The expression of the secretin gene is mainly restricted to a subpopulation of enteroendocrine cells in adult animals and to a fraction of pancreatic β cells during fetal development (38
). Cell-specific expression of many genes relies on the interaction of both cell-type specific and ubiquitously expressed transcription factors with cis
-acting elements of the promoters. Transcription of the secretin gene in secretin-expressing cell lines depends on the presence of an enhancer within 175 bp of the transcription start site (38
). We previously showed that the enhancer activity of the secretin promoter is highly dependent upon the presence of an E-box that binds a cell-specific bHLH protein, BETA2, together with one of its ubiquitously expressed heterodimeric partners, E12 or E47 (22
In this paper, we have further characterized the secretin enhancer and identified three additional mutationally sensitive elements. These include two GC-rich sequences that bind to Sp1 and related proteins, including Sp3, as well as the Finb/RREB-1 binding site. The widespread distribution of Sp1, Sp3, and Finb/RREB-1 in most tissues makes it unlikely that they direct cell type-specific expression of secretin in enteroendocrine cells based on their selective expression in those cells. BETA2/NeuroD is the only factor binding to the secretin enhancer with a relatively restricted expression pattern limited to enteroendocrine cells, pancreatic islet cells, developing neurons, and the corticotrophs of the anterior pituitary. The secretin gene is the only target gene for BETA2 identified thus far that is known to bind to Finb/RREB-1. The combination of these factors binding to the enhancer as an enhanceosome complex may contribute to cell type-specific expression of the secretin gene, potentially along with other yet-to-be-identified factors that bind at some distance from this proximal enhancer.
RREB-1 was first identified as a 756-amino-acid protein containing four zinc fingers that bound to a distal Ras-responsive element of the human calcitonin gene (37
). However, an open reading frame extended upstream from the methionine residue assigned as the first codon, suggesting that RREB-1 was part of a larger protein. The isolation of a cDNA encoding the much larger 1,668-amino-acid protein Finb, containing 15 zinc finger domains, confirmed that RREB-1 was contained within a larger protein. Finb was cloned from a cDNA expression library from human breast carcinoma cell lines by virtue of its ability to bind to a regulatory element of the human c-erbB2
gene promoter (5
). Differences between several Finb clones suggested that the mRNA might undergo alternate splicing with the generation of multiple forms of the protein.
The identified DNA sequences of the RREB-1/Finb binding site exhibit considerable divergence. The DNA-binding sites for Finb/RREB-1 in the calcitonin and c-erbB2
genes share only 8 of 14 nucleotides with the minimal sequence in the secretin gene upstream element and 3 of 14 bases with each other (Table ). Similarly, the secretin gene upstream element shared only 4 of 10 positions with a consensus binding site sequence identified by cyclic amplification and selection of target sequences. One possible explanation for the divergence in binding sites is that DNA-binding specificity is context specific depending on the surrounding sequences and/or the presence of other nearby DNA-bound proteins. The two zinc fingers nearest to the C terminus of RREB-1 bound with similar affinities to two different sequences, an RREB-1 binding site and the sequence GGTCCT (42
). The isolation of the hamster homologue of Finb containing the same two C-terminal zinc finger motifs further suggested that the DBD of Finb resides within these two zinc fingers. Of note, these two zinc fingers were highly conserved in the Drosophila
zinc finger protein encoded by the hindsight
Comparison of Finb/RREB-1 binding sites in different promotersa
The function of Finb is not known. However, transcripts for this gene have been identified in all tissues examined in both mammals and avian species with the exception of the brain (5
). Hindsight, a related zinc finger protein in Drosophila
, is also expressed in several tissues during fly development (41
). Interestingly, analyses of flies with mutant hindsight
alleles revealed that expression of the protein, particularly in aminoserosal cells adjacent to the epidermis, was required to direct epidermal cells to undergo cell shape changes driving germ band retraction (13
). Similarly, hindsight
expression in all tracheal cells during tracheal morphogenesis was required to maintain epithelial integrity and direct assembly of the extracellular structures and to prevent apoptosis (39
Although Finb and RREB-1 cDNAs were isolated on the basis of their ability to bind to the c-erbB2
or calcitonin promoters, respectively, their role in gene transcription was unclear. Neither Finb nor RREB-1 stimulated transcription of reporter genes containing the c-erbB2
or calcitonin promoter sequences. In the case of the calcitonin gene, transcriptional activation occurred only in the presence of activated Ras or Raf. These results suggested that Finb and RREB-1 might not be direct transcriptional activators. Although Finb increased expression of metallothionein and thymidine kinase reporter genes, it is not known whether the observed activation results from the binding of Finb to these promoters or from the regulation of other transcription factors (5
). The role of redox conditions and Ref-1 on Finb function is unclear in vivo. Overexpression of Ref-1 had no effect on secretin promoter activity either in the absence or the presence of cotransfected Finb (not shown). Ref-1 may function to stabilize the DNA-binding activity of Finb/RREB-1 in vitro by maintaining its conformation during protein fractionation. Using an antipeptide antibody against Finb/RREB-1, we show for the first time the presence of Finb/RREB-1 in DNA-protein complexes generated from unfractionated nuclear extracts. In addition, loss of transcriptional activity in transient transfection experiments upon introduction of mutations into the Finb/RREB-1 binding site suggests that Finb/RREB-1 has a direct role in secretin gene transcription.
