Biology subjects

Majewski, J.

Publications and source records attributed to Majewski, J..

14 recordsLinked to original sources

bioRxiv2026

Developmental pleiotropy revealed by mosaic heterozygous Snrpb deletion underlies CCMS-like axial skeletal defects

Cerebro-Costo-Mandibular Syndrome (CCMS) is a rare congenital disorder due to pathogenic variants in the core spliceosomal gene SNRPB. Affected individuals present with axial skeletal abnormalities, including cleft palate, micrognathia, posterior rib gaps, and a bell-shaped thorax. The molecular basis of these axial defects remains poorly understood, limiting the development of targeted interventions. To study the temporal and molecular requirements of SNRPB during axial development, we generated a mouse model of CCMS using an inducible Cre-lox system to conditionally delete Snrpb. Mosaic deletion of exons 2-3 of Snrpb after gastrulation resulted in the full spectrum of CCMS-like abnormalities, including micrognathia, posterior rib gaps and a reduced thoracic cavity. Despite normal somite morphology and patterning, transcriptomic analysis of E9.5 mutant somites revealed upregulation of p53 pathway genes and mis-expression of retinoic acid (RA) signaling components, consistent with reduced RA signaling. Alternatively spliced genes in Snrpb mutant somites were associated with post-transcriptional regulation, including chromatin modifiers. To test if RA pathway supplementation can rescue axial defects, we performed dietary RA supplementation; however, this failed to rescue mutant phenotypes, suggesting that p53 activation and chromatin dysregulation additionally contribute to disease pathogenesis. Together, these findings establish the first in vivo model of CCMS-associated axial skeletal abnormalities and indicate that Snrpb dysfunction disrupts the early specification of axial skeletal identity without overtly altering somite patterning.

developmental biology↗

bioRxiv2025

Integrated Proteomic and Epigenomic Analysis Reveals IGF2 as a Vulnerability in PRC2-Deficient Malignant Peripheral Nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with limited therapeutic options. Loss of the Polycomb repressive complex 2 (PRC2), via inactivating mutations in SUZ12 or EED, occurs frequently in MPNSTs and is associated with poor prognosis. However, the downstream chromatin and signaling consequences of these mutations remain incompletely understood. Here, we show that PRC2 deficiency in MPNST cells induces coordinated chromatin remodeling, characterized by loss of repressive H3K27me3 and gain of activating marks, including H3K27ac and H3K36me2. Integrative epigenomic, transcriptomic, and proteomic profiling revealed that this chromatin reprogramming activates a fetal-like growth signature centered on insulin-like growth factor 2 (IGF2) and its post-transcriptional regulators, Insulin-like Growth Factor 2 mRNA-Binding Protein (IGF2BP1-3). Functional studies demonstrate that PRC2-deficient cells are selectively dependent on IGF2 for proliferation, and that restoration of SUZ12 suppresses IGF2 expression and reduces growth. Analysis of human MPNST tumors confirms upregulation of the IGF2-IGF2BP axis in PRC2-deficient tumors, highlighting its clinical relevance. Together, these findings link PRC2 loss to activation of fetal growth factor-driven oncogenic signaling and identify IGF2 and its regulatory network as potential vulnerabilities in this aggressive tumor subtype.

cancer biology↗

bioRxiv2025

Cellular Reprogramming of H3K27M Pediatric High-Grade Glioma to Neuron-like State

This study explores the cell fate reprogrammability of H3K27M-mutant pediatric high-grade gliomas (pHGG) using neuronal transdifferentiation as a potential targeted therapy. We treated the BT245 patient-derived glioma cell line with pharmacological combinations targeting neuronal differentiation pathways and performed bulk RNA sequencing to characterize gene expression patterns driving cell fate transitions. Our findings reveal that the drug combinations induce transcriptomic changes consistent with differentiation towards neuronal phenotypes, including the upregulation of synaptic and dendritic signaling genes and the downregulation of malignant signatures. In comparison, astrocytic differentiation media (DM) and H3K27M knockout (KO) promote residual astrocytic phenotypes, suggesting neuronal transdifferentiation as a more effective strategy for mitigating tumor aggressiveness and progression. Differentially expressed genes such as GRIK1, GRIN1, NRXN3, NRXN1, CALB2, SCGN, SLC32A1, SLC1A2, KCNC3, and neurodevelopmental regulators including WNT7A, DLX6, ERBB4, ARX, BCL11B, SEMA3C, and FGFBP3 were identified as key markers regulating the neuron-like lineage transition. This study demonstrates that pHGGs can be phenotypically redirected toward neuronal-like identities through modulating cell fate differentiation programs. These findings advance the concept of differentiation therapy as a promising intervention to reduce phenotypic plasticity and malignancy in pHGG ecosystems. While these are early in vitro findings, the ability to steer and control glioma cells toward stable, less malignant fates offers promising translational potential for patient-centered targeted therapies.

