TY - JOUR
T1 - Human p32, interacts with B subunit of the CCAAT-binding factor, CBF/NF-Y, and inhibits CBF-mediated transcription activation in vitro
AU - Chattopadhyay, Chandrani
AU - Hawke, David
AU - Kobayashi, Ryuji
AU - Maity, Sankar N.
N1 - Funding Information:
We thank Henry Adams for helping with the immunofluorescence studies and Andrea Cervin for technical assistance in Mass Spectrometry. We thank Franc¸oise Coustry for helping with the in vitro transcription reactions and Gayle Nesom for editing the manuscript. This work was supported by National Institutes of Health Grant AR46264 (to S.N.M), and the DNA sequencing facility at The University of Texas M.D. Anderson Cancer center was supported by the National cancer Institute Grant CA16672.
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gkh692
DO - 10.1093/nar/gkh692
M3 - Article
C2 - 15243141
AN - SCOPUS:3042731235
SN - 0305-1048
VL - 32
SP - 3632
EP - 3641
JO - Nucleic acids research
JF - Nucleic acids research
IS - 12
ER -