Biology subjects

Bartkuhn, M.

Publications and source records attributed to Bartkuhn, M..

16 recordsLinked to original sources

bioRxiv2026

PGC-1α Links Estrogen Receptor Signaling to Japanese Encephalitis Virus Replication in Neuronal cell

Acute encephalitis due to the viral causative agents, such as Japanese Encephalitis Virus (JEV), remains a leading cause of viral encephalitis in Asia, contributing to high mortality and long-term neurological sequelae. The host factors that modulate JEV replication are not well understood. In this study, we investigated the modulation of JEV infection by estrogen in vitro and in vivo. The results demonstrated that estrogen enhances JEV replication in neuronal cells via estrogen receptor alpha (ER) by modulating peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1). Since JEV pathogenesis increases cellular production demands, it results in dysregulation of cellular homeostasis, whereas estrogen treatment helps keep cellular health in check. Also, the mouse model exhibited an increased viral burden with protective antibody titers in response to estrogen. Conclusively, this work highlights the mechanistic link of estrogen-driven mechanisms and identifies the ER-PGC-1-axis as a potential target for sex-specific therapeutic interventions.

microbiology↗

bioRxiv2026

Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic inflammation, emphysema, and mild pulmonary hypertension (PH). Oxidative/nitrosative stress are key drivers, but specific mitochondrial mechanisms remain unclear. We show increased expression of the regulatory mitochondrial cytochrome c oxidase subunit 4 isoform 2 (COX4I2) in an early murine model and human COPD. After 8 months of cigarette smoke exposure, Cox4i2-/- mice were completely protected from emphysema but not from PH, associated with reduced nitrosative stress, inflammation, and apoptosis. Using a novel Cox4i2 reporter mouse and in situ hybridization of human lungs, COX4I2 was detected in precapillary ACTA2+ cells and capillary pericytes. COX4I2 promotes mitochondrial reactive oxygen species (mtROS) production in these cells, thereby enhancing neutrophil migration and alveolar type II cell apoptosis, and modulates angiogenesis. In contrast to Cox4i2-/-, mitochondria-targeted antioxidant MitoQ reversed emphysema and PH, suggesting pericyte-specific regulation of COPD pathologies and mtROS inhibition as a therapeutic approach in COPD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/703513v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@83bca4org.highwire.dtl.DTLVardef@d5ebaborg.highwire.dtl.DTLVardef@632d1borg.highwire.dtl.DTLVardef@1267a13_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Human heart organoids reveal a regenerative strategy for mitochondrial disease

The human heart is among the most complex tissues to replicate in vitro, with vascular, neuronal, and immune elements shaping its development and function. Here we describe cardiomorphs, self-organising human cardiac organoids that recapitulate the cellular diversity, structural organisation, vascularisation, and innervation of the myocardium and mature along a developmental trajectory from early cardiogenesis to adult tissue. Using patient-derived cardiomorphs, we establish the first three-dimensional human tissue model of Kearns-Sayre syndrome (KSS), a rare mitochondrial disorder characterised by large-scale mtDNA deletions. KSS-cardiomorphs faithfully reproduce disease-associated metabolic, contractile, and ultrastructural hallmarks. Leveraging this platform, we identify Betaxolol, an FDA-approved selective {beta}1-adrenergic antagonist, as a modulator of mitochondrial quality control. Betaxolol increases intracellular oxygenation, selectively eliminates dysfunctional mitochondria via mitophagy, and promotes biogenesis of functional organelles, restoring contractility in KSS tissues. This dual-action, mutation-agnostic mechanism suggests a therapeutic principle with broad relevance to mitochondrial disease, cardiac pathology, and age-associated decline.

