Biology subjects

Johnsen, S. A.

Publications and source records attributed to Johnsen, S. A..

6 recordsLinked to original sources

bioRxiv2025

KLF5 controls subtype-independent highly interactive enhancers in pancreatic cancer to regulate cell survival

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal cancer with a 5-year survival rate of 13%. Despite recent molecular stratification of tumors into distinct classical and basal-like cell states, most tumors are heterogeneous contain of both subtypes. Therefore, therapeutic approaches targeting one subtype may not be suitable for PDAC therapy. Here, we integrated chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac) and H3K4me3-anchored chromatin topology (HiChIP) to uncover subtype-independent highly interactive enhancers that interact with essential genes in PDAC. Motif analysis revealed these common enhancers were bound by KLF5 with subsequent depletion leading to decreased cell viability via induction of apoptosis. To elucidate the transcriptional and epigenetic mechanisms by which KLF5 functions in PDAC, we employed rapid depletion of KLF5 with dTAG technology and profiled the effects on the open and active chromatin landscape and transcription with nascent RNA and mRNA-seq over time. Enhancer inactivation via KRAB domain Zim3-dCas9 fusion protein confirmed KLF5-bound enhancers regulate target genes, including the anti-apoptotic gene BCL2L1. Multiplex immunofluorescence confirmed co-staining of KLF5 and Bcl-xL in patient samples and overexpression of Bcl-xL rescued the induction of apoptosis after KLF5 depletion. Taken together, this study provides new insights into common mechanisms to target highly heterogeneous PDAC tumors. TeaserKLF5 controls subtype-independent highly interactive enhancers to regulate cell viability in pancreatic cancer.

cancer biology↗

bioRxiv2025

Epigenetic Context Defines the Transcriptional Activity of Canonical and Noncanonical NF-κB Signaling in Pancreatic Cancer

NF-{kappa}B signaling can be subdivided into canonical and noncanonical pathways, culminating in the transcriptional activity of RELA and RELB, respectively. However, the upstream signals that activate these transcription factors and their specific regulatory roles in pancreatic ductal adenocarcinoma (PDAC) remain incompletely understood. Here, we demonstrate that TNF is the primary activator of canonical NF-{kappa}B signaling via RELA, while TWEAK engages noncanonical signaling through RELB in PDAC. Using transcriptome-wide gene expression, genome-wide occupancy profiling, and epigenome mapping, we delineate distinct temporal dynamics and regulatory activities of each pathway. Single-cell RNA-seq analysis and multiplex immunofluorescence staining in primary PDAC samples further reveal extensive and distinct interactions with components of the tumor microenvironment. Motif analysis of RELA- and RELB-bound regions uncovers a particularly strong association of RELB with AP1 elements. Notably, while RELA binds to both open and closed chromatin, RELB exclusively occupies regions with pre-existing chromatin accessibility and AP1 co-binding. Collectively, these findings underscore the distinct and complementary roles of TNF and TWEAK in PDAC, with TNF engaging a broader transcriptional program via RELA and TWEAK selectively targeting genes marked by accessible chromatin and epigenetic activity. This study provides critical insights into the regulatory dynamics of NF-{kappa}B signaling in PDAC, highlighting unique and complementary functions of RELA and RELB in modulating downstream gene expression. SignificanceTNF and TWEAK selectively activate RELA and RELB in pancreatic cancer, revealing that RELB-driven noncanonical signaling depends on chromatin accessibility and AP-1, uncovering distinct, targetable epigenetic dependencies from canonical signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/661561v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@14aa356org.highwire.dtl.DTLVardef@1297741org.highwire.dtl.DTLVardef@1545741org.highwire.dtl.DTLVardef@2c982b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

CRISPR gene and transcriptome engineering (CRISPRgate) improves loss-of-function genetic screening approaches

