Recognition Sites For Transcription Factors Research Articles

The glucocorticoid receptor and c-jun promoters contain AP-1 sites that bind different AP-1 transcription factors

The glucocorticoid receptor (GR) promoter contains several potential transcription factor recognition sites, including a putative AP-1 site. The GR promoter AP-1 site differs from the consensus AP-1 site by a nucleotide substitution, while the c-jun promoter contains a functionally characterized AP-1 site that varies from the consensus AP-1 site by a nucleotide insertion. Electrophoretic mobility shift assays were performed using nuclear extracts from a mouse pituitary tumor cell line (AtT-20) to test the binding capability of the AP-1 proteins, Jun and Fos, to the putative glucocorticoid receptor and the c-jun AP-1 sites. In addition, a comparison of the complexes formed at the GR AP-1 and c-jun AP-1 sites was done using antibodies specific for the Jun and Fos family members. The complexes formed with the GR AP-1 and c-jun AP-1 sites revealed striking differences. The GR AP-1 site formed complexes with both Jun and Fos family members. JunD was the most abundant Jun family member present, followed by JunB. cJun was absent from the complex. The amount of Fra-2 was greater than FosB in GR promoter AP-1 site complexes while Fra-1 was absent. A small amount of cFos may bind to the GR AP-1 site. In contrast to the GR promoter AP-1 site, only Jun family members were involved with complex formation on the c-jun promoter using AtT-20-cell nuclear extract, with JunD binding exceeding that of cJun. These results confirm previous studies suggesting that the c-jun promoter is stimulated solely by Jun family members. They also show preferential binding of Jun family members to different AP-1 sites present in different promoters. Finally, this study supports the hypothesis that the coordinate regulation of GR and c-jun gene regulation is mediated by crosstalk involving a Jun protein.

Characterization of the Mouse Cyclin D3 Gene: Exon/Intron Organization and Promoter Activity

The three D-type cyclins have been shown to be differentially expressed in a number of cell types, suggesting that they play distinct roles in cell cycle regulation in particular cell lineages. We have determined the complete nucleotide sequence (−1681 to + 6582) of the mouse cyclin D3 gene, which encodes a G1 phase cyclin. The gene consists of five exons and four introns, varying in length from 422 to 2472 bp. Primer extension analysis revealed one major transcription initiation site at the position 107 bp 5′ upstream of the translation start. The promoter region lacks both canonical “TATA” and “CAAT” boxes. It contains, however, multiple transcription factor recognition sites, including multiple “GC-rich” sequences to which Sp1 factor binds and sequences recognized by GATA, NF-κB, ATF, E2F, and TRE/AP1 transcription factors, E box binding myogenic factors, and the IL-6 induced-transcription factor, APRF. Promoter activity of the 1681-bp fragment upstream of the transcription initiation site was confirmed by linking it to a reporter gene and subjecting it to transient expression experiments in various cell types. Promoter activity was high in cell lines that expressed high levels of endogenous D3 mRNA, as indicated by Northern blot analyses, and was significantly reduced when the promoter was truncated to −122 bp. The characterization of the mouse cyclin D3 gene and insight into its promoter region will allow further studies defining the molecular events regulating the expression of this cyclin in proliferating and quiescent cells.

Structural Analysis of the Gene Encoding the Murine Interleukin-11 Receptor α-Chain and a Related Locus

In this study the gene for the murine interleukin-11 receptor alpha chain (IL-11Ralpha) has been characterized. The gene spans 9 kilobase pairs of DNA, and the organization of its 14 exons conforms to the pattern observed for other members of the hematopoietin receptor family. Analysis of the 5' end of the cDNA using 5' RACE showed that the first two exons, designated exons 1a and 1b, are spliced to form alternate transcripts. Transcripts initiating from exon 1b were not found in adult tissues but were present in embryonic stem cells. S1 nuclease and 5' rapid amplification of cDNA ends assays demonstrated multiple major and minor sites of transcription initiation for each exon. The putative promoter regions of both exons lacked TATA boxes, although potential recognition sites for several transcription factors including Sp1, AP1, and AP2 were present. A comparison of the murine and human IL-11Ralpha revealed that the 5' sequence upstream of the major site of transcription initiation site for exon 1b is highly conserved. Northern analysis showed that IL-11Ralpha is expressed in many adult murine tissues. A second IL-11Ralpha-like locus containing a sequence homologous to exons 2-13 was also identified.

Characterization of the Promoter Region of the Human Apurinic Endonuclease Gene (APE)

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.

