Abstract
The ubiquitous human POU domain protein, Oct-1, and the related B-cell protein, Oct-2, regulate transcription from a variety of eukaryotic genes by binding to a common cis-acting octamer element, 5'-ATTTGCAT-3'. The binding of Oct-1 and Oct-2 to the functionally important lipoprotein lipase (LPL) promoter octamer site was stimulated by the general transcription factor, TFIIB. Comparative analysis of the LPL, histone H2B (H2B), and herpes simplex virus ICPO gene promoter octamer sites revealed that nucleotide sequences within and flanking the octamer sequence determined the degree of TFIIB-mediated stimulation of Oct-1 DNA binding. TFIIB was found to decrease the rate of dissociation of Oct-1 from the LPL octamer site, whereas it increased the rate of association, as well as decreased the rate of dissociation, of Oct-1 from the H2B octamer site. A monoclonal antibody against TFIIB immunoprecipitated a ternary complex containing TFIIB, Oct-1, and the LPL and H2B octamer binding sites. TFIIB did not alter the DNase I footprints generated by Oct-1 on the LPL and H2B promoters. However, Oct-1 on the TATA-binding protein and TFIIB from footprinting the perfect TATA box sequence located 5' of the LPL, NF-Y binding site. In transfection experiments, transcription from the reporters containing the LPL octamer, and either the SV40 or the yeast transcription factor GAL4-dependent enhancers, initiated at a precise position within the octamer sequence. Transcription from reporters containing the H2B octamer and the SV40 enhancer initiated at several positions within and flanking the octamer site, whereas transcription initiated at a precise position within the octamer from reporters with both the H2B octamer and the GAL4-dependent enhancer. These results suggest that octamers and their flanking sequences play an important role in positioning the site of transcription initiation, and that this could be a function of the interaction of Oct-1 with TFIIB.
Highlights
Functional Analyses of the Octamer, the CCAAT Box, and the Putative TATA Box Region of the lipoprotein lipase (LPL) Promoter-To investigate the functional importance of the octamer, the CCAAT box, and the putative TATA box region, the human proximal LPL promoter fragment spanning nucleotides -480 to + 115 was cloned into the enhancer- and promoter-less chloramphenicol acetyltransferase (CAT) reporter vector pBLCAT3 (Luckow and Schultz, 1987)
Mutating the octamer sequence, or the CCAAT box completely abolished LPL promoter activity. These results suggest that the transcription factors Oct-1 and NF-Y which bind to these elements in vitro (Currie and Eckel, 1992; see below) are required for LPL gene regulation
Our results suggest that TFIIB is one cofactor that is recruited to the octamer motif by Oct-L We speculate that depending on the sequences flanking the octamer site, and the specific set of enhancerbinding factors, recruited TFIIB directs the assembly of a functional preinitiation complex
Summary
The binding of Oct-l and Oct-2 to the functionally important lipoprotein lipase (LPL) promoter octamer site was stimulated by the general transcription factor, TFIIB. A monoclonal antibody against TFlffi immunoprecipitated a ternary complex containing TFIIB, Oct-L, and the LPL and H2B octamer binding sites. Transcription from reporters containing the H2B octamer and the SV40 enhancer initiated at several positions within and flanking the octamer site, whereas transcription initiated at a precise position within the octamer from reporters with both the H2B octamer and the GAL4-dependent enhancer These results suggest that octamers and their flanking sequences play an important role in positioning the site of transcription initiation, and that this could be a function of the interaction of Oct-I with TFlffi. Assembly process is stimulated by transcription activators
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
