Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We uncovered the evolutionarily conserved roles of ATP-dependent SWI/SNF chromatin remodelling complexes in hormonal crosstalk in human and plants, and proposed utilization of Arabidopsis mutants with inactivated subunits of SWI/SNF CRCs for studying evolutionarily conserved and evolutionarily diversified regulatory processes.
• We discovered the occurrence of synthetic lethality between various mutations inactivating subunits of SWI/SNF CRCs.
• We recognized the dual function of SWI/SNF CRCs impairment in cancer development.
• We found that SWI/SNF CRCs are involved in an evolutionarily conserved manner in various regulatory processes including control of metabolism, DNA repair, RNA modifications

Opis badań

The SWI/SNF-type ATP-dependent chromatin remodelling complexes (CRCs) are types of machinery present in cell nuclei of all organisms. There are several classes of SWI/SNF CRCs which differ by their subunit composition. They are responsible for switching genes on and off in a proper moment, tissue or developmental stage. They achieve this due to the control of the access of transcription factors or other regulatory proteins to DNA present in the chromatin. Their function is frequently altered in human cancer cells due to mutations and/or alterations caused by other factors. Partial inactivation of SWI/SNF CRCs leads to the development of various syndromes in human (e.g. Coffin-Siris Syndrome, Nicolaides-Baraitser Syndrome, non-syndromic intellectual disability) and such severe developmental alterations in plants like dwarfism, sterility, abnormal root, leaf and flower development and sometimes lethality (1).

The study performed by various scientists, including our Group, indicated that inactivation of some subunits of various classes of SWI/SNF complexes i.e. central ATPase subunits, SWP73 non-core subunits (2), etc. leads to the occurrence of so-called synthetic lethality. This phenomenon has been observed for the first time in 1922 in fruit fly, where it has been shown that the combination of two viable mutations leads to the lethality. Interestingly, it has been recently proven that synthetic lethality may be used successfully to treat some types of cancer providing an attractive possibility to exploit results gathered by our Group.

In our study, we have shown that SWI/SNF CRCs play an important, conserved in evolution role in hormonal crosstalk by the maintenance of fine-tuning of hormone signalling pathways by precise control of expression of genes belonging to particular pathways (3, 4). Moreover, we proposed that Arabidopsis lines carrying T-DNA insertional mutations in genes encoding SWI/SNF subunits may serve as a perfect model for studying the evolutionarily conserved function of chromatin remodelling complexes in a direction ‘from plants to human’ (4).  Of note, we also designed and patented an innovative platform (based on Arabidopsis mutant line) for identification of new compounds with properties suitable for treatment such human metabolic disorders as type 2 diabetes, etc. As our Laboratory is in possession of a unique collection consisting of about 90000 Arabidopsis T-DNA insertional mutant lines, the study may be easily extended towards other evolutionarily conserved or diversified processes upon necessity.

Given the fact that about 20% of human cancers are characterized by the mutations in genes encoding subunits of SWI/SNF CRCs, we extensively studied the role of SWI/SNF impairment in various cancer types (5). We focused on a systematic assessment of the role of SWI/SNF CRCs function alteration or inactivation in several cancer types including clear cell renal cell carcinoma (6, 7) salivary advanced adenoid cystic carcinoma (8), bladder (9), prostate, breast and uterus cancers.

In the course of our study, we found that SWI/SNF CRCs are involved directly in the control of various important regulatory pathways in the cell i.e. by interactions with their key elements. Basing on our results we conclude that SWI/SNF CRCs may play an important role in the control of metabolic processes via interaction with e.g. TOR (Target of Rapamycin) and AMPK (AMP-activated Protein Kinase) master regulators, but also are involved the regulation of alternative splicing, alternative transcription start site (TSS) selection, RNA modification, DNA repair, ribonucleotide biosynthesis and protein modification. We found that SWI/SNF CRCs may play a vital role in the immune response, host-pathogen interactions and response to environmental cues (10). Last but not least we identified so far unrecognized mechanism of signal transduction involving membrane kinase receptors and SWI/SNF CRCs.

Although we know relatively much about the function of SWI/SNF complexes, there is complete lack of knowledge about that what happens just after the loss of SWI/SNF activity, because there are no good tools suitable to precise, inducible inactivation of their activity. In order to fulfil this gap, we are currently developing the technology based on VHH antibody which will allow us to precisely inactivate subunits of SWI/SNF CRCs in studied models. Furthermore, the developed by us VHHs may serve as precise tools for molecular biology.

