Study On The Function Of SAGA Complex Subunit Spt20 In

SAGA is a highly conserved protein complex in eukaryotes. It plays role in many important cellular processes, including transcriptional activation and mRNA exportation. Different modules of SAGA complex, including acetylation module, Dub module, TATA box-binding module, recruitment module and architecture module, are formed by 21 SAGA subunits. Though a combination of genetic, biochemical and structural studies have shown the functional organization of SAGA complex and its role in transcriptional activation via RNA polymerase Ⅱ, how specific SAGA subunit affects vital physiological pathway and ontogenetic process is yet to be elucidated. This study investigates the biological function of the core structural subunit of SAGA complexSpt20 in vivo.In chapter one, we performed a yeast two hybrid screen by using Spt20, as the bait. Ppb1, catalytic subunit of calcineruin was identified in the screen. Calcineurin is a key regulator of signal transduction. The interaction between Spt20 and Ppb1 was confirmed by yeast two-hybrid assay and co-immunoprecipitation. In S.pombe, ppb△ was hypersensitive to high concentration of Cl-. In contrast, spt20△ was able to resist high concentration of Cl-, which maintained the normal growth of the cell. Fluorescent microscopic analysis showed that Ppb1 was translocated from cytoplasm to nucleus and colocalized with Spt20 upon the increase of extracellular Cl-. Further genetic analysis suggested that spt20+ and ppb1+ stay in the same pathway of regulating Cl- homeostasis and spt20+ functions downstream of ppbl+. Our data suggests Spt20 is involved in the calcineurin-mediated Cl- homeostasis and loss of spt20+ maintains viability of the cell under high concentration of Cl-. The aberrant up-regulation of intracellular Cl- is correlated with diseases like myocardial ischemia reperfusion injury in higher organism. As Spt20 is highly conserved in eukaryotes, it may serve as a potential drug target in Cl- imbalance related diseases.In chapter two, the role of Spt20 in cytokinesis in S.pombe is studied. Cytokinesis is the last step of the cell cycle. It is responsible for daughter cell separation when mitosis completes in eukaryotes. The polyploidy and aneuploidy caused by cytokinesis defects is highly correlated with cancer. Septins are conserved GTP binding protein in eukaryotes. Septin complex subunits Spn1, Spn2, Spn3 and Spn4 form primary septin ring at cell division site during cytokinesis in S.pombe. Mid2 then joins to promote the organization of a stable ring structure. Septin ring serves as a scaffold which provides proper function site for septum degradation enzyme Eng1 and Agn1. The expression level of cytokinesis transcriptional factor ace2+ and its target gene eng1+ and agn1+ is significantly decreased in SAGA subunit deletion mutant spt8△ and spt20△. Overexpressing Ace2 in spt8△ is able to rescue its cytokinesis defect, yet overexpression of Ace2 cannot resuce the cytokinesis defect caused by spt20+ deletion, which indicates that the affects of Spt20 to cytokinesis is not fully denpendent on the transcriptional activity of SAGA complex.Further study shows that septin ring assembly was severely impaired in spt20△. Yet the role of Spt20 in septin ring assembly is independent of the transcriptional activity of SAGA complex, as deletion of enzymatic or structural subunit did not affect normal formation of septin ring. Spt20 directly interact Mid2 and Spn2. Spt20 colocalize with septin ring at division site when SAGA is dissipated. Overexpressing Mid2 could significantly suppress the defect of septin ring assembly in spt20△, suggesting Spt20 functions upstream of Mid2. Therefore, our data identifies Spt20 as a novel regulatory factor required for the assembly of septin ring and suggests that SAGA complex may have dual function in cytokinesis through transcriptional activation and also protein-protein interaction.All above indicate that as a core subunit of SAGA complex Spt20 is not only involved in calcineurin-mediated Cl- homeostasis, but also affects septin ring assembly during late cytokinesis independent of SAGA complex. It reveals a multifunctional role of Spt20 in regulating biological processes. The function of Spt20 in mammalian cells is still yet to be validated.