RREB1
RREB1 is a large C2H2 zinc-finger protein, originally identified as the factor binding the RAS-responsive element of the human calcitonin promoter in medullary thyroid carcinoma cells. It has around fifteen fingers and prefers a long C-rich word, CCCCAAACCACCCC, reported when the protein was cloned and reproduced by the database matrices. It is a downstream effector of RAS and TGF-beta signalling: in the TGF-beta pathway RREB1 partners with SMAD complexes to direct fibrogenic and developmental epithelial-mesenchymal transitions, and it has been implicated in metastatic enhancer priming. The motif's length is its main virtue and its main problem, since much of it is a C-run that overlaps several other GC-rich promoter families, and a fragment of the protein has been reported to bind an unrelated sequence instead.
The factors
RREB1 is a single gene, with no paralogue in the human genome that shares its specificity, and this family therefore has one member and three closely agreeing matrices. The protein is large and carries a long array of C2H2 zinc fingers, on the order of fifteen, which is consistent with the long element the databases recover; as with other very large finger arrays, it is unlikely that all fingers contact DNA in a single binding event. It has appeared under several names: RREB-1, from the RAS-responsive element of the calcitonin promoter 8816445; Finb, from work on a Raf-responsive zinc-finger protein that binds divergent sequences 10390538; and zinc finger protein 368. Multiple splice variants exist and have been catalogued in urologic cancers 21703425. RREB1 is broadly expressed and is not a dedicated tissue-restricted factor.
The motif
The consensus CCCCAAACCACCCC was reported in the paper that cloned RREB-1 as the protein binding the distal RAS-responsive element of the human calcitonin promoter 8816445, and the database matrices reproduce it. They are not independent of that report: JASPAR lists its RREB1 matrix, MA0073.2, as SELEX data from the same paper, the CIS-BP M02665 matrix is effectively identical to it (pairwise similarity 0.998 in this collection), and the Vierstra archetype AC0602 is a slightly shortened version. The full 19-base form CCCCAAACCACCCCCCCCC is C-rich throughout with a short internal A-tract, and the information is carried mainly by the CCCCAAACCA portion; the 3' C-run is low in information and will match almost any C-rich stretch. There is no half-site, no spacing rule and no dimerization requirement described. The one conflicting result came from a four-finger RREB1 fragment (LZ321), which bound a GGTCCT-containing tramtrack site and the calcitonin element with similar affinity but did not bind a consensus RREB1 site 10390538; the secretin promoter study described potentiation of BETA2/NeuroD rather than a new consensus 12482979. The literature and data consensuses therefore agree; what is unsettled is whether the protein, through other parts of its long finger array, also reads unrelated sequences.
Several families in this atlas read C-rich promoter words, and they are easy to confuse. ZNF281/ZNF148 (ZBP-89) read CCCCACCCC; ZBTB7A/B/C read GACCCCCCCC; SP/KLF-type GC boxes are GGGGCGGGGC, that is G-rich on the same strand where RREB1 is C-rich plus CpG-containing; ZBTB17/MIZ1 reads core-promoter GC-rich sites. The RREB1 word's distinguishing feature is the internal AAA: CCCCAAACCACCCC has a purine interruption in the middle of the C-run that GC boxes and ZBTB7 sites lack, and it contains no CpG dinucleotide, which sets it apart from the CpG-island-associated GC-rich families. A hit with no internal A-tract is more likely one of the neighbouring families.
Where it acts
There is less genomic mapping for RREB1 than for most families of comparable size, and no ChIP-derived matrix appears in HOCOMOCO for it at all, which is itself informative. The characterized sites are promoter-proximal: the calcitonin promoter RAS-responsive element, the secretin promoter, and the SLC39A1/hZIP1 zinc-transporter promoter, where RREB1 overexpression down-regulates the gene in prostate cancer 19802870,21360563. In the TGF-beta pathway the interesting occupancy is enhancer-associated and combinatorial: RREB1 and SMAD complexes act together at enhancers controlling SNAI1 and other EMT effectors 31915377, and RREB1 has been placed in the mechanism by which TGF-beta and RAS jointly unmask primed enhancers to drive metastasis 39243762. So the motif can be expected both at C-rich proximal promoters and at signal-dependent enhancers, and in the latter case the relevant unit is a composite RREB1-SMAD arrangement rather than an isolated RREB1 site.
