Biology subjects

Stadtfeld, M.

Publications and source records attributed to Stadtfeld, M..

13 recordsLinked to original sources

bioRxiv2026

Developmental rewiring of EHMT1 function during human cortical neurogenesis

Ubiquitously expressed chromatin regulators orchestrate cell-type-specific gene expression programs, yet how they acquire distinct regulatory functions during development remains fundamentally unresolved. Using temporally controlled protein depletion during human cortical neurogenesis in vitro, we show that the autism-associated histone methyltransferase EHMT1 is developmentally repurposed from epiblast to cortical neurons. Broadly expressed zinc finger proteins recruit EHMT1 to distinct cis-regulatory elements within changing chromatin landscapes associated with lineage-specific transcription factor activity, with extensive remodeling of EHMT1 enhancer occupancy during neural induction dependent on ZNF462. This genomic rewiring is accompanied by a fundamental shift from restraining premature developmental gene expression in pluripotent cells to supporting neuronal programs, with distinct transcriptional consequences of EHMT1 loss across neural progenitors and excitatory and inhibitory neuronal subtypes. Timed depletion and recovery experiments identify the neural progenitor stage as the onset of a developmental period during which EHMT1 is required for faithful neuronal maturation. Remarkably, EHMT1 can re-engage stage-specific targets after prolonged deficiency, allowing extensive transcriptional recovery with only limited molecular scarring. These findings reveal how ubiquitous epigenetic machinery acquires developmental specificity while demonstrating the resilience of developmental gene regulatory programs to prolonged disruption, with implications for neurodevelopmental disorders.

developmental biology↗

bioRxiv2026

CTR1-mediated copper uptake orchestrates metabolic-epigenetic regulation of pathogenic TH17 cells in autoimmune disease

Pathogenic T helper 17 (pTH17) cells are a subset of CD4+ T cells driving autoimmune diseases including multiple sclerosis (MS). Compared to homeostatic TH17 cells and other TH subsets, pTH17 have enhanced mitochondrial function and oxidative phosphorylation (OXPHOS) that supports their differentiation and pathogenic function. Here we identify Copper Transporter 1 (CTR1), encoded by Slc31a1, as essential for copper uptake in CD4+ T cells, OXPHOS and pTH17 cell differentiation and function. While copper levels are known to be higher in the cerebrospinal fluid of patients with MS compared to healthy individuals, and excess copper contributes to oligodendrocyte loss in murine models of MS, the effect of copper on T cell function and pathogenicity in MS are unclear. We demonstrate that deletion of Slc31a1 in CD4+ T cells decreased intracellular copper levels, disrupting mitochondrial respiration and rewiring metabolism. These changes altered the epigenetic landscape of pTH17 cells by impairing DNA demethylation capacity, leading to hypermethylated DNA and altered chromatin accessibility at key binding sites for AP-1 transcription factors essential for pTH17 differentiation. As a result, CTR1-deficient T cells showed defective differentiation into pTH17 cells, with decreased production of IL-17A and expression of TH17 signature genes, while the differentiation of other CD4+ T cell subsets remained largely unaffected. Moreover, T cell-specific deletion of Slc31a1 protected mice from central nervous system (CNS) inflammation in the experimental autoimmune encephalomyelitis (EAE) model of MS by suppressing clonal expansion of autoreactive CD4+ T cells. These findings establish copper as a critical regulator of pTH17 differentiation and function, revealing a previously unknown molecular link between copper homeostasis, metabolism and epigenetic regulation governing pTH17-mediated autoimmunity.

immunology↗

bioRxiv2024

Coordinated repression of totipotency-associated gene loci by histone methyltransferase EHMT2 through binding to LINE-1 regulatory elements

