Novel Transcriptional Potentiation of BETA2/NeuroD on the Secretin Gene Promoter by the DNA-Binding Protein Finb/RREB-1

Abstract

The basic helix-loop-helix protein BETA2/NeuroD activates transcription of the secretin gene and is essential for terminal differentiation of secretin-producing enteroendocrine cells. However, in heterodimeric complexes with its partner basic helix-loop-helix proteins, BETA2 does not appear to be a strong activator of transcription by itself. Mutational analysis of a proximal enhancer in the secretin gene identified several cis-acting elements in addition to the E-box binding site for BETA2. We identified by expression cloning the zinc finger protein RREB-1, also known to exist as a longer form, Finb, as the protein binding to one of the mutationally sensitive elements. Finb/RREB-1 lacks an intrinsic activation domain and by itself did not activate secretin gene transcription. Here we show that Finb/RREB-1 can associate with BETA2 to enhance its transcription-activating function. Both DNA binding and physical interaction of Finb/RREB-1 with BETA2 are required to potentiate transcription. Thus, Finb/RREB-1 does not function as a classical activator of transcription that recruits an activation domain to a DNA-protein complex. Finb/RREB-1 may be distinguished from coactivators, which increase transcription without sequence-specific DNA binding. We suggest that Finb/RREB-1 should be considered a potentiator of transcription, representing a distinct category of transcription-regulating proteins.The expression of secretin, a peptide hormone, is restricted to S-type enteroendocrine cells present primarily in the small intestine and in the colon. The gene is also transiently expressed in normal ␤ cells of developing pancreatic islets as well as in several pancreatic and intestinal endocrine cell lines (38). It was previously shown that 1.6 kb of 5Ј-flanking sequence of the secretin gene conferred tissue-specific, developmentally regulated expression of a reporter gene in transgenic mice (19).Analysis of the secretin gene promoter by transient expression assays identified an enhancer between Ϫ174 and Ϫ53 bp 5Ј of the transcription initiation site. The enhancer was necessary and sufficient for high levels of reporter gene expression in secretin-expressing cell lines but had no activity in cell lines that did not express their endogenous secretin gene (38). Examination of a series of 5Ј-and 3Ј-deletion mutants within the enhancer revealed a stepwise loss of activity with deletions of increasing size, suggesting that the enhancer was comprised of multiple regulatory elements.A consensus E box sequence at Ϫ130 was required for full promoter activity. BETA2/NeuroD, a basic helix-loop-helix (bHLH) transcription factor that was originally identified as a transactivator of the insulin gene (25) and as a neurogenic factor in Xenopus embryos (15), was subsequently shown to bind the secretin gene E-box as a heterodimer with the ubiquitously expressed bHLH protein E12 or E47 (22).BETA2 is expressed in a very limited number of tissues, including neurons, the anterior pituitary, pancreatic islets, and enteroendocrine cells. The insuli...

The Basic Helix-Loop-Helix Transcription Factor NeuroD1 Facilitates Interaction of Sp1 with the Secretin Gene Enhancer

“…Gel shift assays and nuclear extract preparation were performed as described earlier (28). For reconstitution of DNA-protein complexes using recombinant Sp1 (Promega) or in vitro-translated proteins, 0.25 g of poly(dI-dC) was used in each assay.…”

“…Our earlier work suggested that NeuroD1 is a relatively weak yet essential transcriptional activator of the secretin gene (28). The organization of the secretin enhancer bears little similarity to that of the insulin or POMC enhancers, suggesting that the function of NeuroD1 in transcription of the secretin, insulin, and POMC genes in enteroendocrine cells, pancreatic ␤ cells, and pituitary corticotrophs, respectively, may depend on other factors recruited to each enhancer.…”

“…The effect of Finb/ RREB1 on NeuroD1 requires both its binding to the enhancer 5Ј to the E-box and direct physical association with NeuroD1 (28). We and others previously identified positive cis regulatory elements in the secretin gene enhancer that bind to Sp1 (13,28). One of the Sp1 sites identified was immediately contiguous to the 3Ј end of the NeuroD1 binding E-box.…”

“…Mutational analysis revealed that the enhancer consists of four distinct protein binding sites important for transcription. These include two binding sites for Sp1, one sequence motif that binds to the DNA binding protein Finb/RREB1 (28), and an E-box that binds to the basic helix-loop-helix (bHLH) protein NeuroD1 heterodimerized with E12/E47 (20).…”

RREB1 Transcription Factor Splice Variants in Urologic Cancer

“…10 The significance of RREB1 continues to be elucidated as studies have found it to function in either the induction or repression of gene expression. Genes induced by RREB1 include those encoding calcitonin, 11 FSH, 12 MT-IIA, 13 p53, 14 and secretin 15 ; genes repressed by RREB1 include those encoding angiotensinogen, 16 HLA-G, 17 hZIP1, 18 p16, 19 and PSA. 20 RREB1 has also been found to bind nuclear proteins, such as CtBP, 21 NeuroD, 15 and androgen receptor (AR).…”