Biology subjects

Merkenschlager, M.

Publications and source records attributed to Merkenschlager, M..

16 recordsLinked to original sources

bioRxiv2026

Locus-specific spatial and transcriptional dynamics direct T cell lineage choice in the thymus

Decades of investigation have made the choice between CD4 and CD8 T cell lineages in the thymus a key paradigm for mammalian cell commitment and differentiation, yet how transcriptional diversification is coordinated with 3D genome organisation remains largely unexplored. Here, using single-molecule transcript quantification and region capture Pico-C (RCPC), we resolve distinct transcriptional and spatial chromatin dynamics at the lineage-defining loci Cd4 and Cd8. We find early Cd4/Cd8 transcriptional divergence driven by MHC-dependent bias in Cd8a transcript copy number, which unfolds gradually across both CCR7- and CCR7+ differentiation stages. Unexpectedly, Cd4 and Cd8 loci show fundamentally different spatial contact dynamics: transiently reduced mesoscale (10-100kb) contacts at Cd8 provide a permissive chromatin environment throughout the transcriptional diversification process. By contrast, Cd4 undergoes switch-like contact changes limited to the microscale (1kb) late in differentiation, associated with lineage stabilization by ThPOK and/or RUNX3. These results place distinct, locus-specific chromatin environments at the centre of transcriptional diversification during T cell lineage choice.

genetics↗

bioRxiv2026

Increased utrophin expression in healthy and DMD patient derived myoblasts in response to ERK1/2 and EZH2 inhibitor treatment

BackgroundThe X-linked muscle wasting disorder Duchenne muscular dystrophy (DMD) is a progressive and ultimately fatal disease caused by loss of function mutations in the dystrophin (DMD) gene. Upregulation of utrophin (UTRN), an embryonic homologue of dystrophin, has been proposed as a therapeutic option that could ameliorate disease. We previously generated a bioluminescent screen for utrophin-upregulating compounds using a mouse reporter of endogenous utrophin expression and discovered that inhibition of ERK1/2 and EZH2, increases utrophin expression in myoblasts. MethodologyHere we extend this analysis to show that treatment of human myoblasts with the ERK1/2 inhibitor LY3214996 and the EZH2 inhibitor GSK503, increases UTRN expression in primary and immortalised myoblasts derived from healthy volunteers and DMD patients. ResultsShort-term (24 hours) inhibition of ERK1/2 and EZH2 resulted in increased expression of utrophin in proliferating myoblasts. Surprisingly, in patient-derived samples, but not healthy controls, increased UTRN expression was sustained following drug removal and in vitro differentiation. Furthermore, dystrophin deficient myoblasts have altered expression of myogenic transcription factors MYOD1 and MYOG and proliferation marker Ki67, signalling an altered regenerative capacity of these cells, while ERK1/2 inhibition, alone or combined with EZH2i, reversed this transcriptional signature. ConclusionsTreatment with ERK1/2 and EZH2 inhibitors could offer a therapeutic option for DMD by increasing UTRN and MYOD1 expression. We propose that this may compensate for DMD loss and help restore productive muscle differentiation and regeneration.

pharmacology and toxicology↗

bioRxiv2025

A step towards animal models with self-sustained fungal bioluminescence

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.

genetics↗

bioRxiv2024

Three-dimensional chromatin mapping of sensory neurons reveals that cohesin-dependent genomic domains are required for axonal regeneration

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression programme at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C and RNA-seq. We find that cohesin is required for the full induction of the regenerative transcriptional program, by organising 3D genomic domains required for the activation of regenerative genes. Importantly, loss of cohesin results in disruption of chromatin architecture at regenerative genes and severely impaired nerve regeneration. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent chromatin interactions in neuronal regeneration.

neuroscience↗

bioRxiv2024

PBK/TOPK mediates Ikaros, Aiolos and CTCF displacement from mitotic chromosomes and alters chromatin accessibility at selected C2H2-zinc finger protein binding sites

PBK/TOPK is a mitotic kinase implicated in haematological and non-haematological cancers. Here we show that the key haemopoietic regulators Ikaros and Aiolos require PBK-mediated phosphorylation to dissociate from chromosomes in mitosis. Eviction of Ikaros is rapidly reversed by addition of the PBK-inhibitor OTS514, revealing dynamic regulation by kinase and phosphatase activities. To identify more PBK targets, we analysed loss of mitotic phosphorylation events in Pbk-/-preB cells and performed proteomic comparisons on isolated mitotic chromosomes. Among a large pool of C2H2-zinc finger targets, PBK is essential for evicting the CCCTC-binding protein CTCF and zinc finger proteins encoded by Ikzf1, Ikzf3, Znf131 and Zbtb11. PBK-deficient cells were able to divide but showed altered chromatin accessibility and nucleosome positioning consistent with CTCF retention. Our studies reveal that PBK controls the dissociation of selected factors from condensing mitotic chromosomes and contributes to their compaction.

