Concerted activity of host cell factor subregions in promoting stable VP16 complex assembly and preventing interference by the acidic activation domain.
In contrast to our understanding of the roles of Oct-1 and VP16 in VP16-mediated transcriptional activation, virtually nothing is known of the role of the second cellular component, termed host cell factor (HCF), or of its structure-function relationships. We show that the majority of the internal region of HCF, including the repeats involved in HCF cleavage, is dispensable for complex assembly with VP16 and Oct-1. The N-terminal domain of HCF (HCF.N) had only weak VP16 binding and complex promoting activity, while the C-terminal region (HCF.C) had no intrinsic activity. However, the C-terminal region strongly enhanced complex formation and reduced dissociation kinetics when linked to the N-terminal domain (HCF.NC). The potent activity of the HCF.NC fusion in complex assembly was recapitulated in vivo in yeast and mammalian cells. Moreover, HCF.N could promote increased complex formation when the acidic activation domain of VP16 was deleted. Restoration of the activation domain strongly inhibited complex formation with HCF.N, but the addition of the C-terminal domain of HCF restored strong stable complex formation with intact VP16. The results indicate that this C-terminal domain is critically required to alter the presentation of the acidic domain of VP16. Additional results are consistent with the interpretation that this alteration in acidic domain presentation for complex assembly also facilitates the activation function in VP16. The sequence of an HCF homolog from Caenorhabditis elegans shows it to be a natural HCF.NC construct, reinforcing the conclusions from our functional analysis.
LaBoissière
S
S
Marie Curie Research Institute, The Chart, Oxted, Surrey, England.
Walker
S
S
O'Hare
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000235:genetics; D029742:Caenorhabditis elegans Proteins; D015342:DNA Probes / Q000235:genetics; D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D051863:Host Cell Factor C1; D006801:Humans; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D029742:Caenorhabditis elegans Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D017405:Herpes Simplex Virus Protein Vmw65; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D046911:Macromolecular Substances; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D011506:Proteins; D014157:Transcription Factors; C497691:hcf-1 protein, C elegans