Expression of Finb or RREB-1 fused to the DBD of GAL4 failed to activate a GAL4-luciferase reporter, further implying that this protein lacks an intrinsic activation domain. Our results suggest that the ability to potentiate the transcriptional activity of BETA2 may be the major function of Finb/RREB-1 in the regulation of secretin gene expression. This potentiation requires binding of both proteins to the enhancer and the presence of a BETA2 interaction domain in Finb.
The present work suggests that the ability of Finb/RREB-1 to interact with other transcription factors may be more important than its ability to directly activate transcription. RREB-1 could not activate the calcitonin promoter except in the presence of activated Ras or Raf. The dependence on the Ras signaling pathway may indicate that RREB-1 interacts with another transcription factor regulated by protein kinases. However, the nature of the proteins involved in Ras signaling that interact with RREB-1 and the mechanism of transcriptional activation when Ras is activated remain to be elucidated.
We and others have shown that BETA2 functions as a relatively modest transcriptional activator even in the presence of its bHLH partner proteins (23
). The insulin and POMC genes are two genes that are regulated by BETA2 for which the activity of BETA2 is enhanced by PDX-1 and Pitx-1, respectively. The present work provides a third example of how yet another DNA-binding protein, Finb/RREB1, interacts with BETA2 to increase transcriptional activation of the secretin gene, one of the targets of BETA2. Finb/RREB-1 is devoid of intrinsic activating function, unlike the two homeodomain proteins PDX-1 and Pitx-1, which are potent transcriptional activators on their own.
The homeodomain protein PDX-1 interacts with BETA2 and synergistically enhances its transcriptional activity on the insulin promoter (7
). Additional studies suggest that the p300 coactivator may further enhance the synergy between BETA2 and PDX-1 (30
). The functional synergy between PDX-1 and BETA2 does not occur if the binding site for either is disrupted (30
The synergism between BETA2 and the homeodomain protein Pitx-1 on the POMC gene differs from the potentiation of BETA2 by Finb/RREB-1 in several respects (28
). Pitx-1 does not appear to directly interact with BETA2 but instead associates with one of the ubiquitously expressed bHLH dimerization partners of BETA2. A second major difference is that Pitx-1 enhances BETA2-dependent transcription of POMC reporter genes without site-specific DNA binding (28
The activity of a number of transcription factors is synergistically increased by other transcription factors binding to separate sites on DNA. In these cases, the transcriptional activation in the presence of both factors exceeds the product of the transcriptional activities of either factor alone. Potentiation of BETA2 activity by Finb/RREB-1 differs from the many known examples of synergistic activation between two or more DNA-binding proteins, each with its own intrinsic activation functions.
In the present work, we show that Finb/RREB-1 increases the activity of BETA2 despite its lack of an intrinsic activation domain. Transcriptional activity generated by the interaction of an activator with a nonactivator partner is well established for heteromeric complexes binding to a single DNA element or closely spaced half-sites. Increased transcriptional activity arising from interactions between BETA2 and Finb/RREB-1 binding to distinct sites separated by 19 bp appears to be uncommon.
The zinc finger protein Ikaros appears to potentiate activity of several transcription factors that bind to the thymidine kinase promoter, including Sp1 and CTF, even in the absence of a domain that confers activation to a GAL4 DBD, suggesting that potentiation is not related to an intrinsic activation domain (11
). Like Finb/RREB-1, potentiation by Ikaros is absolutely dependent on its ability to bind to DNA. However, the mechanism of transcriptional potentiation by Ikaros appears to differ from that by Finb/RREB-1. Unlike that by Finb/RREB-1, potentiation by Ikaros does not require protein-protein interaction with the transcription factors it potentiates. Instead, the ability of Ikaros protein to potentiate transcription appears to correlate with its localization in pericentromeric heterochromatin.
Finb/RREB-1 appears to modulate the transcription-activating function of the bHLH protein BETA2 by an uncommon mechanism. Most proteins involved in regulation of RNA polymerase II transcription can be classified as general transcription factors, gene-specific transcription factors, or transcription cofactors (coactivators and corepressors). In the present work, we show that Finb/RREB-1 increases the activity of BETA2 yet is not a classical activator of transcription that functions by recruiting its own activation domain to DNA. The function of Finb/RREB-1 is also distinct from that of coactivators, which modulate transcription without site-specific binding to DNA. Thus, we suggest that Finb/RREB-1 should be classified as a potentiator of transcription. Further studies of the regulation of secretin gene transcription and other Finb target genes will provide important new information about the mechanisms controlling eukaryotic gene expression.