cell biology↗

bioRxiv2025

Endothelial expression of ZBTB16 protects against cardiac aging

Background and aimAging significantly increases the risk of cardiovascular diseases, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms mediating EC-dependent cardiac aging and identifies a critical role of Zinc finger and BTB domain-containing protein 16 (ZBTB16). MethodsChromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses were performed on aged hearts to identify age-related regulatory changes. Functional studies using genetic models, assessed cardiac aging phenotypes. In vitro assays examined EC senescence and secretory profiles, while co-culture experiments analyzed the impact of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested the potential for ZBTB16 to mitigate aging-associated dysfunction. ResultsAged hearts exhibited decreased chromatin accessibility and expression of the transcription factor ZBTB16 in both human and mice. Loss of ZBTB16 in young mice, including Zbtb16 haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. Supernatants from ZBTB16-deficient ECs activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified key downstream targets of ZBTB16, including nuclear receptor-interacting protein 1 (NRIP1). ZBTB16 suppressed NRIP1 expression, limiting fibroblast activation and pro-fibrotic signaling. ConclusionsZBTB16 is a key regulator of endothelial function, maintaining vascular niche homeostasis and mitigating aging-associated cardiac dysfunction. Its loss promotes EC senescence and pro-fibrotic signaling, contributing to diastolic dysfunction. Overexpression of ZBTB16 presents a potential therapeutic strategy for preserving cardiac function during aging. These findings establish a novel role for ZBTB16 in endothelial aging and cardiovascular disease prevention. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/681100v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a56e2org.highwire.dtl.DTLVardef@13e23fforg.highwire.dtl.DTLVardef@ad681eorg.highwire.dtl.DTLVardef@8eca8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Depletion of CX3CR1+ macrophages results in disrupted functionality and immune surveillance within epididymis and testis

A finely tuned immune regulation within the epididymis and testis is essential for male reproductive health. This balance is especially critical in the epididymis, where sperm mature and ascending infections frequently disrupt homeostasis, resulting in regionally different immune responses and potential long-term fertility impairments. We previously demonstrated that the epididymis harbors a region-specific immunological scaffold, with CX3CR1+ macrophages as the most prominent epithelium-associated immune cell population. Here, we established a transgenic mouse model to selectively deplete these intraepithelial CX3CR1+ macrophages within the epididymis, resulting in focal epithelial damage and impaired sperm maturation processes essential for proper sperm functionality. Additionally, a mild reduction of the testicular macrophage pool resulted in transient disruptions in spermatogenesis and steroidogenesis. Although the macrophage niche was repopulated after depletion, the newly recruited cells displayed altered phenotypes consistent with persistent sperm alterations. Following infection with uropathogenic Escherichia coli (UPEC), macrophage-depleted mice exhibited exacerbated immune responses - particularly in normally protected proximal epididymal regions - with earlier onset and more severe tissue damage. Transcriptomic analysis revealed a failure to restrain inflammatory responses, especially in genes involved in immune regulation and antibacterial defense, accompanied by elevated immune cell infiltration in infected macrophage-depleted mice. Overall, our findings confirm a crucial role for CX3CR1 macrophages in preserving epithelial integrity and modulating immune responses, supporting a stable tissue environment necessary for efficient organ function of both epididymis and testis. Significance statementMaintaining immune balance in the epididymis is essential for tissue health and protection against ascending infections. Using a transgenic mouse model that allows for selective depletion of CX3CR1 macrophages, this study examines their role in both the epididymis and testis under normal and infectious conditions. The results show that the removal of these macrophages causes localized epithelial damage, changes in immune cell make-up, and increased inflammation in the epididymis after bacterial infection, while also causing mild, reversible problems with spermatogenesis and steroid production in the testis. These findings support the idea that CX3CR1 macrophages contribute to region-specific immune regulation and epithelial stability--key features for keeping the tissue environment suitable for proper sperm development.

immunology↗

bioRxiv2025

Synergistic targeting of cancer cells through simultaneous inhibition of key metabolic enzymes

As cancer cell specific rewiring of metabolic networks creates potential therapeutic opportunities, we conducted a synthetic lethal screen utilizing inhibitors of metabolic pathways. Simultaneous administration of (R)-GNE-140 and BMS-986205 (Linrodostat) preferentially halted proliferation of ovarian cancer cells, but not of their non-oncogenically transformed progenitor cells. While (R)-GNE-140 inhibits lactate dehydrogenase (LDH)A/B and thus effective glycolysis, BMS-986205, in addition to its known inhibitory activity on Indoleamine 2,3-dioxygenase (IDO1), also restricts oxidative phosphorylation (OXPHOS), as revealed here. BMS-986205, which is being tested in multiple Phase III clinical trials, inhibits the ubiquinone reduction site of respiratory complex I and thus compromises mitochondrial ATP production. The energetic catastrophe caused by simultaneous interference with glycolysis and OXPHOS resulted in either cell death or the induction of senescence in tumor cells, with the latter being eliminated by senolytics. The frequent synergy observed with combined inhibitor treatment was comprehensively confirmed through testing on tumor cell lines from the DepMap panel and on human colorectal cancer organoids. These experiments revealed highly synergistic activity of the compounds in a third of the tested tumor cell lines, correlating with alterations in genes with known roles in metabolic regulation and demonstrating the therapeutic potential of metabolic intervention.