Mouse embryonic stem cells (mESCs) and other naive pluripotent stem cells can reverse typical developmental trajectories and, at low frequency, de-differentiate into 2-cell-like cells (2CLCs) that resemble the mammalian embryo during zygotic genome activation (ZGA). This affords the opportunity to reveal molecular principles that govern the pre-implantation stages of mammalian development. We leveraged a multipurpose allele for acute protein depletion and efficient immunoprecipitation to dissect the molecular functions of the chromatin repressor EHMT2, a candidate antagonist of the mESC-to-2CLC transition. This allowed us to define categories of EHMT2 target genes characterized by distinct modes of EHMT2 chromatin engagement and repression. Most notably, EHMT2 directly represses large clusters of co-regulated gene loci that comprise a significant fraction of the 2CLC-specific transcriptome by initiating H3K9me2 spreading from distal LINE-1 elements. EHMT2 counteracts the recruitment of the activator DPPA2/4 to promoter-proximal endogenous retroviral elements (ERVs) at 2CLC genes. EHMT2 depletion elevates the expression of ZGA-associated transcripts in 2CLCs and synergizes with spliceosome inhibition and retinoic acid signaling in facilitating the mESC-to-2CLC transition. In contrast to ZGA-associated genes, repression of germ layer-associated transcripts by EHMT2 occurs outside of gene clusters in collaboration with ZFP462 and entails binding to non-repeat enhancers. Our observations show that EHMT2 attenuates the bidirectional differentiation potential of mouse pluripotent stem cells and define molecular modes for locus-specific transcriptional repression by this essential histone methyltransferase.

developmental biology↗

bioRxiv2024

A mouse organoid platform for modeling cerebral cortex development and cis-regulatory evolution in vitro

Natural selection has shaped the gene regulatory networks that orchestrate the development of the neocortex, leading to diverse neocortical structure and function across mammals, but the molecular and cellular mechanisms driving phenotypic changes have proven difficult to characterize. Here, we develop a reproducible protocol to generate neocortical organoids from mouse epiblast stem cells (EpiSCs) that gives rise to diverse cortical cell types, including distinct classes of excitatory neurons (pre-plate, deep-layer, and upper-layer) and glia (oligodendrocyte precursor cells, myelinating oligodendrocytes, astrocytes, ependymal cells). Cortical organoids develop with similar kinetics to the mouse cortex in vivo and begin to exhibit features of maturation in glia and neuronal cell types relatively rapidly compared to human brain organoids. Using this new protocol, we generated cortical organoids from F1 hybrid EpiSCs derived from crosses between standard laboratory mice (C57BL/6J) and four wild-derived mouse strains from distinct sub-species spanning [~]1M years of evolutionary divergence. This allowed us to comprehensively map cis-acting transcriptional regulatory variation across developing cortical cell types using scRNA-seq. We identify hundreds of genes that exhibit dynamic allelic imbalances during cortical neurogenesis, providing the first insight into the developmental mechanisms underpinning changes in cortical structure and function between mouse strains. These experimental methods and cellular resources represent a powerful new platform for investigating mechanisms of gene regulation in the developing cerebral cortex.

developmental biology↗

bioRxiv2024

Genetic variation modulates susceptibility to aberrant DNA hypomethylation and imprint deregulation in naive pluripotent stem cells

Naive pluripotent stem cells (nPSC) frequently undergo pathological and not readily reversible loss of DNA methylation marks at imprinted gene loci. This abnormality poses a hurdle for using pluripotent cell lines in biomedical applications and underscores the need to identify the causes of imprint instability in these cells. We show that nPSCs from inbred mouse strains exhibit pronounced strain-specific susceptibility to locus-specific deregulation of imprinting marks during reprogramming to pluripotency and upon culture with MAP kinase inhibitors, a common approach to maintain naive pluripotency. Analysis of genetically highly diverse nPSCs from the Diversity Outbred (DO) stock confirms that genetic variation is a major determinant of epigenome stability in pluripotent cells. We leverage the variable DNA hypomethylation in DO lines to identify several trans-acting quantitative trait loci (QTLs) that determine epigenome stability at either specific target loci or genome-wide. Candidate factors encoded by two multi-target QTLs on chromosomes 4 and 17 suggest specific transcriptional regulators that contribute to DNA methylation maintenance in nPSCs. We propose that genetic variants represent candidate biomarkers to identify pluripotent cell lines with desirable properties and might serve as entry points for the targeted engineering of nPSCs with stable epigenomes. HighlightsO_LINaive pluripotent stem cells from distinct inbred mouse strains exhibit variable DNA methylation levels at imprinted gene loci. C_LIO_LIThe vulnerability of pluripotent stem cells to loss of genomic imprinting caused by MAP kinase inhibition strongly differs between inbred mouse strains. C_LIO_LIGenetically diverse pluripotent stem cell lines from Diversity Outbred mouse stock allow the identification of quantitative trait loci controlling DNA methylation stability. C_LIO_LIGenetic variants may serve as biomarkers to identify naive pluripotent stem cell lines that are epigenetically stable in specific culture conditions. C_LI

developmental biology↗

bioRxiv2024

Transcriptional remodeling by OTX2 directs specification and patterning of mammalian definitive endoderm

