Saturday, April 20, 2024
4/20/2024
Abstract Although many studies have demonstrated correlations between cytoskeletal dynamics, genome organization, and gene expression, the underlying mechanisms linking them remain unclear. Recently, we discovered crosstalk between the Wiskott-Aldrich syndrome protein family verprolin homolog (WAVE) regulatory complex (WRC), which promotes actin polymerization, and the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, a transcriptional coactivator. Their relationship is established through the sharing of subunits comprising the SAGA deubiquitinase module, including the deubiquitinase Non-stop. Deubiquitinase module mutations—for example, polyglutamine expansion in ataxin 7 (ATXN7)—lead to a spectrum of phenotypes not explained by SAGA’s transcriptional coactivator function, including nervous system degeneration and blindness. In both the nervous system and the eye, the WRC is an essential promoter of actin polymerization, which is facilitated by the constitutively active enzymatic subunit WAVE. WAVE activity and localization are regulated by the remaining WRC subunits, ensuring spatial and temporal control of actin polymerization. Misregulation of actin polymerizing complexes results in a similar spectrum of phenotypes as seen in SAGA mutants, including nervous system degeneration and blindness. This suggests that SAGA is important in the nervous system and eye because it is required to control the WRC in these tissues. We found that the SAGA deubiquitinase module leaves SAGA to bind the WRC. There, Non-stop deubiquitinates WAVE, increasing its level in both the cytoplasm and the nucleus. Therefore, we hypothesize that SAGA controls WRC complex composition, amount, and location; and it is through these activities that SAGA accomplishes functions we had previously attributed to SAGA alone. This hypotheses will be investigated in three aims. First, we will identify and characterize nuclear WAVE-containing complexes through affinity purification and column chromatography coupled to mass spectrometry. Because WAVE activity is regulated in part by its localization, the locations of these complexes will be determined, and the effects of Atxn7 polyglutamine expansion on complex composition and localization will be tested. Second, interactions between the SAGA deubiquitinase module and WRC will be disrupted in cells and flies to determine which SAGA/WRC functions require them. Lastly, the effects of SAGA deubiquitinase-WRC interactions on blindness and neurodegeneration will be investigated in flies, by disrupting them and measuring phenotypes characteristic of Atxn7 polyglutamine expansion. These studies will provide novel insight on the causes of neurodegeneration and blindness, in addition to the links between transcriptional and cytoskeletal regulatory complexes.
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