molecular biology↗

bioRxiv2024

Conserved helical motifs in the Ikaros IDR mediate NuRD interaction and transcriptional repression

The transcription factor IKZF1/Ikaros is essential for B cell development, and recurrently mutated in human B-ALL. Ikaros has been ascribed both activating and repressive functions via interactions with coactivator and corepressor complexes, but the relative abundance of Ikaros-associated coregulatory complexes and their contribution to Ikaros-mediated gene regulation are not well understood. To address this issue, we performed an unbiased identification of Ikaros-interacting proteins in pre-B cells, and found that Ikaros interacts overwhelmingly with corepressors and heterochromatin-associated proteins. Time-resolved analysis of transcription and chromatin state identified transcriptional repression as the immediate response to Ikaros induction. Transcriptional repression preceded transcriptional activation by several hours, and was accompanied by a rapid loss of chromatin accessibility and reduced levels of H3K27ac particularly at enhancers. Functional characterisation of intrinsically disordered regions in the Ikaros protein identified highly conserved helical motifs that mediate Ikaros association with the NuRD corepressor complex and contribute to the silencing of target genes in pre-B cells and antiproliferative functions of Ikaros in human B-ALL.

molecular biology↗

bioRxiv2023

Bioluminescence imaging of Cyp1a1-luciferase reporter mice demonstrates prolonged activation of the aryl hydrocarbon receptor in the lung

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.

cell biology↗

bioRxiv2022

Loss of H3K9 tri-methylation alters chromosome compaction and transcription factor retention during mitosis

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases (DNMTs) and Polycomb Repressor Complexes (PRCs), bind to chromosomes throughout mitosis and their depletion results in increased chromosome size. Here we show that enzymes that catalyse H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly however, mutants lacking H3K9me3 have unusually small and compact mitotic chromosomes that are associated with increased H3S10ph and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous Suv39h1, or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wildtype versus Suv39h1/Suv39h2 double-null ESCs revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis, and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

molecular biology↗

bioRxiv2021

Hi-C-LSTM: Learning representations of chromatin contacts using a recurrent neural network identifies genomic drivers of conformation

Despite the availability of chromatin conformation capture experiments, understanding the relationship between regulatory elements and conformation remains a challenge. We propose Hi-C-LSTM, a method that produces low-dimensional latent representations that summarize intra-chromosomal Hi-C contacts via a recurrent long short-term memory (LSTM) neural network model. We find that these representations contain all the information needed to recreate the original Hi-C matrix with high accuracy, outperforming existing methods. These representations enable the identification of a variety of conformation-defining genomic elements, including nuclear compartments and conformation-related transcription factors. They furthermore enable in-silico perturbation experiments that measure the influence of cis-regulatory elements on conformation.

genomics↗

bioRxiv2021

Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in primary cortical neurons. Immediate early genes remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the Immediate early gene Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterised by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts.

genomics↗

bioRxiv2020

Epigenetic change induced by in utero dietary challenge provokes phenotypic variability across multiple generations of mice

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, histone H3K9me3, H3K27me3, H4K16ac and DNA methylation1-3. In higher vertebrates, epidemiological and experimental evidence supports similar trans-generational effects4,5 although the mechanisms that underpin these are incompletely understood6-9. We generated a luciferase reporter knock-in mouse for the imprinted Dlk1 locus, to visualise and track epigenetic fidelity across generations. We showed that exposure to high-fat diet (HFD) in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 allele in offspring, coincident with increased DNA methylation at the Dlk1 sDMR. Interestingly, maternal Dlk1 mis-expression was also evident in the next generation (F2), exclusively in animals derived from F1-exposed females. Oocytes from these females showed altered microRNA and gene expression, without any major changes in underlying DNA methylation, and correctly imprinted Dlk1 expression resumed in subsequent generations (F3 onwards). Our results reveal how canonical and non-canonical imprinting mechanisms enable the foetal epigenome to adapt to in utero challenge to modulate the properties of two successive generations of offspring.

developmental biology↗

bioRxiv2020

Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterized the transcriptional profile of cortical neurons from CdLS patients and found deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupted 3-D genome organization and transcriptional control in post-mitotic cortical mouse neurons. The genes affected belonged to similar gene ontology classes and showed significant numerical overlap with those deregulated in CdLS. Interestingly, gene expression was largely rescued by subsequent reconstitution of cohesin function. These experiments show that cohesin is continuously required for neuronal gene expression and provide a tractable approach for addressing mechanisms of neuronal dysfunction in CdLS.

