The 19S proteasome ATPase Sug1 plays a critical role in regulating MHC class II transcription
Introduction
MHC class II gene products encode cell-surface glycoproteins that bind and present antigenic peptides derived from extracellular proteins. Molecules of the MHC class II complex, HLA-DR, HLA-DP and HLA-DQ in humans, present these antigenic peptides specifically to CD4+ T helper lymphocytes, resulting in initiation of T helper cell activation and proliferation. This basic premise forms an essential arm of the adaptive immune response and is critical to the activation of both cellular and antibody mediated immune mechanisms (Glimcher and Kara, 1992). Tight regulation of MHC class II protein expression is necessary for control of the adaptive immune response and for the prevention of autoimmune diseases. MHC class II expression is regulated primarily at the level of transcription of MHC class II genes (Benoist and Mathis, 1990, Reith and Mach, 2001) by a group of cis acting elements, W(or S), X1, X2 and Y boxes commonly referred to as the W–X–Y module, that are highly conserved among promoters of MHC class II genes (Glimcher and Kara, 1992). The W–X–Y module is recognized and bound by the trimeric RFX and NFY complexes (Steimle et al., 1995, Ting and Trowsdale, 2002, Nagarajan et al., 1999, Mantovani, 1999, Masternak et al., 1998) and by CREB (Moreno et al., 1999, Muhlethaler-Mottet et al., 2004). These transcription factors are ubiquitously expressed and, while their binding is insufficient for MHC class II expression (Masternak et al., 2000), they form a platform that recruits the class II transactivator (CIITA) to the proximal promoter, a step required for MHC class II expression (Steimle et al., 1993).
Unlike the requisite MHC class II transcription factors mentioned above, CIITA gene expression proceeds and parallels that of MHC class II (Ting and Trowsdale, 2002). CIITA does not bind directly to the MHC class II promoter, but it is known as the master regulator of MHC class II transcription as its association with the pre-assembled enhanceosome complex coordinates several additional steps leading to transcriptional initiation (Moreno et al., 1999, Zhu et al., 2000, Masternak et al., 2000, Bewry et al., 2007). CIITA directs MHC class II transcription initiation through interactions with the BRG1 ATPase of the hSWI/SNF remodeling complex (Mudhasani and Fontes, 2002) and through promoter recruitment of histone acetyltransferases (p300, CBP, pCAF and SRC-1) (Drozina et al., 2005, Wright and Ting, 2006), components of the basal transcriptional machinery (Fontes et al., 1997, Mahanta et al., 1997) and kinases that phosphorylate the carboxyl terminus of RNA polymerase II (Spilianakis et al., 2003). Phosphorylation of CIITA further increases its interactions with the co-activator CBP/p300, with RFX and with CIITA itself, thus increasing MHC class II promoter activity (Sisk et al., 2003). Therefore, interactions of CIITA with the MHC class II enhanceosome complex, with the basal transcriptional machinery, and with chromatin remodeling enzymes play a critical role in regulating MHC class II transcription.
Localization of the enhanceosome proteins, chromatin remodeling enzymes, basal transcriptional machinery and CIITA at the transcribing MHC class II proximal promoter makes this, as are most promoters, a complex regulatory region. How cells manage to regulate and recruit multiple protein:protein interactions like these has become an increasingly important question. Recent evidence has supported a role for components of the 26S proteasome as mediators of the assembly of transcriptional systems in yeast. The 26S proteasome is a multi-protein complex that regulates the degradation of polyubiquitinated proteins and consists of two basic parts: a 20S proteolytic core complex and a 19S regulatory particle (Baumeister et al., 1998). The 19S can be further divided into base and lid subunits. The base is composed of six homologous AAA ATPases (S4, S6a, S6b, S7, S8 (Sug1) and S10b) and three non-ATPases (S1, S2 and S5a) while the lid is composed of eight non-ATPase subunits (Ciechanover, 1998). Studies in yeast have demonstrated that ATPase components of the 19S proteasome play a role in activating gene transcription. The ATPases associate with actively transcribed genes where they may link promoter recruitment of transcription factors to their eventual degradation by polyubiquitination and the proteasome (Makino et al., 1999, Gonzalez et al., 2002). In yeast, the most novel implications for a role for the proteasome in regulating transcription come from observations that 19S ATPases directly associate with genes that are actively being transcribed. These ATPases, but not other proteins from the 19S lid or the 20S proteolytic core, have been shown to bind to the yeast Gal4 activation domain in vitro, suggesting that