The CRISPR/Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR Gene and Transcriptome Engineering (CRISPRgate), which uses two specific sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgate outperforms conventional CRISPRko, CRISPRi, or CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of EMT and impairs neuronal differentiation in a human iPSC model. In a multiplexed chromatin-focused phenotypic LOF screen, CRISPRgate exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgate increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens. MotivationThe CRISPR technology (CRISPRko/CRISPRi) enables the specific depletion of target genes with fewer off-target effects, facilitating precise investigations of gene function. Despite its benefits, CRISPR applications have limitations. Residual active protein expression mediated by in-frame DNA repair or alternative splicing1-8 as well as strong epigenetic regulation and difficulties in sgRNA design to the transcription start site (TSS)9-12 hinder the full potential of loss-of-function studies using CRISPRko or CRISPRi. We aimed to achieve robust target gene reduction in order to improve the reproducibility of the CRISPR technology by integrating the widely used CRISPRko and CRISPRi approaches into a single application.

biochemistry↗

bioRxiv2022

Epigenetic plasticity via adaptive DNA hypermethylation and clonal expansion underlie resistance to oncogenic pathway inhibition in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. Drug resistance is the major cause for therapeutic failure in PDAC patients with progressive disease. The mechanisms underlying resistance formation are complex and remain poorly understood. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. The effector caspase CASP3 is one of the 114 genes for which transcriptional downregulation inversely correlated with the methylation status of the associated DNA region. CASP3 inactivation in resistant cells led to attenuation of drug-induced apoptosis which could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. Overall, our data provide a context for characterization and targeting of epigenetically mediated resistance mechanisms in PDAC.

cancer biology↗

bioRxiv2021

Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity

Interferons protect from virus infections by inducing hundreds of interferon-stimulated genes (ISG) which orchestrate anti-viral adaptive and innate immunity. Upon viral infection or type I interferon (IFN) stimulation of cell lines, a histone modification, monoubiquitinated histone 2B (H2Bub1), increases at ISG loci, raising the possibility that a specific chromatin state can broadly stimulate IFN immunity in vivo. Here we show that, in the absence of virus infection or elevated interferon levels, mice lacking the relevant deubiquitinase, Usp22, in immune cells have elevated H2Bub1 levels. Hypermonoubiquitinated H2B is physically associated with dozens of ISG loci, and expression of large numbers of ISG is upregulated. This epigenetic state promotes intracellular and systemic immune phenotypes akin to adaptive and innate interferon immunity, and thereby identifies Usp22 as a negative regulator of interferon immunity.

immunology↗

bioRxiv2020

RNF40-dependent epigenetic regulation of actin cytoskeletal dynamics is required for HER2-driven mammary tumorigenesis

The HER2-driven breast cancer subtype displays a particularly aggressive behavior. Alterations of the epigenome are common in cancers and represent attractive novel molecular therapeutic targets. Monoubiquitination of histone 2B (H2Bub1) by its obligate heterodimeric E3 ubiquitin ligase complex RNF20/RNF40 has been described to have tumor suppressor functions and loss of H2Bub1 has been associated with cancer progression. In this study, we utilized human tumor samples, cell culture models, and a mammary carcinoma mouse model with tissue-specific Rnf40 deletion and identified an unexpected tumor-supportive role of RNF40 in HER2-positive breast cancer. We demonstrate that RNF40-driven H2B monoubiquitination is essential for transcriptional activation of RHO/ROCK/LIMK pathway components and proper actin cytoskeleton dynamics through a trans-histone crosstalk with histone 3 lysine 4 trimethylation (H3K4me3). Collectively, this work demonstrates a previously unknown essential role of RNF40 in HER2-positive breast cancer, revealing the RNF20/RNF40/H2Bub1 axis as a possible tumor context-dependent therapeutic target in breast cancer. Statement of significanceHER2-positive breast cancer patients frequently develop resistance to anti-HER2 therapies. Here we demonstrate that RNF20/RNF40-mediated H2B monoubiquitination supports the oncogenic properties of cancer cells of this subtype by regulating actin dynamics. The RNF20/RNF40/H2Bub1 axis may therefore represent an attractive drug target for novel therapies.

cancer biology↗