Biology and disease
RREB1 was found through a differentiation response to RAS in human medullary thyroid carcinoma 8816445. Its best-supported function now is as a RAS effector within TGF-beta signalling: it directs the choice between fibrogenic and developmental EMT programs 31915377, and in mouse models contributes to enhancer priming for metastasis 39243762. Independent lines of work report RREB1 repressing the zinc transporter hZIP1 in prostate cancer, altering p16 expression and proliferation in gastric cancer 34666611, and being subject to splice-variant changes in urologic tumours. The RREB1 locus is also recurrently altered in melanoma, and copy-number gain at RREB1 is used diagnostically in melanocytic-lesion FISH panels 36325821, which is one of the more concrete clinical connections. Common variants in and near RREB1 appear in genome-wide association studies of type 2 diabetes and birth weight 32210733. No Mendelian RREB1 disorder is established.
Reading a hit
Treat the long C-rich word as a family-level signal at best. The 5' CCCCAAACCA segment carries the discriminating information; the 3' C-run does not, so check that your match covers the informative half rather than sliding along a homopolymeric C stretch. A hit without the internal A-tract is probably a GC box or a ZNF281/ZBTB7-type site. There is no paralogue problem, but there is a provenance problem: the JASPAR and CIS-BP matrices are near-duplicates and JASPAR attributes its matrix to the 1996 cloning paper's SELEX data, so agreement between them is not independent confirmation, and there is no ChIP-derived matrix to check against. For signalling questions, look for a nearby SMAD-binding element, because the functionally demonstrated RREB1 activity in TGF-beta responses is composite. A single promoter-proximal hit does not license the conclusion that a gene is RAS- or TGF-beta-responsive.
How this was worked out
- 1996mediumAffinity screening of an expression library, binding-site analysis, reporter assays8816445
RREB-1 is cloned by affinity screening as the protein binding the distal RAS-responsive element of the human calcitonin promoter in medullary thyroid carcinoma cells; its reported consensus is CCCCAAACCACCCC, and overexpression increases RAS- and RAF-induced transactivation of the calcitonin promoter.
Still open then: How a single factor with a long finger array selects sites genome-wide was untested.
- 1999mediumExpression cloning, gel-shift binding with multiple ligands, finger deletion, enhancer reporter assays10390538
A liver cDNA isolated on a Drosophila tramtrack site, GGTCCT, turns out to encode RREB1; the protein binds that site and the calcitonin RRE with similar affinity (Kd 50-60 nM) yet does not bind a consensus RREB1 site, and only the two C-terminal fingers of the fragment are needed, so RREB1 is reported to read divergent sequences.
Still open then: The relationship between the published consensus and the divergent sites was left unexplained.
On the secretin promoter, Finb/RREB-1 has no intrinsic activation domain and does not activate alone; it requires both DNA binding and physical interaction with BETA2/NeuroD to potentiate transcription, which places it between classical activators and coactivators.
RREB-1 is shown to bind a site in the hZIP1 (SLC39A1) promoter region and to repress the zinc transporter in prostate cancer cells, with follow-up work linking its overexpression to the loss of zinc accumulation in prostate tumors.
- 2020highGenome-wide binding and expression analysis, proteomics, carcinoma cells and mouse epiblast progenitors31915377
RREB1 is identified as the RAS effector that partners TGF-beta-activated SMAD factors in epithelial-to-mesenchymal transition: MAPK-activated RREB1 recruits SMADs to SNAI1, and chromatin accessibility then decides whether the resulting program is fibrogenic or developmental.
Still open then: What marks the enhancers that RREB1 can use was not yet defined.
The chromatin basis is worked out: RREB1 localizes to H4K16ac/K20ac-marked, H2A.Z-loaded nucleosomes at enhancers of IL11, PDGFB, HAS2 and SNAI1, priming them for activation by a SMAD4-INO80 remodeling complex on TGF-beta input, which assembles a metastasis program in lung adenocarcinoma.
