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

ABSTRACT

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/TGFß) 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; TGFß Transforming Growth Factor ß; TUNEL TdT-mediated dUTP Nick End Labelling; Wg Wingless; ZNC Zone of Non-Proliferating Cells.