favor ring formation. Thus, which oligomeric form and how RAD52 anneals DNA strands and detects sequence homology is unclear. We combine mass photometry with biochemical assays to quantify oligomeric states of human RAD52 with and without DNA and put forward an alternative mechanism illustrating the critical role of short oligomers for single-stranded DNA annealing. We found that while truncated RAD52 formed undecameric rings at nanomolar concentrations, full-length RAD52 was mostly monomeric at lower nanomolar, physiological concentrations. At higher concentrations, it formed rings with a variable stoichiometry from heptamers to tridecamers. At low concentrations, with hardly any rings present, RAD52 already promoted single-strand annealing. Rings and short oligomers could bind at least two single DNA strands, but if complementary strands were both bound to rings annealing was inhibited. Our findings suggest that single-strand annealing and homology detection is mediated by short oligomers of RAD52 instead of rings.

molecular biology↗

bioRxiv2023

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

neuroscience↗

bioRxiv2023

The BOD1L subunit of the mammalian SETD1A complex sustains the expression of DNA damage repair genes despite restraining H3K4 trimethylation

SETD1A is the histone 3 lysine 4 (H3K4) methyltransferase central to the mammalian version of the highly conserved eight subunit Set1 complex (Set1C) that apparently conveys H3K4 trimethylation (H3K4me3) onto all active Pol II promoters. Accordingly, the Setd1a mouse knock-out dies in early embryogenesis at the epiblast stage and mouse embryonic stem cells (ESCs) die when SETD1A is removed. We report that ESC death is accompanied by loss of expression of DNA repair genes and accumulating DNA damage. BOD1L and BOD1 are homologs of the yeast Set1C subunit, Shg1, and subunits of the mammalian SETD1A and B complexes. We show that the Shg1 homology region binds to a highly conserved central alpha-helix in SETD1A and B. Like mutagenesis of Shg1 in yeast, conditional mutagenesis of Bod1l in ESCs promoted increased H3K4 di- and tri-methylation but also, like loss of SETD1A, loss of expression of DNA repair genes, increased DNA damage and cell death. In contrast to similar losses of DNA repair gene expression, the converse changes in H3K4 methylation after loss of SETD1A or BOD1L implies that H3K4 methylation is not essential for expression of the target DNA repair genes. Because BOD1L becomes highly phosphorylated after DNA damage and acts to protect damaged replication forks, the SETD1A complex, and BOD1L in particular, are key nodes for the DNA damage repair network.

cell biology↗

bioRxiv2022

Single Nucleus RNA Sequencing of Human Pancreatic Islets In Vitro and In Vivo Identifies New Gene Sets and Three β-Cell Subpopulations with Different Transcriptional Profile

Single-cell RNA sequencing (scRNA-seq) has provided valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo and provides limited information on the active transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and affords enhanced information from intronic sequences that can be leveraged to identify actively transcribing genes in islet cell populations. Here, we first sought to compare scRNA-seq and snRNA-seq analysis of human islets in vitro using exon reads or combined exon and intron reads, respectively. Datasets reveal similar human islet cell clusters using both approaches. In the snRNA-seq data, however, the top differentially expressed genes in human islet endocrine cells are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT ({beta} cells), PTPRT, FAP, PDK4, LOXL4 ( cells), LRFN5, ADARB2, ERBB4, KCNT2 ({delta} cells) and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 ({gamma} cells). Notably, these markers also accurately define endocrine cell populations in human islet grafts in vivo. Further, by integrating the information from nuclear and cytoplasmic transcriptomes, we identify three {beta}-cell sub-clusters: an active INS mRNA transcribing cluster ({beta}1), an intermediate INS mRNA-transcribing cluster ({beta}2), and a mature INS mRNA rich cluster ({beta}3). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA rich cluster ({beta}3) becomes the predominant sub-cluster in vivo. In summary, snRNA-seq analysis of human islet cells is a previously unrecognized tool that can be accurately employed for improved identification of human islet cell types and their transcriptional status in vivo.

cell biology↗

bioRxiv2021

Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice

Inflammatory chemokines and their receptors are central to the development of inflammatory/immune pathologies. The apparent complexity of this system, coupled with lack of appropriate in vivo models, has limited our understanding of how chemokines orchestrate inflammatory responses and has hampered attempts at targeting this system in inflammatory disease. Novel approaches are therefore needed to provide crucial biological, and therapeutic, insights into the chemokine-chemokine receptor family. Here, we report the generation of transgenic multi-chemokine receptor reporter mice in which spectrally-distinct fluorescent reporters mark expression of CCRs 1, 2, 3 and 5, key receptors for myeloid cell recruitment in inflammation. Analysis of these animals has allowed us to define, for the first time, individual and combinatorial receptor expression patterns on myeloid cells in resting and inflamed conditions. Our results demonstrate that chemokine receptor expression is highly specific, and more selective than previously anticipated.

immunology↗

bioRxiv2021

Postnatal SETD1B is essential for learning and the regulation of neuronal plasticity genes

Histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Amongst these enzymes SET domain containing 1b (SETD1B) has been linked to syndromic intellectual disability but its role in the postnatal brain has not been studied yet. Here we employ mice that lack Setd1b from excitatory neurons of the postnatal forebrain and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that SETD1B controls the expression of genes with a broad H3K4me3 peak at their promoters that represent neuronal enriched genes linked to learning and memory function. Comparative analysis to corresponding data from conditional Kmt2a and Kmt2b knockout mice suggests that this function is specific to SETD1B. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B-mediated regulation of H3K4me levels in postnatal neurons is critical for cognitive function.

neuroscience↗

bioRxiv2021

Loss of SETD1B results in the redistribution of genomic H3K4me3 in the oocyte

Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content. Graphical AbstractIn oogenesis, SETD1B and CXXC1 target H3K4me3 to actively transcribed gene promoters, while MLL2 targets transcriptionally inactive regions based on underlying CpG composition (upper panel). When SETD1B is ablated, H3K4me3 is lost at a subset of active promoters, resulting in downregulation of transcription (lower panel). Loss of SETD1B alters the activity of MLL2, permitting MLL2 to deposit H3K4me3 at CpG-rich regions, many of which should otherwise be DNA methylated. Thus, it is evident that MLL2 and de novo DNMTs compete for genomic occupancy late in oogenesis, and loss of SETD1B disrupts the balance of these mechanisms. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

developmental biology↗