Dennis Kappei

Telomere homeostasis is a tightly regulated and dynamic process sustained by a network of proteins that organize telomeric chromatin and safeguard chromosome stability. In ALT-positive cells, telomere maintenance relies on recombination-based mechanisms and is modulated by local chromatin states and epigenetic regulation. Here, we show that the telomeric binding protein HOT1 (HMBOX1) requires dimerization – depending both on interaction with the dimerization-mediators DYNLL1/2 and its intrinsic HNFp1 domain – for its stable association with telomeres in vivo . Loss of HOT1 leads to an epigenetic switch from H3K9acK14ac to H3K9me3 at telomeres accompanied by attenuated ALT activity as illustrated by reduced ALT recombination by-products (C-circles) and a decrease in telomere dysfunction-induced foci (TIFs). We further show via binding assays and cross-linking mass spectrometry (XL-MS) analyses that HOT1 directly interacts with telomeric nucleosomes, providing a molecular basis for its involvement in telomeric chromatin organization. Collectively, these findings suggest that HOT1 functions as a homodimer to promote a telomeric chromatin state that is highly permissive for ALT recombination.

BACKGROUND: Heart failure is a leading cause of morbidity and mortality worldwide, particularly among the growing elderly population. In degenerative aging and autoimmune diseases, the cytoplasmic leak of mitochondrial DNA, resulting from mitochondrial cristae compromise, triggers persistent low-grade cellular inflammation through activation of the cGAS (cyclic GMP [guanosine monophosphate]–AMP [adenosine monophosphate] synthase)–STING (stimulator of interferon genes) pathway and the IFN-I (type I interferon) response. However, how and whether mitochondrial architectural components and cardiomyocyte inflammation drive cardiac aging and failure are not yet well understood. METHODS: We investigated the function of STMP1 (short transmembrane mitochondrial protein 1), a 47–amino acid nuclear-encoded mitochondrial-localized peptide featuring a distinctive GxxxGxxxG glycine zipper domain. A mouse with cardiomyocyte-specific knockout of Stmp1 ( Stmp1 -KO) was generated to investigate its role in cardiac function. We profiled the transcriptome, proteome, and metabolome of Stmp1 -KO hearts to determine its functional mechanism of action. Electron microscopy was used to assess the impact of STMP1 depletion and functional rescue after adeno-associated virus 9–mediated gene restoration in the Stmp1 -KO mouse. RESULTS: STMP1 is downregulated specifically in cardiomyocytes, and not other cardiac cell types, in aged mice and humans. Genetic loss of Stmp1 in cardiomyocytes resulted in heart failure in vivo. STMP1 interacts with components of the cristae organizing complexes MICOS (mitochondrial contact site and cristae organizing complex) and SAM (sorting and assembly machinery). Consequent to Stmp1 loss, mitochondrial cristae were destabilized, mitochondrial DNA was mislocalized to the cytosol, and the cGAS–STING pathway was activated, with ensuing cellular inflammation and cardiomyocyte cell death. Restoration of wild-type Stmp1 or STING inhibition significantly rescued cardiac function in vivo. CONCLUSION: Our work reveals a mechanism connecting the micropeptide STMP1 to mitochondrial cristae architecture and cardiomyocyte cellular inflammation, both of which are present as potential drivers of heart failure and cardiac aging.

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 combine ChIP with label-free quantitative mass spectrometry (qChIP-MS) to interrogate local chromatin compositions. We demonstrate the versatility of our approach at telomeres, with transcription factors, in tissue and by dCas9-mediated locus-specific enrichment.

Hepatocellular carcinoma is a devastating malignancy with numerous therapeutic targets to guide treatment strategies against the disease. However, given the limited efficacy of current frontline targeted therapies in prolonging the survival for HCC patients both as single agents and in combination, evaluating the potential of epigenome remodelling as a therapeutic target opens unexplored avenues for the clinical management of HCC. In this study, we identified epigenetic vulnerabilities to expand the repertoire of therapeutic strategies for HCC patients. To identify epigenetic regulators essential in HCC, we integrated the functional responses of six HCC cell lines to genetic perturbation of epigenetic regulators using esiRNA with existing data from publicly available databases. Correlation between phenotypic responses of HCC cells to large-scale genetic knockdown of epigenetic regulators and publicly available datasets narrowed down the pool of epigenetic vulnerabilities in HCC to two prospective epigenetic oncogenes (SUPT7L and SMARCC1) and one prospective epigenetic tumour suppressor (PHF2). Subsequently, PHF2 loss-of-function studies in HCC cells were performed through functional, molecular and proteomic analyses. Deeper investigations into PHF2 further established its functional role in mitigating cancer cell growth in vitro. Molecular and proteomic analyses in PHF2-deficient cells further suggested that PHF2 functionally suppresses cancer growth in part through the regulation of the cytoprotective protein, SRXN1. Further characterisation of PHF2-deficient cells were suggestive of independence from the Keap1-Nrf2 pathway. Collectively, our study suggests that PHF2 acts as a candidate epigenetic tumour suppressor in HCC patients through the downregulation of SRXN1, potentially independent of Nrf2.

