Biology subjects

Kappei, D.

Publications and source records attributed to Kappei, D..

12 recordsLinked to original sources

bioRxiv2025

A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

The DNA damage response (DDR) is a central regulator of cancer cell fate, coordinating both pro-death and pro-survival pathways in response to genotoxic stress. Here, we reveal an unexpected role for the DDR at the cell surface, in mediating immune evasion from complement-dependent cytotoxicity (CDC), an innate immune mechanism exploited by therapeutic monoclonal antibodies (mAbs). In the context of diffuse large B-cell lymphoma (DLBCL), where the anti-CD20 mAb rituximab utilizes CDC, we show that genotoxic chemotherapy induces expression of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59, thereby reducing CDC sensitivity and compromising rituximab activity. In this setting, CD59 emerged as the dominant DDR-induced inhibitor of complement-mediated killing. A high-throughput kinase inhibitor screen identified checkpoint kinase 1 (Chk1) as a critical mediator of this response. Mechanistically, DNA damage activates Chk1, enhancing CD59 transcription via an Sp1-bound promoter. Co-immunoprecipitation mass spectrometry revealed a Chk1 dependent remodelling of Sp1-associated complexes to a transcriptionally active state with recruitment of the histone acetyltransferase KAT2A. These findings expand the role of the DDR in immune resistance at the tumor cell surface, and highlight a negative interaction between chemotherapy and monoclonal antibodies that may require sequential administration or targeting of the Chk1- Sp1-CD59 axis. SignificanceThe DNA Damage Response upregulates complement-protective proteins, extending its role in modulating immune evasion at the cell surface, with direct implications for combinations of chemotherapy and monoclonal antibodies widely used in cancer.

cancer biology↗

bioRxiv2025

The TARZN complex binds de novo enhancer mutations and promotes oncogenic expression in T-ALL

TAL1 is overexpressed in 40-60% of T-cell acute lymphoblastic leukemia (T-ALL) cases and forms an oncogenic core regulatory circuit (CRC) with other transcription factors such as LMO1, LMO2 and GATA3. In 5% of T-ALL cases an insertion of a consensus GT dinucleotide (MuTE) is observed upstream of the TAL1 gene, driving TAL1 overexpression. Using an in vitro reconstitution DNA pull-down assay combined with quantitative mass spectrometry, we identified proteins that preferentially bound to the MuTE sequence and demonstrated that among the candidates the RNA methyltransferase TARBP1 and the zinc finger proteins ZBTB2, ZBTB25 and ZNF639 form a complex that we term TARZN. Interestingly, the TARZN complex also bound to de novo super enhancer sites upstream of the LMO1 and LMO2 genes in T-ALL cells, indicating a putative common mechanism between these different non-coding driver mutations. Furthermore, knock-down of all TARZN members resulted in lower TAL1 protein expression in MuTE-positive but not in MuTE-negative T-ALL cells. Given TARZNs methyltransferase activity and the lack of concomitant TAL1 mRNA level changes, we investigated reduced TAL1 translation and identified reduced neo-synthesised TAL1 protein levels upon TARBP1 knockdown. Overall, these data suggest that the TARZN complex promotes oncogenic expression in T-ALL via co-transcriptional RNA methylation.

cell biology↗

bioRxiv2024

Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction.

The scaffold protein IQGAP3 is highly upregulated in most epithelial cancers. While recent studies have highlighted its pivotal roles in cancer cell proliferation and metastasis, a deeper mechanistic understanding of IQGAP3 is currently lacking. We have here used TurboID to map IQGAP3 proximity partners and identified the Wnt signaling members Axin1 and CK1 as IQGAP3-interacting proteins. Our functional studies demonstrated that overexpression of IQGAP3 increases {beta}-catenin levels, while IQGAP3 depletion reduces {beta}-catenin levels in gastric cancer cells. Mechanistically, IQGAP3 disrupts Axin1-CK1 interaction, thereby inhibiting {beta}-catenin phosphorylation and ultimately leading to its accumulation. Importantly, we discovered that IQGAP3 itself is regulated by Wnt signaling, suggesting its involvement in a positive feedback loop in Wnt/{beta}-catenin signaling through interactions with Axin1 and CK1. These findings identify IQGAP3 as a novel mediator of {beta}-catenin stabilization and underscore its potential as a target for cancer therapy.

cancer biology↗

bioRxiv2023

ZBTB48 is a pioneer factor regulating B-cell-specific CIITA expression

CIITA is the master regulator of MHC II gene expression and hence the adaptive immune response. CIITA expression itself is tightly regulated by three cell type-specific promoters, pI, pIII, and by pIV, and can also be induced by IFN{gamma} in non-immune cells. While key regulatory elements have been identified within these promoters, knowledge of transcription factors regulating CIITA is incomplete. Here, we demonstrate that the telomere-binding protein and transcriptional activator ZBTB48 directly binds to both the critical activating elements within CIITA pIII and is essential for its gene expression. ZBTB48 establishes open chromatin at CIITA pIII upstream of activating H3K4me3 modifications both priming CIITA transcription for IFN{gamma}-induction and ensuring constitutive expression in primary murine B cells. Hence, ZBTB48 acts as a molecular on-off-switch for B-cell-specific CIITA expression.

immunology↗

bioRxiv2023

An isoform-resolution transcriptomic atlas of colorectal cancer from long-read single-cell sequencing

