AABBSSTTRRAACCTT Phosducin (Pdc) is a highly conserved acidic phosphoprotein, which plays an important role in the regulation of G-protein signalization in intact retina. It binds to Gβγ dimer of heterotrimeric G-protein transducin thereby decreases the pool of available transducin resulting in modulation of signal. Function of phosducin is negatively regulated by its phosphorylation followed by interaction with the 14-3-3 protein. Besides this established way of regulation, we were interested in other putative interaction partners of phosducin, like SUG1 and CRX. SUG1 is a subunit of 26S proteasome with a large scale of biological functions, especially a degradation of many transription factors. Its role in regulation of phosducin is still unclear, but is probably involved in targeting of phosducin to 26S proteasome for its degradation. Subsequently, we prepared four different expression constructs of full-length protein in order to find the best expression and purification strategy. These results suggest that all purified fusion proteins of SUG1 form stable and soluble high molecular weight oligomers. This behaviour was confirmed by dynamic light scattering and analytical ultracentrifugation measurements. In addition, this observation is consistent with previous studies of its bacterial counterpart, PAN...

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

Major Histocompatibility Complex (MHC) class II molecules are indispensable arms of the im-mune system that present extracellular antigens to CD4+T cells and initiate the adaptive immune response. MHC class II expression requires recruitment of a master regulator, the class II trans-activator (CIITA). How this master transcriptional regulator is recruited, stabilized and degraded is unknown. The 26S proteasome, a master regulator of protein degradation, is a multi-subunit complex composed of a 20S core particle capped on one or both ends by 19S regulatory particles. Previous findings have linked CIITA and MHC class II transcription to the ubiquitin proteasome system (UPS) as mono-ubiquitination of CIITA increases its transactivity whereas poly-ubiquitination targets CIITA for degradation. Increasing evidence indicates individual ATPase subunits of the 19S regulator play non-proteolytic roles in transcriptional regulation and histone modification. Our initial observations indicate proteasome inhibition decreases CIITA transac-tivity and MHC class II expression without affecting CIITA expression levels. Following cyto-kine stimulation, the 19S ATPase Sug1 associates with CIITA and with the MHC class II enhan-ceosome complex. Absence of Sug1 reduces promoter recruitment of CIITA and proteasome inhibition fails to restore CIITA binding, indicating Sug1 is required for CIITA mediated MHC class II expression. Furthermore, we identify a novel N-terminal 19S ATPase binding domain (ABD) within CIITA. The ABD of CIITA lies within the Proline/Serine/Threonine (P/S/T) re-gion of CIITA and encompasses a majority of the CIITA degron sequence. Absence of the ABD increases CIITA half-life, but blocks MHC class II surface expression, indicating that CIITA requires interaction with the 19S ATPases for both its deployment and destruction. Finally, we identify three degron proximal lysine residues, lysines (K): K315, K330 and K333, and a phosphorylation site, serine (S), S280, located within the CIITA degron, that regulate CIITA ubiquitination, stability and MHC class II expression. These are the first lysine residues identified as sites of CIITA ubiquitination that are essential for MHC class II expression. These observations increase our understanding of the role of the UPS in modulating CIITA mediated MHC class II transcription and will facilitate the development of novel therapies involving manipulation of MHC class II gene expression.

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

Major histocompatibility complex molecules (MHCII) are cell surface glycoproteins that present extracellular antigens to CD4+ T lymphocytes and initiate adaptive immune responses. Apart from their protective role, overexpression of MHCII contributes to autoimmune disorders where the immune system attacks our own tissues. Autoimmune diseases are characterized by self-reactive responses to autoantigens, promoting tissue damage, inflammation mediated by proinflammatory cytokines, autoreactive lymphocytes, and autoantibodies. MHCII molecules are tightly regulated at the level of transcription by Class II transactivator (CIITA). CIITA associates with an enhanceosome complex at MHCII promoters and regulates the expression of MHCII. It is thus crucial to understand the regulation of CIITA expression in order to regulate MHCII in autoimmune diseases. Our lab has shown that the 19S ATPases of the 26S proteasome associate with MHCII and CIITA promoters and play important roles in gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear. Here, we define novel roles of the 19S ATPases Sug1, S7, and S6a in expression of CIITApIV genes. These ATPases are recruited to CIITApIV promoters and coding regions, interact with the elongation factor PTEFb, and with Ser5 phosphorylated RNA Pol II. Both the generation of CIITApIV transcripts and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by knockdown of 19S ATPases. Alternatively, inflammation is also suppressed via the Regulator of G-protein signaling 10 (RGS10) in microglial cells which express high levels of RGS10 and promote homeostasis in the central nervous system. However, chronic activation of microglial cells leads to release of cytokines which cause neuroinflammation. Our investigation of roles played by RGS10 in chronically activated microglial cells indicates that RGS10 binds to promoters of IL-1β, and TNF-α and regulates these genes, while the molecular mechanism remains to be investigated. Together, our observations indicate roles for the UPS in modulating gene expression and for RGS10 in regulating proinflammatory cytokines in microglial cells, each of which provides novel therapeutic targets to combat inflammation in autoimmune and neurodegenerative diseases.