components of the 19S complex, and not the 26S holoenzyme, are intimately involved with transcription (Gonzalez et al., 2002). In addition, the 26S proteasome associates with RNA polymerase II (RNA Pol II) (Gillette et al., 2004) and is critical for efficient RNA Pol II mediated elongation (Ferdous et al., 2001). Specific inhibition of activity of the 19S subunit decreases elongation while inhibition of the 20S subunit increases elongation, indicating that a balance between these two subunits and their activities may be important for transcriptional regulation in yeast (Gillette et al., 2004, Ferdous et al., 2001, Ferdous et al., 2002). While most research to date has been performed in yeast systems, several recent publications have highlighted the significance of the 19S in regulating transcription in mammalian cells. The 19S ATPase Sug1 is recruited to the viral transcription factor E1A and enhances transcription from viral promoters during adenoviral infection of human cells (Rasti et al., 2006). Sug1 also associates with the p53 transcription factor and is recruited to p53 responsive p21waf1 promoters in a manner that correlates with p53 recruitment (Zhu et al., 2007). Finally, the HIV-1 trans-activator protein, Tat, regulates the conversion of the 26S proteasome into separate 19S and 20S components by recruiting a proteasome-associated protein, PAAF-1, which results in decreased proteasome activity and increased association of multiple 19S, but not 20S, subunits with the HIV-1 promoter (Lassot et al., 2007).
While the evidence for 19S involvement in mammalian transcription is tantalizing, many unanswered questions remain. It is primarily unclear if 19S subunits play a role in recruiting transcription factors to promoters and, if so, how these factors are targeted for recruitment. To understand the role of the proteasome system in regulating transcription of MHC class II genes, we investigated the role of the 19S ATPase subunit Sug1 in regulating CIITA activity, promoter recruitment and MHC class II transcription. Our data show that Sug1 associates with CIITA and with the MHC class II proximal promoter. Sug1 plays important roles in regulating CIITA activity and MHC class II expression, in part by regulating promoter recruitment of CIITA. The 20S proteasome is also strongly recruited to the proximal promoter, but only following prolonged cytokine stimulation, suggesting that the entire 26S proteasome is recruited to the MHC class II proximal promoter at various times during active transcription. Our data show for the first time that CIITA, the master regulator of MHC class II transcription, is in turn regulated by components of the master regulator of protein destruction, the 26S proteasome. These data define novel roles for proteasomal subunits in transcriptional programs regulated by CIITA and offer further insights into the function of the proteasome in mammalian transcription.
Section snippets
Cell culture
HeLa cells (human epithelial) from ATCC (Manassas, VA) were maintained using high-glucose Dulbecco modified Eagle (DMEM) medium (Mediatech Inc., Herndon, VA) supplemented with 10% FCS, 50 U/ml of penicillin, 50 μg/ml of streptomycin and 2 mM of l-glutamine. The cells were maintained at 37 °C with 5% CO2. Raji cells (Burkitts lymphoma-derived cell line) from ATCC were maintained in Roswell Park Memorial Institute (RPMI) Media (Mediatech) supplemented with 10% FCS, 50 U/ml of penicillin, 50 μg/ml of
Proteasome inhibition reduces CIITA-mediated MHC class II transcription
If components of the 26S proteasome positively regulate MHC class II gene transcription in a degradation-independent manner as has been proposed in yeast, then inhibition of the 26S proteasome should decrease, rather than increase, transcription. We used transient transfection to test the effect of the potent 26S proteasome inhibitor MG132 on the activation of the human MHC class II promoter, HLA-DRA, associated with a luciferase (Luc) reporter gene by CIITA. As shown in Fig. 1A, transfection
Discussion
Our data has provided evidence suggesting a model in which 19S and 20S components of the 26S proteasome associate with the MHC class II proximal promoter at different stages of transcription where they both positively and negatively regulate CIITA mediated transcription. In support of this model, ChIP analysis demonstrates that low levels of the 19S ATPase Sug1 rapidly associate with the MHC class II proximal promoter within 4 h of cytokine stimulation. Following 4 h of cytokine stimulation,
Acknowledgements
This work is supported by grants from the National Multiple Sclerosis Society, The Georgia Cancer Coalition and Georgia State University. We thank Dr. A. Wani (Department of Radiology, The Ohio State University, Columbus, OH) for generously providing us with the Myc-Sug1 construct.
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