Biology subjects

Elsafi Mabrouk, M. H.

Publications and source records attributed to Elsafi Mabrouk, M. H..

7 recordsLinked to original sources

bioRxiv2026

Reversing PROTAC-induced ASH2L degradation reactivates proliferation in senescent cells

Nucleosomes control access to gene promoters. Histone H3 lysine 4 tri-methylation, catalyzed by 6 KMT2 complexes, correlates with accessible promoters and gene expression. The catalytic activity of KMT2 enzymes depends on an obligatory core complex with ASH2L being an essential subunit. We find that PROTAC induced depletion of ASH2L reduces H3K4me3, deregulates gene expression and prevents proliferation. Upon prolonged ASH2L loss, cells develop a senescent phenotype, a process linked to aging and disease. Competing the PROTAC reactivates ASH2L, reestablishes H3K4me3 at promoters and reverts gene expression changes. Cells reenter the cell cycle and resume proliferation, thereby reverting senescence. Structure-function studies demonstrate that these molecular and cellular consequences are primarily due to the loss of ASH2L functions associated with KMT2 complexes. Together, these findings indicate that stress inflicted by the loss of KMT2 catalytic activities promotes a reversible senescence phenotype, suggesting that the functions of KMT2 complexes are implicated in aging. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/722411v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@10ace2forg.highwire.dtl.DTLVardef@667c81org.highwire.dtl.DTLVardef@78039dorg.highwire.dtl.DTLVardef@13563be_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Age-associated Tissue Organisation Shifts Retinal Pigment Epithelium Actomyosin Plasticity in Phagocytosis

Tissue homeostasis relies on mechanical feedback loops balanced by cell loss and proliferation. However, maintaining this balance becomes particularly challenging in postmitotic tissues, where alternative mechanisms replace compensatory proliferation. In the postmitotic retinal pigment epithelium (RPE), these mechanisms include significant structural adaptations over time. Yet, how these adaptations relate to epithelial mechanical homeostasis and age-associated functional decline remains poorly understood. To establish the relationship between structural changes, mechanical homeostasis and function, we developed an in vitro reductionistic model mimicking age-related reduction in RPE cell density. Inducing large-scale apoptosis in postmitotic stem cell-derived RPE monolayers recapitulates structural hallmarks of aged tissue, such as reduced cell height, shortened microvilli and cytoskeletal reorganisation. This new structure acquires a new mechanical equilibrium, evidenced by tissue stiffening and enhanced junctional contractility. Functionally, the monolayers display impaired vision-supporting phagocytosis of photoreceptor outer segments. Mechanistically, modulation of actin nucleators, Arp2/3 and formins, demonstrates that apicolateral monolayer deformation is critical for phagocytosis and may be compromised in aged RPE. Our findings suggest that a shift in mechanical homeostasis due to cell loss is a major driver of age-related RPE functional decline. Importantly, we show that structural remodelling in ageing alone can compromise tissue function, independent of other stressors.

cell biology↗

bioRxiv2024

IFNα Inhibits JAK2V617F-Driven Neoplastic Vasculogenesis and Endothelial-to-Mesenchymal Transition in a 3D iPSC-Based Bone Marrow Niche Model

