Sargunas, P. | Search bioRxiv

bioRxiv2026

Uhrf1 loss disrupts Ctcf-associated chromatin organization during early mouse embryogenesis

UHRF1 is a chromatin-binding protein essential for maintaining DNA methylation and histone modification states, yet its integrated role in vivo remains incompletely understood. To define its function, we generated conditional Uhrf1 knockout embryonic stem cells (ESCs) and embryos. Uhrf1-/- ESCs exhibited near-complete loss of 5mC and 5hmC but maintained pluripotency, whereas Uhrf1-null embryos developed normally until E8.5 and then failed to develop further by E9.5, phenocopying Dnmt1 loss. Single-cell multi-omic (ME-seq) profiling of E8.5 embryos revealed impaired lineage stabilization, widespread hypomethylation, and disrupted chromatin architecture. Uhrf1 loss was associated with altered CTCF-associated chromatin signal, broad remodeling of chromatin contacts, altered cis-regulatory relationships, and reduced predicted BMP-related ligand-receptor communication, particularly within neural crest populations. These findings identify Uhrf1 as a central regulator that tightly couples DNA methylation maintenance to 3D genome organization during gastrulation, thereby, directing early lineage specification and positioning Uhrf1 as a pivotal mediator of epigenetic information transfer during early embryogenesis. HighlightsO_LIUhrf1 knockout ESCs show global loss of 5mC/5hmC but maintain pluripotency. C_LIO_LIUhrf1-null embryos develop normally until E8.5 but die by E9.5 with severe defects. C_LIO_LISingle-cell multi-omics revealed disrupted chromatin, transcription, and lineage stability upon knockout. C_LIO_LILoss of Uhrf1 alters CTCF-associated chromatin signal, predicted BMP-related communication, and cis-regulatory relationships. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738670v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@15bf31dorg.highwire.dtl.DTLVardef@1b601ecorg.highwire.dtl.DTLVardef@386018org.highwire.dtl.DTLVardef@12ce99f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Multispecific nanobody degraders co-deplete membrane receptors and enable targeted delivery of diverse payloads

Targeting membrane receptors underlies the success of antibody-drug conjugates (ADCs), yet single-receptor formats can be limited by heterogeneous expression, compensatory signaling, and variable internalization. Here we developed Multivalent Interchangeable Nanobody Degradation System (MINDS), a modular nanobody-Fc chassis that co-engages multiple membrane receptors, promotes their lysosomal co-depletion, and enables delivery of diverse intracellular payloads. As a proof of concept, we generated Tritazumab, a trispecific nanobody-Fc targeting three oncogenic receptors EGFR, cMET, and TfR1. Tritazumab incorporates a high-affinity, non-transferrin-competing anti-TfR1 nanobody that drives efficient uptake and lysosomal trafficking, enabling coordinated depletion of all three receptors. Across non-small cell lung cancer models, Tritazumab achieved rapid and sustained multi-receptor surface loss with picomolar degradation potency, reaching near-maximal depletion within approximately 1.5 hours. Conjugation of Tritazumab to MMAE preserved receptor binding and produced substantially greater antiproliferative activity and improved tumor selectivity relative to clinical ADCs in matched cell models, along with potent in vivo tumor growth inhibition and acceptable tolerability in a xenograft model. Extending the platform beyond cytotoxic payloads, a BRD4 molecular glue conjugate improved the selectivity window by > 100-fold and showed marked in vivo efficacy, while an EZH2-targeting PROTAC conjugate achieved an approximately 1,000-fold increase in intracellular degradation potency relative to the free PROTAC. These findings establish MINDS as a modular multispecific degrader-payload platform that integrates receptor co-depletion to enhance anticancer selectivity and efficacy.

cancer biology↗