Nucleic Acid Sequence and DNase Hypersensitive Sites of the 5′ Region of the Mouse Progesterone Receptor Gene

To allow study of the control of the promoter of the mouse progesterone receptor gene and its specific elements, we have isolated nine individual clones and selectively sequenced the 5′ region, which includes 5′ promoter sequence and the non-coding region. Sequence comparison was performed versus the progesterone receptor genes of other species. Moreover, potential estrogen response elements, progesterone response elements, and other transcription factor recognition sites were determined by sequence analysis. In addition, since it has been confined that chromatin structures are important for regulation of gene expression, we also examined them in different estrogen target organs using the DNase hypersensitivity assay. A different pattern of the DNase hypersensitivity sites was detected between uterus and mediobasal hypothalamus and these sites were close to specific recognition elements.

A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

The p41 DNA-binding protein of human herpesvirus 6 is an apparent processivity factor important for viral DNA replication. The p41 promoter was characterized to understand how this processivity factor is regulated. A single transcription start site and a functional TATA box are located 48 and 74 bp, respectively, upstream of the start codon. A reporter construct containing 1,027 bp of the sequence upstream of the p41 start codon was inactive in uninfected T cells but functioned as a strong promoter in human herpesvirus 6-infected cells. Mutational analysis identified a 21-bp element (the EA site) which is located at -73 to -52 bp relative to the transcription start site and is essential for promoter activity. The ability of the EA site to stimulate transcription optimally appears to be strictly dependent upon its distance from the p41 basal promoter. The EA site contains three overlapping sequences, a CAAT-enhancer-binding protein (C/EBP) transcription factor recognition site and two repeat elements. Mobility shift assays using the EA site identified four binding activities (C1 to C4). C1 and C2 are present in both uninfected and infected cells and do not contain C/EBP factors. In infected cells, point mutation of the EA site abrogates C1 and C2 binding activities and destroys transcriptional activity of the p41 promoter. C3 and C4 are present in uninfected cells only and were found to contain C/EBP factors. These findings indicate that in infected cells, transcriptional stimulation of the p41 promoter by the EA site requires C1 and C2 binding activities. These results further suggest that transcriptional activity may also depend upon the elimination of C3 and C4 binding activities.

Characterization of the human gene encoding LBR, an integral protein of the nuclear envelope inner membrane

We have characterized the human gene encoding LBR, an integral protein of the nuclear envelope inner membrane. Restriction mapping shows that the transcription unit spans approximately 35 kilobases. A transcription start site is located approximately 4 kilobases 5' to the translation initiation codon, and an RNA splice of 3863 bases occurs in the 5'-untranslated region to generate mature HeLa cell mRNA. 5' to the identified transcription start site are two CCAAT sequences and potential recognition sites for several transcription factors including Sp1, AP-1, AP-2, and NF-kB. There are 13 protein coding exons in the LBR gene. LBR's nucleoplasmic domain is encoded by exons 1-4, and its hydrophobic domain, with eight putative transmembrane segments, is encoded by exons 5-13. The hydrophobic domain is homologous to three yeast polypeptides, suggesting that this higher eukaryotic gene could have evolved from recombination between a gene that encoded a soluble nuclear protein and a membrane protein gene similar to those in yeast. These results are the first to demonstrate the structural organization of a vertebrate gene encoding an integral membrane protein of the nuclear envelope that may be a member of a family of polypeptides conserved in evolution.

Structure of the rat gene encoding the mitochondrial benzodiazepine receptor

The gene encoding the rat mitochondrial benzodiazepine receptor (MBR) was cloned and characterized. Hybridization of a previously cloned cDNA for MBR to genomic Southern blots indicated that the gene was probably present at one copy per haploid genome. Rapid amplification of cDNA ends with rat adrenal RNA was used to obtain 47 nt of additional sequence upstream from our previously cloned MBR cDNA proving to be a crucial step in cloning the first exon of this gene. The MBR gene is comprised of four exons spanning approx. 10 kb. The first intron, contained within a 8-kb stretch of this gene, is located within the 5′-untranslated sequence, whereas the remaining two introns are much shorter (641 and 854 bp) and interrupt the coding sequence. The third intron contains sequences homologous to rodent B1 repetitive elements and a novel sequence closely resembling part of a repetitive element belonging to the Alu family in humans. The transcription start point was mapped by S1 nuclease protection assays suggesting that the first exon is just 56 bp in length. The sequence upstream from this region contains three GC boxes but lacks other known consensus recognition sites for sequence-specific transcription factors.

Multiple Basal Promoter Elements Determine the Level of Human c-fos Transcription

Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.

Multiple basal promoter elements determine the level of human c-fos transcription.

Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.