O_LIMouse and human pluripotent cells with multipurpose degron alleles establish versatile platforms to dissect cell type-specific functions of the pleiotropic transcription factor OTX2 C_LIO_LIOTX2 controls molecular programs required for anterior-posterior patterning of the developing gut C_LIO_LIOTX2 establishes and maintains chromatin accessibility at distinct distal gene regulatory elements in definitive endoderm C_LIO_LIOTX2 functions as a patterning factor across different germ layers and species C_LI The molecular mechanisms that drive essential developmental patterning events in the mammalian embryo remain poorly understood. To generate a conceptual framework for gene regulatory processes during germ layer specification, we analyzed transcription factor (TF) expression kinetics around gastrulation and during in vitro differentiation. This approach identified Otx2 as a candidate regulator of definitive endoderm (DE), the precursor of all gut-derived tissues. Analysis of multipurpose degron alleles in gastruloid and directed differentiation models revealed that loss of OTX2 before or after DE specification alters the expression of core components and targets of specific cellular signaling pathways, perturbs adhesion and migration programs as well as de-represses regulators of other lineages, resulting in impaired foregut specification. Key targets of OTX2 are conserved in human DE. Mechanistically, OTX2 is required to establish chromatin accessibility at candidate enhancers, which regulate genes critical to establishing an anterior cell identity in the developing gut. Our results provide a working model for the progressive establishment of spatiotemporal cell identity by developmental TFs across germ layers and species, which may facilitate the generation of gut cell types for regenerative medicine applications.

developmental biology↗

bioRxiv2023

Systematic mapping and modeling of 3D enhancer-promoter interactions in early mouse embryonic lineages reveal regulatory principles that determine the levels and cell-type specificity of gene expression

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or "hubness" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors. HIGHLIGHTS- Cell lines representing early embryonic lineages undergo drastic enhancer remodeling and fine-scale 3D chromatin reorganization - Highly interacting 3D hubs strongly enrich for highly expressed, cell-type specific and essential genes - 3D chromatin features greatly improve prediction of cell-type specific gene expression compared to 1D promoter features - In silico and experimental perturbations identify novel enhancers regulating the expression of two or more genes in early embryonic lineages

molecular biology↗

bioRxiv2021

Bioactive isoprenoids guide migrating germ cells to the embryonic gonad

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in the germline lifecycle. Here we investigated the role of these potent signaling molecules in embryonic germ cell migration, using JHs in Drosophila melanogaster as a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using an in vivo biosensor, we found JH signaling is first active near primordial germ cells (PGCs) as they migrate to the developing somatic gonad. Through in vivo and in vitro assays, we found that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were reduced or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization to the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids, a JH-related family of signaling isoprenoids, has many roles in development and reproduction. We found that retinoic acid, like JH, was sufficient to impact PGC migration in vitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration. HighlightsO_LIJuvenile hormones (JH) are necessary & sufficient for Primordial Germ Cell (PGC) migration. C_LIO_LIJH signaling acts directly in and around migrating PGCs prior to its endocrine function. C_LIO_LICompensatory feedback sensitive to JH receptor function maintains JH homeostasis. C_LIO_LIJH-like retinoic acids may have similar roles during mammalian germ cell migration. C_LI

developmental biology↗

bioRxiv2020

The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II atpro-differentiation genes

