Degradation, Promoter Recruitment and Transactivation Mediated by the Extreme N-Terminus of MHC Class II Transactivator CIITA Isoform III

Abstract

Multiple relationships between ubiquitin-proteasome mediated protein turnover and transcriptional activation have been well documented, but the underlying mechanisms are still poorly understood. One way to induce degradation is via ubiquitination of the N-terminal α-amino group of proteins. The major histocompatibility complex (MHC) class II transactivator CIITA is the master regulator of MHC class II gene expression and we found earlier that CIITA is a short-lived protein. Using stable and transient transfections of different CIITA constructs into HEK-293 and HeLa cell lines, we show here that the extreme N-terminal end of CIITA isoform III induces both rapid degradation and transactivation. It is essential that this sequence resides at the N-terminal end of the protein since blocking of the N-terminal end with an epitope-tag stabilizes the protein and reduces transactivation potential. The first ten amino acids of CIITA isoform III act as a portable degron and transactivation sequence when transferred as N-terminal extension to truncated CIITA constructs and are also able to destabilize a heterologous protein. The same is observed with the N-terminal ends of several known N-terminal ubiquitination substrates, such as Id2, Cdt1 and MyoD. Arginine and proline residues within the N-terminal ends contribute to rapid turnover. The N-terminal end of CIITA isoform III is responsible for efficient in vivo recruitment to the HLA-DRA promoter and increased interaction with components of the transcription machinery, such as TBP, p300, p400/Domino, the 19S ATPase S8, and the MHC-II promoter binding complex RFX. These experiments reveal a novel function of free N-terminal ends of proteins in degradation-dependent transcriptional activation.

Genetic variants at the 16p13 locus confer risk for eosinophilic esophagitis

“…Genotype-dependent expression of both CLEC16A and DEXI have been identified [ 56 , 57 ], and chromatin looping from the EoE risk variants shared with type 1 diabetes has demonstrated looping back to the promoter of DEXI [ 84 , 85 ]. CIITA acts as main positive transcriptional regulator of the class II major histocompatibility complex genes [ 86 – 88 ]. While not expressed in esophageal epithelial cell cultures (Fig.…”

Expression of HLA‐DQA1 and HLA‐DQB1 genes in B lymphocytes, monocytes and whole blood

“…N‐terminal acidic domain of CIITA functions as transcription activation domain, and a large number of proteins shown to interact with CIITA employ the residues in or near this domain for binding (Harton & Ting, ). Beaulieu, Leon Machado, Ethier, Gaudreau, & Steimle () documented that N‐terminal domain of CIITA isoform III is responsible for faster turnover (i.e. lower levels) and higher transactivation potential of isoform III compared to other protein isoforms.…”

“…lower levels) and higher transactivation potential of isoform III compared to other protein isoforms. Increased activation potential was mostly due to more efficient interaction with transcription machinery and promoter‐binding protein (Beaulieu et al., ). Therefore, it is possible that promoter polymorphisms, cell type‐specific CIITA isoforms and maybe some other, yet unidentified, cell type‐specific factors interact and generate observed complexity of HLA class II allele expression.…”

Protein arginine methyltransferase 1 (PRMT1) represses MHC II transcription in macrophages by methylating CIITA

“…More recently, Beaulieu et al . have reported that the extreme N-terminus of CIITA is responsible for its rapid degradation: removal of the first 10 amino acids stabilizes CIITA but paradoxically decreases its transcriptional activity34. Of note, this part of CIITA contains two arginine residues but it is not clear whether either one could be methylated by PRMT1.…”