cell biology↗

bioRxiv2019

Systematic identification of factors bound to isolated metaphase ESC chromosomes reveals a role for chromatin repressors in compaction

Epigenetic information is transmitted from mother to daughter cells through mitosis. To identify trans-acting factors and cis-acting elements that might be important for conveying epigenetic memory through cell division, we isolated native (unfixed) chromosomes from metaphase-arrested cells using flow cytometry and performed LC-MS/MS to determine the repertoire of chromosome-bound proteins. Quantitative proteomic comparisons between metaphase-arrested cell lysates and chromosome-sorted samples revealed a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers (such as PRC2 and DNA methyl-transferases) and proteins governing chromosome architecture. We showed that deletion of PRC2, DNMT1/3a/3b or Mecp2 provoked an increase in the size of individual mitotic chromosomes consistent with de-condensation, as did experimental cleavage of cohesin complexes. These data provide a comprehensive inventory of chromosome-bound factors in pluripotent stem cells at mitosis and reveal an unexpected role for chromatin repressor complexes in preserving mitotic chromosome compaction.

cell biology↗

bioRxiv2019

Chromatinization of Escherichia coli with archaeal histones

Nucleosomes restrict DNA accessibility throughout eukaryotic genomes, with repercussions for replication, transcription, and other DNA-templated processes. How this globally restrictive organization emerged from a presumably more open ancestral state remains poorly understood. Here, to better understand the challenges associated with establishing globally restrictive chromatin, we express histones in a naive bacterial system that has not evolved to deal with nucleosomal structures: Escherichia coli. We find that histone proteins from the archaeon Methanothermus fervidus assemble on the E. coli chromosome in vivo and protect DNA from micrococcal nuclease digestion, allowing us to map binding footprints genome-wide. We provide evidence that nucleosome occupancy along the E. coli genome tracks intrinsic sequence preferences but is disturbed by ongoing transcription and replication. Notably, we show that higher nucleosome occupancy at promoters and across gene bodies is associated with lower transcript levels, consistent with local repressive effects. Surprisingly, however, this sudden enforced chromatinization has only mild repercussions for growth, suggesting that histones can become established as ubiquitous chromatin proteins without interfering critically with key DNA-templated processes. Our results have implications for the evolvability of transcriptional ground states and highlight chromatinization by archaeal histones as a potential avenue for controlling genome accessibility in synthetic prokaryotic systems.

systems biology↗

bioRxiv2019

STATegra: a comprehensive multi-omics dataset of B-cell differentiation in mouse

Multi-omics approaches use a diversity of high-throughput technologies to profile the different molecular layers of living cells. Ideally, the integration of this information should result in comprehensive systems models of cellular physiology and regulation. However, most multi-omics projects still include a limited number of molecular assays and there have been very few multi-omic studies that evaluate dynamic processes such as cellular growth, development and adaptation. Hence, we lack formal analysis methods and comprehensive multi-omics datasets that can be leveraged to develop true multi-layered models for dynamic cellular systems. Here we present the STATegra multi-omics dataset that combines measurements from up to 10 different omics technologies applied to the same biological system, namely the well-studied mouse pre-B-cell differentiation. STATegra includes high-throughput measurements of chromatin structure, gene expression, proteomics and metabolomics, and it is complemented with single-cell data. To our knowledge, the STATegra collection is the most diverse multi-omics dataset describing a dynamic biological system.

bioinformatics↗

bioRxiv2018

Building gene regulatory networks from single-cell ATAC-seq and RNA-seq using Linked Self-Organizing Maps

Rapid advances in single-cell assays have outpaced methods for analysis of those data types. Different single-cell assays show extensive variation in sensitivity and signal to noise levels. In particular, scATAC-seq generates extremely sparse and noisy datasets. Existing methods developed to analyze this data require cells amenable to pseudo-time analysis or require datasets with drastically different cell-types. We describe a novel approach using self-organizing maps (SOM) to link scATAC-seq and scRNA-seq data that overcomes these challenges and can generate draft regulatory networks. Our SOMatic package generates chromatin and gene expression SOMs separately and combines them using a linking function. We applied SOMatic on a mouse pre-B cell differentiation time-course using controlled Ikaros over-expression to recover gene ontology enrichments, identify motifs in genomic regions showing similar single-cell profiles, and generate a gene regulatory network that both recovers known interactions and predicts new Ikaros targets during the differentiation process. The ability of linked SOMs to detect emergent properties from multiple types of highly-dimensional genomic data with very different signal properties opens new avenues for integrative analysis of single-cells.

genomics↗