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ~1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ZBTB11 and ZFP131 maintain promoter-proximally paused Polymerase II at pro-differentiation genes in ESCs. ZBTB11 or ZFP131 loss leads to NELF pausing factor release, an increase in H3K4me3, and transcriptional upregulation of genes associated with all three germ layers. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency. One-sentence summaryA Transcription Factor-wide CRISPR screen identifies ZBTB11 and ZFP131 maintaining pluripotency by pausing POL II at pro-differentiation genes

developmental biology↗

bioRxiv2020

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus

Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the DNA (de)methylation machinery. HIGHLIGHTSO_LIThe IG-DMR is a bipartite element with distinct allele-specific functions C_LIO_LIA non-canonical enhancer within the IG-DMR prevents DNA methyltransferase activity C_LIO_LITargeted epigenome editing allows induction of specific imprinting phenotypes C_LIO_LICRISPRi reveals a functional hierarchy between DMRs that dictates imprint stability C_LI

developmental biology↗

bioRxiv2020

A susceptibility locus on chromosome 13 profoundly impacts the stability of genomic imprinting in mouse pluripotent stem cells

Cultured pluripotent cells accumulate detrimental epigenetic alterations, including DNA methylation changes at imprinted genes known as loss-of-imprinting (LOI). Despite the substantial biomedical relevance of this phenomenon, the molecular cause of this epigenetic instability in pluripotent cells remains unknown. While the occurrence of LOI is generally considered a stochastic phenomenon, here we document a strong genetic determinant that segregates mouse pluripotent cells into epigenetically stable and unstable cell lines. Unstable lines exhibit hypermethylation at Dlk1-Dio3 and select other imprinted loci, which is associated with impaired developmental potential. Stimulation of demethylases by ascorbic acid prevents LOI and can preserve developmental potential. Susceptibility to LOI greatly differs between commonly used mouse strains, which we utilize to map a causal region on chromosome 13 with Quantitative Trait Locus (QTL) analysis. Our observations identify a strong genetic determinant of locus-specific epigenetic abnormalities in pluripotent cells and provide a non-invasive way to suppress them. This highlights the importance of considering genetics in conjunction with culture conditions for assuring the quality of pluripotent cells for biomedical applications.

cell biology↗

bioRxiv2019

Context-dependent requirement of H3K9 methyltransferase activity during cellular reprogramming to iPSCs

Methylation of histone 3 at lysine 9 (H3K9) is widely regarded as a major roadblock for cellular reprogramming and interference with associated methyltransferases such as EHMT1 and EHMT2 (also known as GLP and G9A, respectively) increases the efficiencies at which induced pluripotent stem cells (iPSCs) can be derived. Activation of histone and DNA demethylases by ascorbic acid (AA) has become a common approach to facilitate the extensive epigenetic remodeling required for iPSC formation, but possible functional interactions between the H3K9 methylation machinery and AA-stimulated enzymes remain insufficiently explored. Here we show that reduction of EHMT1/2 activity counteracts iPSC formation in an optimized reprogramming system in the presence of AA. Mechanistically, EHMT1/2 activity under these conditions is required for efficient downregulation of somatic genes and transition into an epithelial state. Of note, transient inhibition of EHMT1/2 during reprogramming yields iPSCs that fail to efficiently give rise to viable mice, suggesting persistent molecular defects in these cells. Genetic interference with the H3K9 demethylase KDM3B ameliorated the adverse effect of EHMT1/2 inhibition on iPSC formation. Together, our observations document novel functions of H3K9 methyltransferases during iPSC formation and suggest that the balancing of AA-stimulated enzymes by EHMT1/2 supports efficient and error-free iPSC reprogramming to pluripotency.

developmental biology↗

bioRxiv2018

KLF4 binding during reprogramming is involved in 3D architectural rewiring and transcriptional regulation of enhancer hubs

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors (TFs), as is strikingly exemplified by reprogramming somatic cells to pluripotent stem cells (PSCs) via expression of OCT4, KLF4, SOX2 and cMYC. How TFs orchestrate the complex molecular changes around their target gene loci in a temporal manner remains incompletely understood. Here, using KLF4 as a paradigm, we provide the first TF-centric view of chromatin reorganization and its association to 3D enhancer rewiring and transcriptional changes of linked genes during reprogramming of mouse embryonic fibroblasts (MEFs) to PSCs. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in the connectivity of enhancers, while disruption of individual KLF4 binding sites from PSC-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying TF during a controlled cell fate transition and offers novel insights into the order and nature of molecular events that follow TF binding.

molecular biology↗