Multiple myeloma (MM) is the second most common hematological malignancy that displays diverse genetic heterogeneity leading to treatment resistance. Recurrent mutations causing hyperactivation of the non-canonical NF-ĸB pathway are highly prevalent in relapsed, refractory MM patients, but the precise mechanisms driving chemoresistance are poorly understood. Here, we identify a long non-coding RNA termed PLUM, that is overexpressed in NF-ĸB mutant high-risk MM subtypes and patients who are refractory to VRd treatment regimen. Mechanistically, PLUM interacts with Polycomb Repressive Complex 2 to regulate its stability and histone methyltransferase activity, modulating the expression of tumor suppressor genes, FOXO3 and ZFP36, to activate the unfolded protein response (UPR). Importantly, disruption of PLUM-EZH2 interaction using steric antisense oligonucleotides re-sensitizes myeloma cells to drug treatment in vivo, correlating with the loss of PRC2 stability and H3K27 trimethylation activity. These findings indicate that PLUM facilitates formation of PRC2 complex and enhances EZH2 activity, modulating the myeloma epigenome to mediate chemoresistance. Hence, targeting PLUM-EZH2 interactions may represent a clinically potent strategy for the treatment of relapsed, refractory MM.

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 CK1α as IQGAP3-interacting proteins. Our functional studies demonstrated that overexpression of IQGAP3 increases β-catenin levels, while IQGAP3 depletion reduces β-catenin levels in gastric cancer cells. Mechanistically, IQGAP3 disrupts Axin1-CK1α interaction, thereby inhibiting β-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/β-catenin signaling through interactions with Axin1 and CK1α. These findings identify IQGAP3 as a novel mediator of β-catenin stabilization and underscore its potential as a target for cancer therapy.

DNA methylation has fundamental implications for vertebrate genome evolution by influencing the mutational landscape, particularly at CpG dinucleotides. Methylation‐induced mutations drive a genome‐wide depletion of CpG sites, creating a dinucleotide composition bias across the genome. Examination of the standard genetic code reveals CpG to be the only facultative dinucleotide; it is however unclear what specific implications CpG bias has on protein coding DNA. Here, we use theoretical considerations of the genetic code combined with empirical genome‐wide analyses in six vertebrate species—human, mouse, chicken, great tit, frog, and stickleback—to investigate how CpG content is shaped and maintained in protein‐coding genes. We show that protein‐coding sequences consistently exhibit significantly higher CpG content than noncoding regions and demonstrate that CpG sites are enriched in genes involved in regulatory functions and stress responses, suggesting selective maintenance of CpG content in specific loci. These findings have important implications for evolutionary applications in both natural and managed populations: CpG content could serve as a genetic marker for assessing adaptive potential, while the identification of CpG‐free codons provides a framework for genome optimization in breeding and synthetic biology. Our results underscore the intricate interplay between mutational biases, selection, and epigenetic regulation, offering new insights into how vertebrate genomes evolve under varying ecological and selective pressures.

DNA damage responses primarily coordinate intracellular processes, but their effects on the cell surface remain poorly understood. We uncovered a DNA damage response at the cell surface that regulates complement dependent cytotoxicity (CDC), a key mechanism of antibody-mediated tumor cell killing. We found that several mCRPs, such as CD46, CD55, and CD59, are upregulated in diffuse large B-cell lymphoma (DLBCL) cells following chemotherapy. These mCRPs protect cells from CDC, including CDC triggered by anti-CD20 immunotherapy. Chemotherapy pre-treatment reduced the efficacy of rituximab-induced CDC, with CD59 exerting a dominant function. A high-throughput kinase screen revealed that Chk1 inhibition restored CDC sensitivity. Mechanistically, chemotherapy-induced CD59 expression was Chk1-dependent and regulated at an Sp1-driven promoter site. Inhibition of Sp1 with Mithramycin A abrogated chemotherapy-induced CD59 transcription. Co-immunoprecipitation mass spectrometry revealed that DNA damage releases transcriptional repressors from Sp1, an effect reversed by Chk1 inhibition. These findings define a Chk1-Sp1-CD59 signaling axis linking genotoxic stress to complement modulation. Our study reveals potential antagonism between chemotherapy and antibody-mediated CDC and highlights consideration in sequencing combination therapies and a therapeutic potential of Chk1 inhibitors in maintaining CDC efficacy during chemoimmunotherapy. Short Summary Rituximab-based chemoimmunotherapy is the first-line treatment for DLBCL. Our study reveals that chemotherapy induces resistance to rituximab-mediated cell killing. We identify this resistance as driven by a Chk1-Sp1-CD59 signaling axis and provide a therapeutic rationale for targeting this pathway to enhance chemoimmunotherapy efficacy.

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 TARZN’s 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.

The class-II transactivator (CIITA) is the master regulator of MHC class-II gene expression and hence the adaptive immune response. Three cell type-specific promoters (pI, pIII, and pIV) are involved in the regulation of CIITA expression, which can be induced by IFN-γ in non-immune cells. While key regulatory elements have been identified within these promoters, our understanding of the transcription factors regulating CIITA expression is incomplete. Here, we demonstrate that the telomere-binding protein and transcriptional activator ZBTB48 directly binds to both critical activating elements within the B-cell-specific promoter CIITA pIII. ZBTB48 knockout impedes the CIITA/MHC-II expression program induced in non-APC cells by IFN-γ, and loss of ZBTB48 in mice silences MHC-II expression in pro-B and immature B cells. Transcriptional regulation of CIITA by ZBTB48 is enabled by ZBTB48-dependent chromatin opening at CIITA pIII upstream of activating H3K4me3 marks. We conclude that ZBTB48 primes CIITA pIII by acting as a molecular on-off-switch for B-cell-specific CIITA expression.