Biology subjects

Knotz, C.

Publications and source records attributed to Knotz, C..

4 recordsLinked to original sources

bioRxiv2026

Alternative lengthening of telomeres stratifies complex karyotype sarcomas into distinct genomic and transcriptomic states

Complex karyotype sarcomas (CKS) are heterogeneous mesenchymal malignancies that typically lack recurrent actionable oncogenic drivers and remain therapeutically challenging. Loss of ATRX is a recurrent feature of CKS and defines a particularly high-risk subgroup. ATRX loss is also associated with activation of the alternative lengthening of telomeres (ALT) pathway, and ALT-positive sarcomas have been linked to poor clinical outcomes. However, the molecular underpinnings underlying ALT-status-dependent differences in CKS, as well as the therapeutic vulnerabilities associated with ALT, remain poorly defined. By integrating C-circle-based ALT detection across 776 sarcoma samples with multi-modal sequencing of five CKS subtypes, we find that ALT activity is associated with enriched hallmarks of genomic instability. ALT-positive transcriptomes are dominated by a coordinated DNA damage response and mitotic program, in contrast to oncogenic signaling pathways that drive TERT activation in ALT-negative tumors. Long-read sequencing reveals telomere repeat clusters and telomere-mediated healing at structural breakpoints in ALT-positive tumors. These events also occur on extrachromosomal DNA (ecDNA), linking ALT activity to ecDNA biology. Together, our findings position ALT status as an important stratifying feature of CKS and identify ALT-associated transcriptional programs as potential therapeutic targets.

cancer biology↗

bioRxiv2026

Single-stranded telomeric repeats segregate into spatial compartments within ALT-associated PML bodies

Alternative lengthening of telomeres (ALT)-associated PML bodies (APBs) concentrate single-stranded (ss) telomeric DNA and RNA species that are critical for recombination-based telomere maintenance. However, how these species are organized inside APBs has remained invisible at microscopic resolution. Here, we map the nanoscale topology of APB components using 3D MINFLUX super-resolution microscopy combined with multiplexed exchange DNA-PAINT labeling at [~]3 nm localization precision. We discover that ssC-rich and ssG-rich telomeric repeats occupy distinct spatial compartments within a partially open, [~]70 nm thick PML protein shell: ssC-rich repeats concentrate at the inner shell surface, while ssG-rich repeats distribute broadly through the interior alongside TRF1-marked double-stranded telomeric chromatin. The ssG-rich signal is predominantly DNA and frequently colocalizes with POT1 assemblies. Additional TRF1 clusters outside the shell indicate multi-telomere association. Together, these structural constraints motivate a model of ALT in which t-loop resolution generates a C-circle template that drives rolling-circle amplification of telomeric repeats. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/724127v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@fdb6a2org.highwire.dtl.DTLVardef@e185c9org.highwire.dtl.DTLVardef@168b12org.highwire.dtl.DTLVardef@44bd0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI3D MINFLUX combined with exchange DNA-PAINT resolves APB components at {bsim}3 nm isotropic precision, enabling direct visualization of molecular organization beyond the reach of conventional super-resolution microscopy. C_LIO_LIPML forms a partially open, [~]70 nm thick spherical shell in APBs with reduced molecular density compared to canonical PML nuclear bodies, providing the structural scaffold and coordinate system for mapping internal APB organization. C_LIO_LIDouble-stranded telomeric chromatin (TRF1-marked) fills the APB interior, with additional TRF1 domains outside the shell indicative of multi-telomere clustering at individual APBs. C_LIO_LIssC-rich repeats concentrate in the inner part of the PML shell while ssG-rich DNA repeats distribute broadly through the interior, revealing strand-specific spatial segregation. C_LIO_LIThe ssG-rich single-stranded repeats in APBs predominantly comprise DNA and partially colocalize with POT1, connecting their spatial distribution to single-stranded telomeric DNA substrates. C_LIO_LIThe spatial organization of APB components motivates a mechanistic model in which t-loop processing generates C-circle templates for rolling-circle amplification, while multi-telomere clustering facilitates inter-telomeric recombination. C_LI

cell biology↗

bioRxiv2024

HP1 binding creates a local barrier against transcription activation and persists during chromatin decondensation

Mouse pericentric repeats form transcriptionally silent and compacted domains known as chromocenters. These prototypic heterochromatin compartments are marked by heterochromatin protein 1 (HP1). However, its contributions to chromocenter structure and function remain debated. We investigated the role of HP1 by recruiting the activators VP16, p65, and VPR to mouse fibroblast chromocenters and analyzed its silencing activity with a transcription reporter. Upon chromocenter decondensation and transcription activation, interactions of HP1 with chromatin and H3K9 trimethylation remained stable, suggesting stoichiometric binding rather than higher-order assembly. HP1-mediated repression required promoter-proximal binding and effectively suppressed VP16-triggered activation but not the stronger activation by VPR. These observations are explained by a 1D lattice binding model, which conceptualizes chromocenters as arrays of repeat units that can independently switch between silenced and activated states. Our findings provide a quantitative framework that explains how chromocenter organization responds to transcriptional activation while maintaining local heterochromatin features. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/627308v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@83816corg.highwire.dtl.DTLVardef@1a603c6org.highwire.dtl.DTLVardef@41d8b4org.highwire.dtl.DTLVardef@fa4d0d_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHP1 represses transcription at mouse chromocenters and an ectopic reporter when competing with transcriptional activators VP16, p65 and VPR C_LIO_LIHP1 repression requires promoter-proximal binding and effectively counteracts the weak activator VP16 C_LIO_LIThe strong activator VPR overcomes HP1-mediated repression while HP1 remains bound to chromatin C_LIO_LIHP1 binding and H3K9me3 persist during chromocenter decondensation and transcriptional activation C_LIO_LIA 1D lattice binding model explains how independent repeat units transition between silenced and activated states without requiring phase separation C_LI

cell biology↗

bioRxiv2022

Epigenetic signals that direct cell type specific interferon beta response in mouse cells

The antiviral response induced by type I interferon (IFN) via the JAK-STAT signaling cascade activates hundreds of IFN-stimulated genes (ISGs). While this response occurs essentially in all human and mouse tissues it varies between different cell types. However, the linkage between the underlying epigenetic features and the ISG pattern of a given cell is not well understood. We mapped ISGs, binding sites of the STAT1 and STAT2 transcription factors and chromatin features in three different mouse cell types (embryonic stem cells, neural progenitor cells and embryonic fibroblasts) before and after treatment with IFN{beta}. The analysis included gene expression, chromatin accessibility and histone H3 lysine modification by acetylation (ac) and mono-/tri-methylation (me1, me3). A large fraction of ISGs and STAT binding sites were cell type specific with promoter binding of a STAT1-STAT2 complex (STAT1/2) being a key driver of ISG induction. Furthermore, STAT1/2 binding to putative enhancers at intergenic and intronic sites induced ISG expression as inferred from a chromatin co-accessibility analysis. STAT1/2 binding was dependent on the chromatin context and positively correlated with pre-existing H3K4me1 and H3K27ac marks in an open chromatin state while the presence of H3K27me3 had an inhibitory effect. Thus, chromatin features present before stimulation represent an additional regulatory layer for the cell type specific antiviral response.

molecular biology↗