title: Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of the ARC/Mediator complex.
abstract: The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.
authors.affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA. sara.wezensky@myportal.montana.edu
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Wezensky
authors.forename: Tracey S
authors.initials: TS
authors.lastname: Hanks
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Wilkison
authors.forename: Mary Cloud
authors.initials: MC
authors.lastname: Ammons
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Siemsen
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Gauss
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism; D056892:Mediator Complex / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers / Q000235:genetics; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D004268:DNA-Binding Proteins; D007335:Insulin-Like Growth Factor II; D056892:Mediator Complex; D046912:Multiprotein Complexes; D019255:NADPH Oxidases; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D000099232:Proto-Oncogene Protein Spi-1; C416944:MED15 protein, human; C114598:PLAGL2 protein, human; C527764:NCF2 protein, human