Abstract
The Polycomb group (PcG) and Trithorax group (TrxG) proteins are key epigenetic regulators controlling the silenced and active states of genes in multicellular organisms, respectively. In Drosophila, PcG/TrxG proteins are recruited to the chromatin via binding to specific DNA sequences termed polycomb response elements (PREs). While precise mechanisms of the PcG/TrxG protein recruitment remain unknown, the important role is suggested to belong to sequence-specific DNA-binding factors. At the same time, it was demonstrated that the PRE DNA-binding proteins are not exclusively localized to PREs but can bind other DNA regulatory elements, including enhancers, promoters, and boundaries. To gain an insight into the PRE DNA-binding protein regulatory network, here, using ChIP-seq and immuno-affinity purification coupled to the high-throughput mass spectrometry, we searched for differences in abundance of the Combgap, Zeste, Psq, and Adf1 PRE DNA-binding proteins. While there were no conspicuous differences in co-localization of these proteins with other functional transcription factors, we show that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. We further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap–Adf1, Psq–Dsp1, and Pho–Spps can interact in the yeast two-hybrid assay. Overall, our data suggest that Combgap, Psq, Zeste, and Adf1 are associated with the protein complexes implicated in different regulatory activities and indicate their potential multifunctional role in the regulation of transcription.
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Data availability
All relevant data are within the paper and its Supporting Information. The ChIP-seq data generated for this work are accessible through the GEO Series accession number GSE200213. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [96] partner repository with the dataset identifier PXD029459 and https://doi.org/10.6019/PXD029459.
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Acknowledgements
Experiments of LC–MS fingerprinting were carried out using the equipment provided by the Institute of Bioorganic Chemistry core facility (CKP IBCH). We thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, IGB RAS for the use of their equipment. We thank Judith Kassis for providing the CgA22 fly line.
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The work was supported by the Russian Science Foundation (RSF) Grant no.18-74-10091.
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Conception and design of the project: DC, PG, ME. Plasmid cloning—DC, NEV, LVF, DL, AG, MYM, VM, ME. Antigen expression, antibodies production, purification and verification—DC, NEV, LVF, DL, MYM, ME. ChIP-seqs and X-ChIP—MYM, NEV, ME. Purification and analysis of protein complexes—ME, LVF, DC. LC–MS—RHZ. Interactome data analysis—DC and ME. IP/Western blotting—NEV, MYM, LVF, AG, ME. Y2H assay—DC, LVF, DL, AG. Data visualization—DC, NEV, MYM, ME. The initial draft of this manuscript was written by—DC and ME, reviewed and edited by—DC, NEV, MYM, VM, PG, RHZ, ME. All authors read and approved the final manuscript.
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Animal handling for the antibody production was carried out strictly according to the procedures outlined in the NIH (USA) Guide for the Care and Use of Laboratory Animals. The protocols used were approved by the Committee on Bioethics of the Institute of Gene Biology, Russian Academy of Sciences. All procedures were performed under the supervision of a licensed veterinarian, under conditions that minimize pain and distress. Rabbits were purchased from a licensed specialized nursery, Manihino. Soviet chinchilla rabbits used in the study are not endangered or protected. Only healthy rabbits, certified by a licensed veterinarian were used. The rabbits were individually housed in standard size, stainless steel rabbit cages, and provided an ad libitum access to alfalfa hay, commercial rabbit food pellets, and water. The appetite and behavior of each rabbit was monitored daily by a licensed veterinarian. Body weight and temperature of each rabbit were evaluated prior to and daily following the immunization. No animals became ill or died at any time prior to the experimental endpoint. At the end of the study period all rabbits were euthanized by intravenous injection of barbiturate anesthetics.
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Chetverina, D., Vorobyeva, N.E., Mazina, M.Y. et al. Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap-, Zeste-, Psq-, and Adf1-associated proteins. Cell. Mol. Life Sci. 79, 353 (2022). https://doi.org/10.1007/s00018-022-04383-2
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DOI: https://doi.org/10.1007/s00018-022-04383-2