The vascular niche is a critical regulator of hematopoiesis and disease progression in myeloproliferative neoplasms (MPN). The presence of JAK2V617F+ endothelial cells (EC) in MPN patients and their association with cardiovascular complications highlight the need to understand and therapeutically target this compartment. Using patient-specific induced pluripotent stem cells (iPSC) harboring JAK2WT or the MPN-driver JAK2V617F (heterozygous, JAK2V617FHET, or homozygous, JAK2V617FHOM), we identified zygosity-dependent transcriptional profiles in iPSC-derived EC (iEC) at baseline and following interferon-alpha (IFN) treatment. JAK2V617FHET iEC exhibited an endothelial-to-mesenchymal transition (EndMT) signature, while JAK2V617FHOM iEC showed suppression of translation and ribosome biogenesis. Leveraging iPSC-based 3D assembloids that mimic the bone marrow (BM) niche, we showed that JAK2V617F-driven EndMT is inhibited by tyrosine kinase inhibitors and IFN. In both JAK2V617F-driven polycythemia vera and TPO-driven myelofibrosis murine models, scRNA-seq analysis of the BM vascular niche consistently revealed inflammatory and EndMT-associated signatures in arterial and arteriolar EC. Notably, dysregulation of ribosome- and translation-related pathways emerged in the myelofibrosis model and at advanced disease stages in JAK2V617F-driven polycythemia vera, indicating progressive vascular remodeling with disease evolution. Chronic pegylated IFN treatment in vivo effectively reversed these pathological changes. IFNs anti-EndMT activity was further validated in BM biopsies from MPN patients undergoing IFN therapy. This is the first study to define MPN stage-dependent vascular remodeling and zygosity-specific endothelial effects of JAK2V617F, and to directly link IFN-mediated EndMT inhibition as a novel antifibrotic mechanism. Our 3D assembloids provide a translational platform for mechanistic studies and therapeutic targeting of the BM microenvironment in MPN. Bullet PointsO_LIArterial vascular remodeling emerges as a novel hallmark of MPN, characterized by TNF-inflammation, ribosomal dysregulation and EndMT. C_LIO_LIIFN restores neoplastic endothelial dysfunction, highlighting its role as a vascular niche-modulating and anti-fibrotic agent in MPN. C_LI

cancer biology↗

bioRxiv2024

Epigenetic editing at individual age-associated CpGs affects the genome-wide epigenetic aging landscape

Aging is reflected by genome-wide DNA methylation changes, but it is largely unclear how these epigenetic modifications are regulated. In this study, we explored the possibility to interfere with epigenetic clocks by epigenetic editing at individual CpG sites. CRISPR-guided approaches (dCas9-DNMT3A and CRISPRoff) facilitated targeted methylation at an age-associated genomic region in PDE4C that remained stable for more than three months. Furthermore, epigenetic editing evoked many genome-wide off-target effects, which were highly reproducible and enriched at other age-associated CpGs - thus, they are not random off-target effects, but seem to resemble coregulated epigenetic bystander modifications. 4C chromatin conformation analysis at age-associated sites revealed increased interactions with bystander modifications and other age-associated CpG sites. Subsequently, we multiplexed epigenetic modifications in HEK293T and primary T cells at five genomic regions that become either hypermethylated or hypomethylated upon aging. While epigenetic editing at age-hypomethylated CpGs appeared less stable, it also resulted in a clear enrichment of bystander modifications at other age-associated CpGs. Conversely, epigenetic clocks tend to be accelerated up to ten years after targeted DNA methylation, particularly at hypermethylated CpGs. These results demonstrate that targeted epigenome editing can modulate the epigenetic aging network in its entirety and thereby interfere with epigenetic clocks.

genetics↗

bioRxiv2024

Substrate elasticity does not impact on DNA methylation changes during differentiation of pluripotent stem cells

Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness impacts on differentiation of induced pluripotent stem cells (iPSCs) and if this is also reflected by epigenetic modifications. We have therefore cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Youngs modulus (0.2 kPa, 16 kPa, or 64 kPa) to investigate the sequel on growth and differentiation towards endoderm, mesoderm, and ectoderm. Immunofluorescence and gene expression of canonical differentiation markers was hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of undifferentiated iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP, we observed epigenetic differences, particularly upon mesodermal differentiation. Taken together, stiffness of PDMS-substrates did not impact on directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters.

cell biology↗

bioRxiv2022

YAP1 is essential for self-organized differentiation of pluripotent stem cells

The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. It has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human induced pluripotent stem cells (iPSCs) remains largely unclear. To gain better insight into the function of YAP1 in these early cell-fate decisions, we generated iPSC lines with YAP1 knockout (YAP-/-) with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. In YAP-/- iPSCs the expression of several YAP1 targets changed and NODAL, which is an important regulator of cell differentiation, was upregulated. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies toward endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in self-organized embryoid bodies (EBs) germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and in tendency also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the non-directed self-organization of EBs, and this is at least partly attributed to activation of the NODAL pathway.

cell biology↗

bioRxiv2020

The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 {micro}m from the outer rim of iPSC colonies. Single- cell RNA-sequencing demonstrated that OCT4high subsets have pronounced up-regulation of the TGF-{beta} pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interactions were found to be relevant for the self-organization. Interestingly, after 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

cell biology↗