cancer biology↗

bioRxiv2025

Deciphering human heart failure with preserved ejection fraction (HFpEF) at single cell resolution

BACKGROUNDHeart failure with preserved ejection fraction (HFpEF) is a complex and growing condition, representing over half of all heart failure cases. Despite its high morbidity and mortality, its heterogeneity and limited therapeutic options pose significant challenges. Understanding the molecular mechanisms driving HFpEF is essential for the development of new therapies to improve patient outcomes. METHODSWe performed single-nucleus RNA sequencing of nuclei obtained from endomyocardial biopsies of six patients with HFpEF. The obtained dataset was integrated with a dataset of 12 healthy human hearts and their transcriptomic differences were analyzed. RESULTSAfter quality control and integration of the datasets, nine major cardiac cell types were annotated. HFpEF cardiomyocytes were characterized by a reduction in genes associated with aerobic respiration and fatty acid metabolism and showed an upregulation of RHOA/ROCK1 signaling, which was validated using immunofluorescence staining in human HFpEF myocardial sections. Endothelial cells exhibited signs of increased apoptosis, SEMA3 signaling and signs of reduced VEGFA signaling as well as a reactivation of a fetal gene signature. In line with a prominent role of cardiac fibrosis in HFpEF, we observed increased signs of fibroblast activation and proliferation, and reduced signs of IFN{gamma} signaling in HFpEF which was most pronounced in activated fibroblasts. Treatment of human cardiac fibroblast with rhIFN{gamma} resulted in decreased collagen contents. Macrophages from HFpEF myocardium showed a pro-inflammatory transcriptomic signature and showed increased expression of MHC-II molecules. This was associated with signs of an increased IFN{gamma} response. CONCLUSIONOur results provide insights into the transcriptional diversity of HFpEF recapitulating structural, functional, and molecular hallmarks of the disease and provide mechanistic insights which might represent therapeutic targets and biomarkers to improve outcome of patients with HFpEF. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIWe provide a single-nucleus RNA sequencing (snRNA-Seq) dataset from human HFpEF myocardium and demonstrate feasibility of snRNA-Seq from endomyocardial biopsies C_LIO_LIThe snRNA-Seq data confirms signs of known molecular hallmarks of HFpEF, such as metabolic changes, inflammation and fibrosis C_LIO_LIWe identify signs of regulating cellular mechanisms underlying these hallmarks, such as cytoskeleton remodeling via RhoA/ROCK1 in cardiomyocytes, and differential interferon gamma signaling in stromal and immune cells C_LI What are the clinical implications?O_LIWe provide several cell type-specific cellular mechanisms which might serve as biomarkers or therapeutic targets in the treatment of HFpEF C_LI

cell biology↗

bioRxiv2024

Exploring the Antifibrotic Potential of the heparan sulfate mimetic OTR4120: Insights from Preclinical Models

RationaleIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive deposition of extracellular matrix (ECM), resulting in lung function impairment. Heparan sulfate mimetics (HSm) have been suggested to have potential antifibrotic effects by regulating ECM. This study aims to investigate the impact of a specific HSm named OTR4120 on fibrotic processes in ex vivo, in vitro and in vivo models. MethodsHuman Precision Cut Lung Slices (hPCLS) treated with a fibrotic cocktail alone or with OTR4120 were evaluated using second-harmonic imaging microscopy (SHIM) to assess collagen deposition. Human embryonic fibroblast WI-38 cell line and primary fibroblasts obtained from human donors were differentiated into myofibroblasts (MYF) using TGF-{beta}1 and treated with OTR4120 or a control vehicle. Gene expression analysis for MYF markers was performed using quantitative PCR (qPCR). Protein expression of MYF markers was evaluated using immunofluorescence techniques. Bulk-RNA sequencing analysis on WI-38 cells cultured under different experimental conditions was conducted. Finally, the therapeutic effects of OTR4120 on a bleomycin-induced fibrosis mouse model were investigated. ResultsSHIM analysis on OTR4120-treated hPCLS showed a decrease in collagen deposition. OTR4120 treatment of primary fibroblasts and WI-38 cells exposed to TGF-{beta}1 significantly reduced the expression of MYF markers. Bulk-RNA sequencing analysis on OTR4120-treated WI-38 cells showed significant impacts on fibrosis-related processes. Therapeutic application of OTR4120 in vivo to bleomycin-induced fibrosis mice resulted in enhanced fibrosis resolution. ConclusionOTR4120 has potential therapeutic benefits as an antifibrotic agent in the context of lung fibrosis. Further investigations are necessary to understand the precise mechanism through which OTR4120 exerts its antifibrotic effects.