At the beginning of 2020, when the SARS-CoV-2 outbreak appeared we decided to employ the techniques and methodologies available in the laboratory, and in collaboration with Maria Sklodowska-Curie National Research Institute of Oncology started the collaborative project focused on the construction of VHH and M13 bacteriophage-based SARS-CoV-2-inactivating compound.

Our plans mainly focus on the further investigation of SWI/SNF CRCs role in the main regulatory processes in studied model organisms. We are interested in a better understanding of the role of SWI/SNF CRCs and other chromatin-related types of machinery in cancer development and progression with special focus on the exploitation of our results and knowledge to propose/design innovative methods of cancer treatment. Given the possession of ready-to-use pipeline for designing and identification of VHH and/or VHH-M13 bacteriophage-based compounds against various pathogenic diseases, our additional long term plan is to use this approach and skills upon necessity in case of future outbreaks appearance.

• Bibliography

1. Sarnowski et al. Plant Cell. 2005. doi: 10.1105/tpc.105.031203
2. Sacharowski et al. Plant Cell. 2015. doi: 10.1105/tpc.15.00233.
3. Sarnowska et al. Plant Phys. 2013. doi: 10.1104/pp.113.223933.
4. Sarnowska et al. Trends Plant Sci. 2016. doi: 10.1016/j.tplants.2016.01.017.
5. Jancewicz et al. Epigenetics Chromatin. 2019. doi: 10.1186/s13072-019-0315-4.
6. Sarnowska et al. Am J Cancer Res. 2017.
7. Swiatek et al. IUBMB Life. 2020. doi: 10.1002/iub.2281.
8. Jagielska et al. J Cancer Res Clin Oncol. 2019. doi: 10.1007/s00432-018-2783-5.
9. Stachowiak et al. IUBMB Life. 2020. doi: 10.1002/iub.2254.
10. Gratkowska-Zmuda et al. Int J Mol Sci. 2020. doi: 10.3390/ijms21030762.

Metodologia

• We use state-of-the-art genetics and molecular biology techniques including classical genetics, reverse genetics, phenotyping, chromatin immunoprecipitation (ChIP-qPCR, ChIP-seq), Micrococcus nuclease protection assay (MNase-qPCR, MNase-seq), whole-genome bisulfite sequencing (WGBS), etc.
• We use innovative recombineering and turbo-recombineering methods enabling modification of large plant genomic DNA fragments.
• We perform M13 bacteriophage VHH library screens for identification of VHHs specifically recognizing target proteins.
• We are in possession of the collection of 90000 T-DNA insertional Arabidopsis mutants enabling rapid, PCR-based identification of mutant line with an inactivated gene of interest. This is the largest private Arabidopsis T-DNA collection all over the world.

Wybrane publikacje

• The SWI/SNF ATP-Dependent Chromatin Remodeling Complex in Arabidopsis Responds to Environmental Changes in Temperature-Dependent Manner. Gratkowska-Zmuda DM, Kubala S, Sarnowska E, Cwiek P, Oksinska P, Steciuk J, Rolicka AT, Zaborowska M, Bucior E, Maassen A, Franzen R, Koncz C, Sarnowski TJ. Int J Mol Sci. 2020. doi: 10.3390/ijms21030762.
• SWI/SNF chromatin remodeling complex and glucose metabolism are deregulated in advanced bladder cancer. Stachowiak M, Szymanski M, Ornoch A, Jancewicz I, Rusetska N, Chrzan A, Demkow T, Siedlecki JA, Sarnowski TJ*, Sarnowska E*. IUBMB Life. 2020. doi: 10.1002/iub.2254. *equal contribution
• Evaluation of the role of downregulation of SNF5/INI1 core subunit of SWI/SNF complex in clear cell renal cell carcinoma development. Sarnowska E, Szymanski M, Rusetska N, Ligaj M, Jancewicz I, Cwiek P, Skrodzka M, Leszczynski M, Szarkowska J, Chrzan A, Stachowiak M, Steciuk J, Maassen A, Galek L, Demkow T, Siedlecki JA, Sarnowski TJ. Am J Cancer Res. 2017.
• The Role of SWI/SNF Chromatin Remodeling Complexes in Hormone Crosstalk. Sarnowska E, Gratkowska DM, Sacharowski SP, Cwiek P, Tohge T, Fernie AR, Siedlecki JA, Koncz C, Sarnowski TJ. Trends Plant Sci. 2016. doi: 10.1016/j.tplants.2016.01.017.
• SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development. Sacharowski SP, Gratkowska DM, Sarnowska EA, Kondrak P, Jancewicz I, Porri A, Bucior E, Rolicka AT, Franzen R, Kowalczyk J, Pawlikowska K, Huettel B, Torti S, Schmelzer E, Coupland G, Jerzmanowski A, Koncz C, Sarnowski TJ. Plant Cell. 2015. doi: 10.1105/tpc.15.00233.

