Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins.
Oct-4 is a transcription factor that is specifically expressed in mouse embryonic stem cells and in cell lines derived thereof. In these cells, Oct-4 activates transcription from remote binding sites due to as of yet unknown co-activators. Expression of Oct-4 in differentiated cells is not sufficient to activate transcription from a distance, rather it requires the co-expression of co-activators such as the adenoviral oncoprotein E1A. In this paper, we used phage display to identify Oct-4-interacting proteins. We first analyzed the interaction between Oct-4 and E1A in order to optimize the biochemical conditions that enable Oct-4-specific interactions with other interacting proteins. A panning approach was used to enrich Oct-4 interacting phages from a pool of excess unspecific phages. The biochemical conditions established in our interaction assays were then used to screen a P19 EC cell cDNA expression library in M13 filamentous phage. A number of phage clones displaying portions of unknown and known transcription factors were obtained, from which the HMG-1 transcription factor was identified. HMG-1, and the closely related factor HMG-2, interact with Oct-4 when co-expressed in mammalian cells. In addition, HMG-1 was found to cooperate with Oct-4 in P19 EC cells. These results provide the first evidence of a non-viral factor that enhances Oct-4 distance-dependent transactivation in stem cells.
Butteroni
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C
Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69126, Germany.
De Felici
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M
Schöler
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HR
Pesce
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M
D017500:Adenovirus E1A Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005854:Germ Cells / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3; D009928:Organ Specificity; D019151:Peptide Library; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D050814:Octamer Transcription Factor-3; D019151:Peptide Library; C494174:Pou5f1 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors