Lisa-Marie Appel

Max Perutz Labs

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Dea Slade

Max Perutz Labs

Vedran Franke

Max Delbrück Center

Altuna Akalin

Max Delbrück Center

Johannes Benedum

Medical University of Vienna

Kristina Djinović‐Carugo

University of Vienna

Melania Bruno

Eunice Kennedy Shriver National Institute of Child Health and Human Development

Irina Grishkovskaya

Research Institute of Molecular Pathology

Lena Walch

Vienna Biocenter

Martin Leeb

Max Perutz Labs

Anna Vlasova

Research Institute of Molecular Pathology

Cooperative Institutions

Vienna Biocenter

Medical University of Vienna

Max Perutz Labs

University of Vienna

Comprehensive Cancer Center Vienna

Research Institute of Molecular Pathology

MedAustron

Institute of Molecular Biotechnology

Austrian Academy of Sciences

Central European Institute of Technology

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Cytosolic nucleic acid sensors and interferon beta-1 activation drive radiation-induced anti-tumour immune effects in human pancreatic cancer cells

Frontiers in Immunology (2024)

Sylvia Gruber Ava Kleinwächter K. Popova Alexandra Kneringer Lisa-Marie Appel

Introduction Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths worldwide with limited treatment options due to extensive radiation and chemotherapy resistance. Monotherapy with immune checkpoint blockade showed no survival benefit. A combination of immunomodulation and radiotherapy may offer new treatment strategies, as demonstrated for non-small cell lung cancer. Radiation-induced anti-tumour immunity is mediated through cytosolic nucleic acid sensing pathways that drive the expression of interferon beta-1 (IFNB1) and proinflammatory cytokines. Methods Human PDAC cell lines (PANC-1, MIA PaCa-2, BxPC-3) were treated with X-rays and protons. Immunogenic cell death was measured based on HMGB1 release. Cytosolic dsDNA and dsRNA were analysed by immunofluorescence microscopy. Cell cycle progression, MHC-I and PD-L1 expression were determined by flow cytometry. Galectin-1 and IFNB1 were measured by ELISA. The expression levels and the phosphorylation status of the cGAS/STING and RIG-I/MAVS signalling pathways were analysed by western blotting, the expression of IFNB1 and proinflammatory cytokines was determined by RT-qPCR and genome-wide by RNA-seq. CRISPR-Cas9 knock-outs and inhibitors were used to elucidate the relevance of STING, MAVS and NF-κB for radiation-induced IFNB1 activation. Results We demonstrate that a clinically relevant X-ray hypofractionation regimen (3x8 Gy) induces immunogenic cell death and activates IFNB1 and proinflammatory cytokines. Fractionated radiation induces G2/M arrest and accumulation of cytosolic DNA in PDAC cells, which partly originates from mitochondria. RNA-seq analysis shows a global upregulation of type I interferon response and NF-κB signalling in PDAC cells following 3x8 Gy. Radiation-induced immunogenic response is regulated by STING, MAVS and NF-κB. In addition to immunostimulation, radiation also induces immunosuppressive galectin-1. No significant changes in MHC-I or PD-L1 expression were observed. Moreover, PDAC cell lines show similar radiation-induced immune effects when exposed to single-dose protons or photons. Conclusion Our findings provide a rationale for combinatorial radiation-immunomodulatory treatment approaches in PDAC using conventional photon-based or proton beam radiotherapy.

Proinflammatory cytokine

TLR3

10.3389/fimmu.2024.1286942

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The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation

Nature Communications (2023)

Johannes Benedum Vedran Franke Lisa-Marie Appel Lena Walch Melania Bruno

Abstract Transcription is regulated by a multitude of activators and repressors, which bind to the RNA polymerase II (Pol II) machinery and modulate its progression. Death-inducer obliterator 3 (DIDO3) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here, we show that DIDO3 and PHF3 form a complex that bridges the Pol II elongation machinery with chromatin and RNA processing factors and tethers Pol II in a phase-separated microenvironment. Their SPOC domains and C-terminal intrinsically disordered regions are critical for transcription regulation. PHF3 and DIDO exert cooperative and antagonistic effects on the expression of neuronal genes and are both essential for neuronal differentiation. In the absence of PHF3, DIDO3 is upregulated as a compensatory mechanism. In addition to shared gene targets, DIDO specifically regulates genes required for lipid metabolism. Collectively, our work reveals multiple layers of gene expression regulation by the DIDO3 and PHF3 paralogues, which have specific, co-regulatory and redundant functions in transcription.

RNA polymerase II

Transcription

10.1038/s41467-023-43724-y

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Citations (3)

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

Nature Communications (2021)

Lisa-Marie Appel Vedran Franke Melania Bruno Irina Grishkovskaya Aistė Kasiliauskaitė

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

CTD

10.1038/s41467-021-26360-2

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Citations (40)

Brillouin light scattering anisotropy microscopy for imaging the viscoelastic anisotropy in living cells

Nature Photonics (2024)

Hamid Keshmiri Domagoj Cikes Marketa Samalova Lukas Schindler Lisa-Marie Appel

Brillouin zone

Brillouin Spectroscopy

10.1038/s41566-023-01368-w

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Citations (11)

The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators

Nature Communications (2023)

Lisa-Marie Appel Vedran Franke Johannes Benedum Irina Grishkovskaya Xué Strobl

Abstract The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3’end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m 6 A, the most abundant RNA modification. RBM15 positively regulates m 6 A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

RNA polymerase II

Transcription

CTD

Phosphoserine

WW domain

10.1038/s41467-023-35853-1

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Citations (16)

PHF3 Regulates Neuronal Gene Expression Through the New Pol II CTD Reader Domain SPOC

SSRN Electronic Journal (2020)

Lisa-Marie Appel Vedran Franke Melania Bruno Irina Grishkovskaya Aistė Kasiliauskaitė

T he C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a new CTD-binding factor that negatively regulates transcription and mRNA stability. The PHF3 SPOC domain preferentially binds to CTD repeats phosphorylated on Serine-2 and PHF3 tracks with Pol II across the length of genes. PHF3 competes with TFIIS for Pol II binding through its TFIIS-like domain (TLD), thus inhibiting TFIIS-mediated rescue of backtracked Pol II. PHF3 knock-out or PHF3 SPOC deletion in human cells result in gene upregulation and a global increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 is a prominent effector of neuronal gene regulation at the interface of transcription elongation and mRNA decay.

CTD

10.2139/ssrn.3543843

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Imaging the microscopic viscoelastic anisotropy in living cells

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Hamid Keshmiri Domagoj Cikes Marketa Samalova Lukas Schindler Lisa-Marie Appel

Abstract Maintaining and modulating the mechanical anisotropy is essential for biological processes. How this is achieved on the microscopic scale in living soft matter is however not always clear. Here we introduce Brillouin Light Scattering Anisotropy Microscopy (BLAM) for mapping the high-frequency viscoelastic anisotropy inside living cells. Following proof-of-principle experiments on muscle myofibers, we apply this to study two fundamental biological processes. In plant cell walls we show how a phase-transition driven switch between anisotropic-isotropic wall properties may lead to asymmetric growth. In mammalian cell nuclei we uncover a spatio-temporally oscillating elastic anisotropy correlated to chromatin condensation, with long range orientational correlations that may provide a dynamic framework for coordinating intra-nuclear processes. Our results highlight the direct and indirect role the high-frequency mechanics can play in providing dynamic structure that lead to the regulation of diverse fundamental processes in biological systems, and offer a means for studying these. BLAM should find diverse biomedical and material characterization applications.

Active matter

Living cell

Brillouin zone

Living matter

10.1101/2023.05.28.542585

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Citations (1)

The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Lisa-Marie Appel Irina Grishkovskaya Johannes Benedum Vedran Franke Anton A. Polyansky

Abstract The heptarepeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulation factors as well as RNA capping, splicing and 3’end processing factors. The SPOC domain of PHF3 was recently identified as a new CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP (SPEN) SPOC-CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with the m6A writer and reader proteins. Our findings establish the SPOC domain as a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

Phosphoserine

CTD

Transcription

RNA polymerase II

WW domain

10.1101/2022.02.26.482114

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SPOC domain proteins in health and disease

Genes & Development (2023)

Lisa-Marie Appel Johannes Benedum Magdalena Engl Sebastian Platzer Alexander Schleiffer

Since it was first described >20 yr ago, the SPOC domain (Spen paralog and ortholog C-terminal domain) has been identified in many proteins all across eukaryotic species. SPOC-containing proteins regulate gene expression on various levels ranging from transcription to RNA processing, modification, export, and stability, as well as X-chromosome inactivation. Their manifold roles in controlling transcriptional output implicate them in a plethora of developmental processes, and their misregulation is often associated with cancer. Here, we provide an overview of the biophysical properties of the SPOC domain and its interaction with phosphorylated binding partners, the phylogenetic origin of SPOC domain proteins, the diverse functions of mammalian SPOC proteins and their homologs, the mechanisms by which they regulate differentiation and development, and their roles in cancer.

10.1101/gad.350314.122

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Citations (7)

PHF3 regulates neuronal gene expression through the new Pol II CTD reader domain SPOC

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Lisa-Marie Appel Vedran Franke Melania Bruno Irina Grishkovskaya Aistė Kasiliauskaitė

SUMMARY The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a new CTD-binding factor that negatively regulates transcription and mRNA stability. The PHF3 SPOC domain preferentially binds to CTD repeats phosphorylated on Serine-2 and PHF3 tracks with Pol II across the length of genes. PHF3 competes with TFIIS for Pol II binding through its TFIIS-like domain (TLD), thus inhibiting TFIIS-mediated rescue of backtracked Pol II. PHF3 knock-out or PHF3 SPOC deletion in human cells result in gene upregulation and a global increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 is a prominent effector of neuronal gene regulation at the interface of transcription elongation and mRNA decay.

RNA polymerase II

Transcription

CTD

10.1101/2020.02.11.943159

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Citations (6)