Identification of two factors required for transcription of the ovalbumin gene.

Abstract

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and a-actin genes. In contrast, the simian virus 40 early genes, the 3-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-H factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.The interactions between specific DNA sequences, regulatory factors, and RNA polymerase II play an important role in modulating gene expression at the level of transcription. Many studies with DNA templates containing specific mutations have identified the cis-acting elements in DNA such as the conserved TATA box and the regions further upstream which are necessary for the efficient and accurate initiation of transcription. Recently, the GGGCGG sequence (GC box) located upstream of various genes was found to be important for their expression since deletion or mutation of this sequence resulted in a reduction or inactivation of their expression (14,22,23). Furthermore, Tjian and co-workers (7-10, 16, 19) have isolated a protein factor, SP1, which binds to the GC box and is essential for the transcription of this class of genes. Similarly, the CAAT box sequence (GGTCAATCT consensus) which is cis linked and located around 80 base pairs upstream from the RNA initiation site of several genes (1, 11) has also been found to be important for its expression (4, 6, 18). In this case, a protein binding to that region has been identified in our laboratory and others, but its function has not been defined (12,17,27).We have recently demonstrated that the distal promoter region of the chicken ovalbumin gene contains a duplicate GTCAAA box (GGTGTCAAAGGTCAAACT) which is essential fo...

Orphan Nuclear Receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) Protein Negatively Regulates Bone Morphogenetic Protein 2-induced Osteoblast Differentiation through Suppressing Runt-related Gene 2 (Runx2) Activity

“…COUP-TFs have been shown to affect gene expression and development by multiple mechanisms, some of which require DNA binding and some of which do not. COUP-TFII can activate or repress gene expression by binding to COUP-TF motifs, such as those found in the ovalbumin gene (27). Also, COUP-TFII can influence gene expression by binding to other transcription factors (6).…”

Suppression of ischemia‐induced fos expression and AP‐1 activity by an antisense oligodeoxynucleotide to c‐fos mRNA

“…interaction in nuclear extract was measured using 32 P-radiolabeled consensus sequences of AP-1, CREB, and SP-1 as described previously [51,52]. This assay was established as a method to detect alterations of the AP-1 binding activity in a previous publication [9].…”

Transcriptional regulation of the human hepatic lipase (LIPC) gene promoter

“…ARP-1 is widely expressed in multiple tissues during embryonic development, and like Hnf4a, disruption of the Arp-1 gene is embryonic lethal (42). HNF4a activates transcription by binding as a homodimer to DR1 sequences (43,44), whereas ARP-1 can bind to either DR1 or DR4 and activate (45,46) or repress (44) transcription by various direct and indirect actions (47). ARP-1 has also been shown to specifically influence transcriptional activation by HNF4a either negatively or positively, depending on the promoter context.…”