Współpraca

• Csaba Koncz, Max-Planck Institute for Plant Breeding Research, Cologne, Germany
• George Coupland, Max-Planck Institute for Plant Breeding Research, Cologne, Germany
• Mien-Chie Hung, University of Texas MD Anderson Cancer Center, Houston, USA
• Louis Staudt, Lymphoid Malignancies Branch, National Cancer Institute, NIH, Bethesda, USA
• Alisdair Fernie, Max-Planck for Plant Molecular Physiology, Potsdam-Golm, Germany
• Aviah Zilberstein, University of Tel Aviv, Tel Aviv, Israel
• Richard Bayliss, University of Leeds, UK
• Martin Dreyling, LMU, Munich, Germany
• Seth Davis, Department of Biology, University of York, York, UK
• Apinunt Udomkit, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
• Laszlo Szabados PhD, Biological Research Centre HAS, Szeged, Hungary
• Zoltan Magyar PhD, Biological Research Centre HAS, Szeged, Hungary
• Franziska Turck PhD, Max-Planck Institute for Plant Breeding Research, Cologne, Germany
• Bruno Huettel PhD, Genome-Centre, Max-Planck Institute for Plant Breeding Research, Cologne, Germany
• Alfonso Urbanucci, PhD, Oslo University Hospital, Norway
• Janusz Siedlecki, Elżbieta Sarnowska DSc, PhD, prof. NIO, Sergiusz Markowicz DSc, PhD, prof. NIO, Department of Molecular and Translational Oncology, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
• Zygmunt Pojda, Department of Regenerative Medicine, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
• Beata Jagielska PhD, Department of Oncology and Internal Medicine, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
• Piotr Sobiczewski DSc, PhD, prof. NIO, Department of Gynecologic Oncology, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
• Jan Walewski, Department of Lymphoid Malignancies Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
• Tomasz Rutkowski DSc, PhD, prof. NIO, I Radiation and Clinical Oncology Department Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice, Poland
• Radoslaw Zagożdżon DSc, PhD, prof. WUM, Department of Clinical Immunology, Department of Immunology, Transplantology, and Internal Diseases, Medical University of Warsaw, Poland
• Michał Szymański PhD, Department of Urology and Urological Oncology, Central Clinical Hospital of Ministry of The Interior and Administration in Warsaw, Warsaw, Poland
• Tomasz Pawłowski, Institute of Dendrology, Polish Academy of Sciences, Kórnik, Poland

Nagrody i wyróżnienia

• Szymon Kubala. Prize for the best original research article in the 2017-2019 period. 2019. Polish Society of Plant Experimental Biology, Poland
• Tomasz Sarnowski. Research Project Award. 2018. Polpharma Scientific Foundation, Poland
• Tomasz Sarnowski, Paweł Ćwiek, Jarosław Steciuk, Anna Maassen. Award for the best scientific publication. 2018. Polish Society of Clinical Oncology, Poland.
• Sebastian Sacharowski. START Fellowship. 2016. Foundation for Polish Science, Poland.
• Szymon Kubala. Fellowship for Outstanding Young Scientists. 2016. Ministry of Science and Higher Education, Poland.
• Paulina Oksińska. Diamond Grant. 2015. Ministry of Science and Higher Education, Poland.
• Paweł Ćwiek. Diamond Grant. 2013. Ministry of Science and Higher Education, Poland.
• Dominika Gratkowska-Żmuda. Diamond Grant. Ministry of Science and Higher Education, Poland.
• Tomasz Sarnowski. Marie Sklodowska-Curie Actions: European Reintegration Grant 2010. Research Executive Agency (REA) of the European Commission, Belgium.