cell biology↗

bioRxiv2024

Adventitial fibroblasts direct smooth muscle cell-state transition in pulmonary vascular disease

Pulmonary vascular remodeling is a progressive pathological process characterized by functional alterations within pulmonary artery smooth muscle cells (PASMC) and adventitial fibroblasts (PAAF). Mechanisms driving the transition to a diseased phenotype remain elusive. Utilizing a combination of transcriptomic and proteomic profiling, along with phenotyping of source-matched cells from healthy controls and individuals with idiopathic pulmonary arterial hypertension (IPAH), our investigation uncovered that while PASMC and PAAF retained their original cellular identities, they acquired distinct disease-associated states. Though both cell types exhibited reduced mitochondrial content and hyperpolarization, IPAH-PASMC displayed heightened glycosaminoglycan production and downregulation of contractile machinery, contrasting a hyperproliferative phenotype of IPAH-PAAF. We investigated the involvement of cellular crosstalk in regulating cell state dynamics and identified pentraxin-3 and hepatocyte growth factor as potential modulators of PASMC phenotypic transition orchestrated by PAAF. Our findings contribute to a deeper understanding of pulmonary vascular mesenchyme dynamics in disease pathogenesis.

cell biology↗

bioRxiv2024

The proximity-based protein interaction landscape of the transcription factor p65 NF-kappaB/RELA and its gene-regulatory logics

The protein interactome of p65 / RELA, the most active subunit of the transcription factor (TF) NF-{kappa}B, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins, we identified by biotin tagging > 350 RELA interactors from untreated and IL-1-stimulated cells, including many TFs (47 % of all interactors) and > 50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. According to point mutants of p65, the interactions primarily require intact dimerization rather than DNA binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identified gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3 / TFEB or S100A8 / A9. The remarkably large, dynamic and versatile high resolution interactome of RELA and its gene-regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns. HighlightsO_LIIdentification of > 350 largely dimerization-dependent interactors of p65 / RELA by miniTurboID C_LIO_LIThe interactome is dominated by transcription factors and epigenetic regulator complexes C_LIO_LIFunctional validation of 38 high confidence interactors by targeted siRNA screen C_LIO_LIIdentification of genetic networks regulated by RELA and six of its interactors in the IL-1 response C_LI

molecular biology↗

bioRxiv2023

In vivo binding free energy landscape reveals kinetic control of transcription factor function

Transcription factors (TFs) such as the central DNA-binding hub in Notch signal transduction, RBPJ, bind to specific DNA sequences to regulate gene transcription. How the efficiency of gene regulation depends on the TF-DNA binding kinetics and cofactor interactions is mostly unknown. We determined the DNA binding kinetics and the transcriptional activity of RBPJ and several mutant variants by live-cell single-molecule tracking and reporter assays, and measured their genome-wide chromatin occupation by ChIP-Seq. We observed that cofactor binding, in addition to DNA binding, was required for target site specificity. Importantly, the target site search time of RBPJ was longer than its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired DNA binding, e.g. by mutation K195E related to Adams-Oliver-Syndrome, modulated not only dissociation, but also association to target sites. Impaired cofactor binding mainly altered the rates of unspecific binding and target site association. For other TFs, we also observed longer search than residence times, indicating that kinetic rather than thermodynamic stability of DNA binding might be a general feature of TFs in vivo. We propose that an effective in vivo binding energy landscape of TF-DNA interactions constitutes an instructive visualization of TF-DNA binding kinetics and the changes upon mutations.

biophysics↗

bioRxiv2023

Comprehensive Genomic Features indicative for Notch Responsiveness

Transcriptional specificity is often determined by transcription factor levels and/or chromatin context. In the Notch signal transduction pathway, transcription factor RBPJ is the central component and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While the RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq making use of Notch-dependent T cells. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSS), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed in different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. This approach is potentially applicable to other transcription factors regulating signal-induced gene sets. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness.

genomics↗

bioRxiv2023

ZNF512B binds RBBP4 via a variant NuRD interaction motif and aggregates chromatin in a NuRD complex-independent manner

