title: Human p32, interacts with B subunit of the CCAAT-binding factor, CBF/NF-Y, and inhibits CBF-mediated transcription activation in vitro.
abstract: To understand the role of the CCAAT-binding factor, CBF, in transcription, we developed a strategy to purify the heterotrimeric CBF complex from HeLa cell extracts using two successive immunoaffinity chromatography steps. Here we show that the p32 protein, previously identified as the ASF/SF2 splicing factor-associated protein, copurified with the CBF complex. Studies of protein-protein interaction demonstrated that p32 interacts specifically with CBF-B subunit and also associates with CBF-DNA complex. Cellular localization by immunofluorescence staining revealed that p32 is present in the cell throughout the cytosol and nucleus, whereas CBF is present primarily in the nucleus. A portion of the p32 colocalizes with CBF-B in the nucleus. Interestingly, reconstitution of p32 in an in vitro transcription reaction demonstrated that p32 specifically inhibits CBF-mediated transcription activation. Altogether, our study identified p32 as a novel and specific corepressor of CBF-mediated transcription activation in vitro.
authors.affiliation: Department of Molecular Genetics, M.D. Anderson Cancer Center and Genes, Graduate School of Biomedical Sciences, The University of Texas, Houston, TX 77030, USA.
authors.forename: Chandrani
authors.initials: C
authors.lastname: Chattopadhyay
authors.forename: David
authors.initials: D
authors.lastname: Hawke
authors.forename: Ryuji
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Sankar N
authors.initials: SN
authors.lastname: Maity
mesh_terms: D023081:CCAAT-Binding Factor / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002352:Carrier Proteins; D002467:Cell Nucleus / Q000737:chemistry; D002846:Chromatography, Affinity; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D021122:Protein Subunits / Q000378:metabolism; D011951:Receptors, Complement / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation
chemical_list: C087299:C1QBP protein, human; D023081:CCAAT-Binding Factor; D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D021122:Protein Subunits; D011951:Receptors, Complement; D011994:Recombinant Proteins; D012097:Repressor Proteins; C032402:complement 1q receptor