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Phosphorylation of Drosophila Brahma on CDK-phosphorylation sites is important for cell cycle regulation and differentiation

journal contribution
posted on 2022-03-25, 03:20 authored by Siti Nur Ain Roesley, John La-MarcaJohn La-Marca, Andrew Deans, Lisa Mckenzie, Randy Suryadinata, Peter BurkePeter Burke, Marta Portela-EstebanMarta Portela-Esteban, Hongyan Wang, Ora Bernard, Borislav SarcevicBorislav Sarcevic, Helena RichardsonHelena Richardson
The SWI/SNF ATP-dependent chromatin-remodeling complex is an important evolutionarily conserved regulator of cell cycle progression. It associates with the Retinoblastoma (pRb)/HDAC/E2F/DP transcription complex to modulate cell cycle-dependent gene expression. The key catalytic component of the SWI/SNF complex in mammals is the ATPase subunit, Brahma (BRM) or BRG1. BRG1 was previously shown to be phosphorylated by the G1-S phase cell cycle regulatory kinase Cyclin E/CDK2 in vitro, which was associated with the bypass of G1 arrest conferred by BRG1 expression. However, it is unknown whether direct Cyclin E/CDK2-mediated phosphorylation of BRM/BRG1 is important for G1-S phase cell cycle progression and proliferation in vivo. Herein, we demonstrate for the first time the importance of CDK-mediated phosphorylation of Brm in cell proliferation and differentiation in vivo using the Drosophila melanogaster model organism. Expression of a CDK-site phospho-mimic mutant of Brm, brm-ASP (all the potential CDK sites are mutated from Ser/Thr to Asp), which acts genetically as a brm loss-of-function allele, dominantly accelerates progression into the S phase, and bypasses a Retinoblastoma-induced developmental G1 phase arrest in the wing epithelium. Conversely, expression of a CDK-site phospho-blocking mutation of Brm, brm-ALA, acts genetically as a brm gain-of-function mutation, and in a Brm complex compromised background reduces S phase cells. Expression of the brm phospho-mutants also affected differentiation and Decapentaplegic (BMP/TGFβ) signaling in the wing epithelium. Altogether our results show that CDK-mediated phosphorylation of Brm is important in G1-S phase regulation and differentiation in vivo. Abbreviations: A-P: Anterior-Posterior; BAF: BRG1-associated factor; BMP: Bone Morphogenetic Protein; Brg1: Brahma-Related Gene 1; Brm: Brahma; BSA: Bovine Serum Albumin; CDK: Cyclin dependent kinase dpp: decapentaplegic; EdU: 5-Ethynyl 2'-DeoxyUridine; EGFR: Epidermal Growth Factor Receptor; en: engrailed; GFP: Green Fluorescent Protein; GST: Glutathione-S-Transferase; HDAC: Histone DeACetylase; JNK: c-Jun N-terminal Kinase; Mad: Mothers Against Dpp; MAPK: Mitogen Activated Protein Kinase; MB:: Myelin Basic Protein; nub: nubbin; pH3: phosphorylated Histone H3; PBS: Phosphate Buffered Saline; PBT: PBS Triton; PFA: ParaFormAldehydep; Rb: Retinoblastoma protein; PCV: Posterior Cross-Vein; Snr1: Snf5-Related 1; SWI/SNF: SWitch/Sucrose Non-Fermentable; TGFβ: Transforming Growth Factor β; TUNEL: TdT-mediated dUTP Nick End Labelling; Wg: Wingless; ZNC: Zone of Non-Proliferating Cells.


This work and BS was supported by an Australian Research Council (ARC) Discovery grant [DP0771366] and an National Health and Medical Research Council (NHMRC) Project grant [APP1011320] to BS and HER, and by funds from St Vincent’s Institute of Medical Research, the Peter MacCallum Cancer Centre and La Trobe University. SNAR was supported by Melbourne International Fee Remission and Melbourne International Research Scholarships from the University of Melbourne. JELM was supported by an ARC Discovery grant to HER [DP 170102549]. AJD is a Victorian Cancer Agency fellow. Support was provided by the Victorian Government’s OIS Program. LM, PB and RS were supported by the ARC Discovery grant [DP0771366] and/or the NHMRC Project grant [APP1011320]. MP was supported by a Cancer Council Victoria grant to HER [APP1041817]. HW was supported by Singapore National Medical Research Council [NMRC/CBRG/0082/2015]. HER was supported by an National Health and Medical Research Council (NHMRC) Fellowship level B [APP1020056], and funds from the La Trobe Institute of Molecular Science and La Trobe University.


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Cell Cycle






20p. (p. 1559-1578)


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