cancer biology↗

bioRxiv2024

ChIPbinner: An R package for analyzing broad histone marks binned in uniform windows from ChIP-Seq or CUT&RUN/TAG data

BackgroundThe decreasing costs of sequencing, along with the growing understanding of epigenetic mechanisms driving diseases, have led to the increased application of chromatin immunoprecipitation (ChIP-Seq), Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and Cleavage Under Targets and Tagmentation (CUT&TAG) sequencing - which are designed to map DNA or chromatin-binding proteins to their genome targets - in biomedical research. Existing software tools, namely peak-callers, are available for analyzing data from these technologies, although they often struggle with diffuse and broad signals, such as those associated with broad histone post-translational modifications (PTMs). ResultsTo address this limitation, we present ChIPbinner, an open-source R package tailored for reference-agnostic analysis of broad PTMs. Instead of relying on pre-identified enriched regions from peak-callers, ChIPbinner divides (bins) the genome into uniform windows. Thus, users are provided with an unbiased method to explore genome-wide differences between two samples using scatterplots, principal component analysis (PCA), and correlation plots. It also facilitates the identification and characterization of differential clusters of bins, allowing users to focus on specific genomic regions significantly affected by treatments or mutations. We demonstrated the effectiveness of this tool through a case study assessing H3K36me2 depletion following NSD1 knockout in head and neck squamous cell carcinoma, highlighting the advantages of ChIPbinner in detecting broad histone mark changes over existing software. ConclusionsBinned analysis provides a more holistic view of the genomic landscape, allowing researchers to uncover broader patterns and correlations that may be missed when solely focusing on individual peaks. ChIPbinner offers researchers a convenient tool to perform binned analysis. It improves on previously published software by providing a clustering approach that is independent of each bins differential enrichment status, while also offering additional features for downstream analysis of these differentially enriched bins.

bioinformatics↗

bioRxiv2024

H3K36 Methylation - a Guardian of Epigenome Integrity

H3K36 methylation is emerging as a key epigenetic modification with strong implications in genetic disease and cancer. However, the mechanisms through which H3K36me impacts the epigenome and asserts its functional consequences are far from understood. Here, we use mouse mesenchymal stem cell lines with successive knockouts of the H3K36 methyltransferases: NSD1, NSD2, SETD2, NSD3, and ASH1L, which result in progressive depletion of H3K36me and its complete absence in quintuple knockout cells, to finely dissect the role of H3K36me2 in shaping the epigenome and transcriptome. We show that H3K36me2, which targets active enhancers, is important for maintaining enhancer activity, and its depletion results in downregulation of enhancer-dependent genes. We demonstrate the roles of H3K36me2/3 in preventing the invasion of gene bodies by the repressive H3K27me modifications. Finally, we observe a previously undescribed relationship between H3K36me and H3K9me3: Following the depletion of H3K36me2, H3K9me3 is redistributed away from large heterochromatic domains and towards euchromatin. This results in a drastic decompartmentalization of the genome, weakening the boundaries between active and inactive compartments, and a catastrophic loss of long-range inter-compartment interactions. By studying cells totally devoid of H3K36 methyltransferase activity, we uncover a broad range of crucial functions of H3K36me in maintaining epigenome integrity.

genomics↗

bioRxiv2024

Etiology of Craniofacial and Cardiac Malformations in a Mouse Model of SF3B4-Related Syndromes

Pathogenic variants in SF3B4 are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related Syndromes, associated with malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighbouring non NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities showed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. We identified several key transcription factors and histone modifiers involved in craniofacial and cardiac development with increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities.

developmental biology↗

bioRxiv2023

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights{square} The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions. {square}The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs. {square}Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture. {square}The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

molecular biology↗

bioRxiv2023

Systematic perturbations of SETD2, NSD1, NSD2, NSD3 and ASH1L reveals their distinct contributions to H3K36 methylation

BackgroundMethylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development, disease, and cancer, how the molecular agents collectively shape the H3K36me landscape is unclear. ResultsWe use a mouse mesenchymal stem cell model to perturb the H3K36me deposition machinery and infer the activities of the five most prominent players: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and is predominantly deposited at intergenic regions by NSD1, and partly by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 deposits most H3K36me3, it also deposits H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting other H3K36 methyltransferases (K36MTs) prime gene bodies with lower methylation states ahead of transcription. Through a reductive approach, we uncover the distribution patterns of NSD3- and ASH1L-catalyzed H3K36me2. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks on active promoters and enhancers. Meanwhile, the activity of ASH1L is restricted to the regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor. ConclusionsWithin genes, SETD2 deposits both H3K36me2/3, while the other K36MTs are capable of depositing H3K36me1/2 independently of SETD2 activity. For the deposition of H3K36me1/2, we find a hierarchy of K36MT activities where NSD1>NSD2>NSD3>ASH1L. While NSD1 and NSD2 are responsible for most genome-wide propagation of H3K36me2, the activities of NSD3 and ASH1L are confined to active regulatory elements.

