title: 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.
authors.affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Qc, Canada.
authors.forename: Yves B
authors.initials: YB
authors.lastname: Beaulieu
authors.affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Qc, Canada.
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Leon Machado
authors.affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Qc, Canada.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Ethier
authors.affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Qc, Canada.
authors.forename: Luc
authors.initials: L
authors.lastname: Gaudreau
authors.affiliation: Département de biologie, Université de Sherbrooke, Sherbrooke, Qc, Canada.
authors.forename: Viktor
authors.initials: V
authors.lastname: Steimle
mesh_terms: D000595:Amino Acid Sequence; D005786:Gene Expression Regulation; D005802:Genes, MHC Class II; D057809:HEK293 Cells; D059805:HLA-DR alpha-Chains / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D059805:HLA-DR alpha-Chains; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D020033:Protein Isoforms; D015534:Trans-Activators