Roles of the Ubiquitin-Proteasome System and Mono-ubiquitination in Regulating MHC class II Transcription

Major Histocompatibility Complex (MHC) class II molecules are indispensable arms of the immune 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 transactivator (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 polyubiquitination 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 transactivity and MHC class II expression without affecting CIITA expression levels. Following cytokine stimulation, the 19S ATPase Sug1 associates with CIITA and with the MHC class II enhanceosome 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) region 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. INDEX WORDS: Major Histocompatibility Complex class II, Class II transactivator, Transcription regulation, 26S proteasome, 19S proteasome, Ubiquitination, Degradation, 19S ATPases, Sug1 ROLES OF THE UBIQUITIN-PROTEASOME SYSTEM AND MONO-UBIQUITINATION IN REGULATING MHC CLASS II TRANSCRIPTION