genomics↗

bioRxiv2021

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

CDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with cancer in two families. Characterization of these mutants showed they retain APC/C activation activity but show reduced binding to BUBR1, a component of the SAC. Expression of L151R and N331K promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. CRISPR/Cas9 was used to generate mice carrying N331K. Homozygous mice carrying N331K were non-viable, however, heterozygotes displayed accelerated oncogenicity in Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor promoting genes in humans.

cancer biology↗

bioRxiv2021

Snrpb, the gene mutated in CCMS, is haploinsufficient and required for proper morphogenesis and splicing

Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5 splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations.

developmental biology↗

bioRxiv2020

Epigenome dysregulation resulting from NSD1 mutation in head and neck squamous cell carcinoma

Epigenetic dysregulation has emerged as an important mechanism of oncogenesis. To develop targeted treatments, it is important to understand the epigenetic and transcriptomic consequences of mutations in epigenetic modifier genes. Recently, mutations in the histone methyltransferase gene NSD1 have been identified in a subset of head and neck squamous cell carcinomas (HNSCCs) - one of the most common and deadly cancers. Here, we use whole (epi)genome approaches and genome editing to dissect the downstream effects of loss of NSD1 in HNSCC. We demonstrate that NSD1 mutations are directly responsible for loss of intergenic H3K36me2 domains, followed by loss of DNA methylation, and gain of H3K27me3 in the affected genomic regions. We further show that those regions are enriched in cis-regulatory elements and that subsequent loss of H3K27ac correlates with reduced expression of their target genes. Our analysis identifies genes and pathways affected by the loss of NSD1 and paves the way to further understanding the interplay among chromatin modifications in cancer.

genomics↗

bioRxiv2020

Lipid membrane templated misfolding and self-assembly of intrinsically disordered tau protein

The aggregation of the intrinsically disordered tau protein into highly ordered {beta}-sheet fibrils is implicated in many neurodegenerative disorders. Fibrillation mechanism remains unresolved, particularly early events that trigger tau misfolding and assembly. We investigated the role membrane plays in modulating aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation mutant hTau40/3Epi. Despite being charged and soluble, tau proteins were also highly surface active and favorably interacted with anionic, but not zwitterionic, lipid monolayer at the air/water interface. Membrane binding induced macroscopic tau phase separation and {beta}-sheet-rich tau oligomer formation. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein taus inherent surface activity and favorable electrostatic interactions drive tau-membrane association, inducing tau phase separation that is accompanied by misfolding and self-assembly of disordered tau into {beta}-sheet-rich oligomers, which subsequently seed fibrillation and deposition into diseased tissues.

biophysics↗

bioRxiv2019

3DGV: Immersive Exploration of 3D Genome Structures using Virtual Reality

The analysis of 3D genomic data is expected to revolutionize our understanding of genome organization and regulatory mechanisms. Yet, the complex spatial organization of this information can be difficult to interpret with 2D viewers. Virtual Reality (VR) technologies offer an opportunity to rethink our methods to visualize and navigate 3D objects. In this paper, we introduce the Virtual Reality 3D Genome Viewer (3DGV), an open platform to experiment and develop VR solutions to explore 3D genome structures. Availabilityhttp://3dgv.cs.mcgill.ca/

bioinformatics↗

bioRxiv2017

CYRI1-mediated inhibition of RAC1 signalling restricts Salmonella Typhimurium infection

Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defence mechanisms through bacterial effectors that target key host proteins implicated in restricting infection. Thus, to gain insight into host-pathogen interactions governing Salmonella infection, a thorough understanding of host defence mechanisms is needed. To tackle this, we performed an in vivo genome-wide ENU mutagenesis screen to uncover novel host defence proteins. Through this screen we identified an uncharacterised protein, which we name CYRI1 (CYFIP-related RAC1 Interacting protein 1) that serves as a Salmonella resistance factor. We show that CYRI1 binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerisation. However, unlike CYFIP proteins, CYRI1 negatively regulates RAC1-driven actin cytoskeleton remodelling, thereby attenuating processes such as phagocytosis and cell migration. This, in turn, enables CYRI1 to counteract Salmonella at various stages of infection, including bacterial entry into epithelial cells, internalisation into myeloid-derived phagocytes as well as phagocyte-mediated bacterial dissemination. Together, this outlines a novel host defence mechanism that is crucial for determining bacterial fate.

cell biology↗