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. In recent years, short-read single-cell RNA sequencing (scRNA-seq) has been instrumental in deciphering tumor cell heterogeneities. However, these studies only enable gene-level expression quantification but neglect alterations in transcript structures, which arise from alternative end processing or splicing, and are frequently observed in cancer. In this study, we integrated short- and long-read scRNA-seq of CRC patient samples to build the first isoform-resolution CRC transcriptomic atlas. We identified 394 dysregulated transcript structures in tumor epithelial cells, including 299 resulting from various combinations of multiple splicing events. Secondly, we characterized genes and isoforms associated with epithelial lineages and subpopulations that exhibit distinct prognoses. Finally, we built an algorithm that integrated novel peptides derived from predicted ORFs of recurrent tumor-specific transcripts with mass spectrometry data and identified a panel of recurring neoepitopes that may aid the development of neoantigen-based cancer vaccines.

cancer biology↗

bioRxiv2023

ChIP-MS reveals the local chromatin composition by label-free quantitative proteomics

Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versatility of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.

biochemistry↗

bioRxiv2023

E4F1 and ZNF148 are transcriptional activators of the A57C and wildtype TERT promoter

Point mutations within the TERT promoter are the most recurrent somatic non-coding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at C146T and C124T and rarer at A57C, with the latter originally described as a familial case but subsequently shown also to occur somatically. All three mutations create de novo ETS (E-twenty-six specific) binding sites and result in the reactivation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we employed a systematic proteomics screen to identify transcription factors preferentially binding to the C146T, C124T and A57C mutations. While we confirmed binding of multiple ETS factors to the mutant C146T and C124T sequences, we identified E4F1 as an A57C-specific binder and ZNF148 as a TERT WT binder that is excluded from the TERT promoter by the C124T allele. Both proteins are activating transcription factors that bind specifically to the A57C and wildtype (at position 124) TERT promoter sequence in corresponding cell lines and upregulate TERT transcription and telomerase activity. Our work describes new regulators of TERT gene expression with possible roles in cancer.

cancer biology↗

bioRxiv2022

ZNF524 directly interacts with telomeric DNA and supports telomere integrity

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

cell biology↗

bioRxiv2021

Formation and recycling of an active epigenetic mark mediated by cell cycle-specific RNAs

The mechanisms by which epigenetic modifications are established in gene regulatory regions of active genes remain poorly understood. The data presented show that the establishment and recycling of a major epigenetic mark, the acetylated form of the replacement histone H2A.Z, is regulated by cell cycle-specific long noncoding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEARs (S Phase EArly RNAs), are induced in early S phase: their expression precedes that of the downstream genes on which they exert their regulatory action. SPEARs drive the modification and deposition of the acetylated form of histone H2A.Z by bringing together the replacement histone and the histone acetyl transferase TIP60. This widespread bimodal pathway constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, thereby providing a unifying explanation for the accuracy and persistence of epigenetic marks on chromatin.

genetics↗

bioRxiv2021

PLK1 inhibition selectively kills ARID1A deficient cells through uncoupling of oxygen consumption from ATP production

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2-M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knocked-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/ expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of a key subunit of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer. Statement of significanceCurrently, no predictive biomarkers have been identified for PLK1 inhibitors in cancer treatment. We show that ARID1A loss sensitizes cells to PLK1 inhibitors through a previously unrecognized vulnerability in mitochondrial oxygen metabolism.

cancer biology↗

bioRxiv2020

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Telomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

cell biology↗

bioRxiv2018

Programmed DNA elimination of germline development genes in songbirds

Genomes can vary within individual organisms. Programmed DNA elimination leads to dramatic changes in genome organisation during the germline-soma differentiation of ciliates1, lampreys2, nematodes3,4, and various other eukaryotes5. A particularly remarkable example of tissue-specific genome differentiation is the germline-restricted chromosome (GRC) in the zebra finch which is consistently absent from somatic cells6. Although the zebra finch is an important animal model system7, molecular evidence from its large GRC (>150 megabases) is limited to a short intergenic region8 and a single mRNA9. Here, we combined cytogenetic, genomic, transcriptomic, and proteomic evidence to resolve the evolutionary origin and functional significance of the GRC. First, by generating tissue-specific de-novo linked-read genome assemblies and re-sequencing two additional germline and soma samples, we found that the GRC contains at least 115 genes which are paralogous to single-copy genes on 18 autosomes and the Z chromosome. We detected an amplification of [≥]38 GRC-linked genes into high copy numbers (up to 308 copies) but, surprisingly, no enrichment of transposable elements on the GRC. Second, transcriptome and proteome data provided evidence for functional expression of GRC genes at the RNA and protein levels in testes and ovaries. Interestingly, the GRC is enriched for genes with highly expressed orthologs in chicken gonads and gene ontologies involved in female gonad development. Third, we detected evolutionary strata of GRC-linked genes. Developmental genes such as bicc1 and trim71 have resided on the GRC for tens of millions of years, whereas dozens have become GRC-linked very recently. The GRC is thus likely widespread in songbirds (half of all bird species) and its rapid evolution may have contributed to their diversification. Together, our results demonstrate a highly dynamic evolutionary history of the songbird GRC leading to dramatic germline-soma genome differences as a novel mechanism to minimise genetic conflict between germline and soma.

evolutionary biology↗