The SWI/SNF protein BAF60b is ubiquitinated through a signalling process involving Rac GTPase and the RING finger protein Unkempt

摘要

The SWI/SNF chromatin remodelling complexes are important regulators of transcription; they consist of large multisubunit assemblies containing either Brm or Brg1 as the catalytic ATPase subunit and a variable subset of approximately 10 Brg/Brm-associated factors (BAF). Among these factors, BAF60 proteins (BAF60a, BAF60b or BAF60c), which are found in most complexes, are thought to bridge interactions between transcription factors and SWI/SNF complexes. We report here on a Rac-dependent process leading to BAF60b ubiquitination. Using two-hybrid cloning procedures, we identified a mammalian RING finger protein homologous to drosophila Unkempt as a new partner of the activated form of RacGTPases and demonstrated that mammalian Unkempt specifically binds to BAF60b and promotes its ubiquitination in a Rac1-dependent manner. Immunofluorescence studies demonstrated that Unkempt is primarily localized in the cytoplasmic compartment, but has the ability to shuttle between the nucleus and the cytoplasm, suggesting that the Rac- and Unkempt-dependent process leading to BAF60b ubiquitination takes place in the nuclear compartment. Ubiquitinated forms of BAF60b were found to accumulate upon treatment with the proteasome inhibitor MG132, indicating that BAF60b ubiquitination is of the degradative type and could regulate the level of BAF60b in SWI/SNF complexes. Our observations support the new idea of a direct connection between Rac signalling and chromatin remodelling. Structured digital abstract MINT-7543606: Rac1 (uniprotkb: P63000) physically interacts ( MI:0915) with Unkempt (uniprotkb: B1GXI8) by two hybrid ( MI:0018) MINT-7543198: Unkempt (uniprotkb: B1GXI8) physically interacts ( MI:0915) with Rac1 (uniprotkb: P63000) by pull down ( MI:0096) MINT-7543251: Unkempt (uniprotkb: B1GXI8) physically interacts ( MI:0915) with BAF60b (uniprotkb: B4DV56) by pull down ( MI:0096) MINT-7543745: Unkempt (uniprotkb: B1GXI8) physically interacts ( MI:0915) with BAF60b (uniprotkb: B4DV56) by two hybrid ( MI:0018) MINT-7543182: Ubiquitin (uniprotkb: P61864) physically interacts ( MI:0915) with Unkempt (uniprotkb: B1GXI8) by pull down ( MI:0096) MINT-7543805: Ubiquitin (uniprotkb: P61864) physically interacts ( MI:0915) with Unkempt (uniprotkb: Q6RUT6) by pull down ( MI:0096) MINT-7543760: Ubiquitin (uniprotkb: P61864) physically interacts ( MI:0915) with BAF60b (uniprotkb: B4DV56) by pull down ( MI:0096)