The evolutionarily conserved histone variant H2A.Z plays a crucial role in various DNA-based processes but the underlying mechanisms by which it acts are not completely understood. Recently, we identified the zinc finger protein ZNF512B as an H2A.Z-, HMG20A- and PWWP2A-associated protein. Here, we report that ZNF512B binds the nucleosome remodeling and deacetylase (NuRD) complex. We discover a conserved amino acid sequence within ZNF512B that resembles the NuRD-interaction motif (NIM) previously identified in FOG-1 and other transcriptional regulators. By solving the crystal structure of this motif bound to the NuRD component RBBP4 and by applying several biochemical assays we demonstrate that this internal NIM is both necessary and sufficient for robust NuRD binding. Transcriptome analyses and reporter assays identify ZNF512B as a repressor of gene expression that can act in both NuRD-dependent and -independent ways. Surprisingly, high levels of ZNF512B expression lead to nuclear protein and chromatin aggregation foci that form independent of the interaction with the NuRD complex but depend on the zinc finger domains of ZNF512B. Our study has implications for diseases in which ZNF512B expression is deregulated, such as cancer and neurodegenerative diseases, and hint at the existence of more proteins as potential NuRD interactors.

molecular biology↗

bioRxiv2023

Functional diversity of the TP53 mutome revealed by saturating CRISPR mutagenesis

The tumor suppressor gene TP53 is the most frequently mutated gene in various cancers. Unlike other tumor suppressors, TP53 is mostly hit by missense mutations, of which more than 2,000 have been described in cancer patients. To take advantage of TP53 mutation status for personalized therapy, a deeper knowledge of the functional ramifications of specific mutations is required as evidence of the functional heterogeneity of mutant p53 proteins mounts. Here, we report on a CRISPR-based saturation mutagenesis screen of 9,225 variants expressed from the endogenous TP53 gene locus of a cancer cell. By tracking changes in the abundance of individual variants in response to specific p53-pathway stimulation, we were able to construct high-resolution functional activity maps of the TP53 mutome, covering [~]94.5% of all cancer-associated missense mutations. The results demonstrate the impact of individual mutations on tumor cell fitness with unprecedented precision and coverage, even revealing underlying mechanisms such as apoptosis. The high discriminatory power also resolves subtle loss-of-function phenotypes and highlights a subset of mutants as particularly promising targets for pharmacological reactivation. Moreover, the data offer intriguing insight into the role of aberrant splicing and nonsense-mediated mRNA decay in clearing truncated proteins due to not only nonsense, frameshift, and splice-site mutations but also missense and synonymous mutations. Surprisingly, no missense mutation provided an immediate proliferative advantage over a null mutation. Nonetheless, cells with a missense, but not null mutations, acquired pro-metastatic properties after prolonged growth in mice, emphasizing the significance of mutant p53-directed clonal evolution in the progression of tumors towards metastasis.

cancer biology↗

bioRxiv2022

The H2A.Z.1/PWWP2A/NuRD-associated protein HMG20A controls early head and heart developmental transcription programs

Abstract/SummarySpecialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as direct histone variant H2A.Z interactor involved in mitosis and cranial-facial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes including NuRD, and show that it localizes to genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental defects are pheno-copied in HMG20A-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CMs). Accordingly, loss of HMG20A caused striking deregulation of transcription programs involved in epithelial- mesenchymal transition (EMT) and cardiac differentiation, thereby providing insights into the regulatory circuits controlled by HMG20A. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of the intricate developmental transcription programs that guide NCC and cardiomyocyte differentiation.

molecular biology↗

bioRxiv2022

The structure, binding, and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3

The highly conserved Notch pathway transmits signals between neighboring cells to elicit distinct downstream transcriptional programs. In given contexts, Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant Notch signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The canonical pathway signals through the transcription factor CSL (RBPJ in mammals), which forms a transcriptional activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified the malignant brain tumor (MBT) family member L3MBTL3 as a bona fide RBPJ binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here we define the RBPJ-interacting domain (RBP-ID) of L3MBTL3 and report the 2.06 [A] crystal structure of the complex formed between RBPJ, the RBP-ID of L3MBTL3 and DNA. The structure reveals the molecular interactions underlying L3MBTL3 complexation with RBPJ, which we comprehensively analyze with a series of L3MBTL3 and RBPJ mutations that span the binding interface. Compared to other RBPJ-binding proteins, we find that L3MBTL3 interacts with RBPJ via an unusual binding motif, which is sensitive to mutations throughout its RBPJ-interacting region. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.

biochemistry↗