Still unresolved
- Whether RREB1 has one binding specificity is unsettled: the 1996 cloning paper reported the consensus CCCCAAACCACCCC that the in vitro matrices now reproduce, while a 1999 study found that a four-finger fragment of the protein bound a dissimilar GGTCCT-containing site and the calcitonin element with comparable affinity but did not bind a consensus RREB1 site at all.
- Which of the roughly fifteen fingers contact DNA is unknown: a fragment containing four fingers bound two unrelated ligands through its two C-terminal fingers, so the long array may provide alternative reading heads rather than one continuous interface, which would explain the reported sequence promiscuity.
- Where RREB1 binds genome-wide is poorly documented for a factor of this prominence: no ChIP-derived matrix exists in HOCOMOCO, and the three available matrices are near-duplicate in vitro products, so it is not known how much endogenous occupancy falls on the C-rich word versus on composite RREB1-SMAD enhancers.
- Whether RREB1 acts as an activator, a repressor or a potentiator is context-dependent in a way that is not understood: it potentiates BETA2/NeuroD without an intrinsic activation domain, represses hZIP1, and with SMAD4 activates EMT and fibrogenic genes.
The consensus is the one reported when RREB-1 was cloned, and the matrices reproduce it, but the support is narrower than three matrices suggest: JASPAR attributes its matrix to that paper's SELEX data and the CIS-BP matrix is a near-duplicate, so their agreement is not independent evidence, and no ChIP-derived matrix is available to cross-check. A later study of a four-finger fragment found binding to an unrelated GGTCCT site and none to the consensus. The word is long but much of its length is a low-information C-run that will match many GC-rich promoters, overlapping the ZNF281, ZBTB7, ZBTB17 and SP/KLF families; only the CCCCAAACCA portion discriminates. Functionally demonstrated activity in TGF-beta signalling is composite with SMAD, not standalone.
References
- Thiagalingam A, De Bustros A, Borges M, et al. RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas. Mol Cell Biol 1996;16:5335-45. PMID 8816445
- Zhang L, Zhao J, Edenberg HJ. A human Raf-responsive zinc-finger protein that binds to divergent sequences. Nucleic Acids Res 1999;27:2947-56. PMID 10390538
- Ray SK, Nishitani J, Petry MW, et al. Novel transcriptional potentiation of BETA2/NeuroD on the secretin gene promoter by the DNA-binding protein Finb/RREB-1. Mol Cell Biol 2003;23:259-71. PMID 12482979
- Milon BC, Agyapong A, Bautista R, et al. Ras responsive element binding protein-1 (RREB-1) down-regulates hZIP1 expression in prostate cancer cells. Prostate 2010;70:288-96. PMID 19802870
- Zou J, Milon BC, Desouki MM, et al. hZIP1 zinc transporter down-regulation in prostate cancer involves the overexpression of ras responsive element binding protein-1 (RREB-1). Prostate 2011;71:1518-24. PMID 21360563
- Nitz MD, Harding MA, Smith SC, et al. RREB1 transcription factor splice variants in urologic cancer. Am J Pathol 2011;179:477-86. PMID 21703425
- Su J, Morgani SM, David CJ, et al. TGF-β orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1. Nature 2020;577:566-571. PMID 31915377
- Deng YN, Xia Z, Zhang P, et al. Transcription Factor RREB1: from Target Genes towards Biological Functions. Int J Biol Sci 2020;16:1463-1473. PMID 32210733
- Gao Q, Wu Y, Lu C, et al. Knockdown of RREB1 inhibits cell proliferation via enhanced p16 expression in gastric cancer. Cell Cycle 2021;20:2465-2475. PMID 34666611
- O'Hern K, Barney R, Chambers M, et al. A novel method to assess copy number variation in melanoma: Droplet digital PCR for precise quantitation of the RREB1 gene in formalin-fixed, paraffin-embedded melanocytic neoplasms, a proof-of-concept study. J Cutan Pathol 2023;50:169-177. PMID 36325821
- Lee JH, Sánchez-Rivera FJ, He L, et al. TGF-β and RAS jointly unmask primed enhancers to drive metastasis. Cell 2024;187:6182-6199.e29. PMID 39243762
3 motifs in this family — JASPAR 1 · CIS-BP 1 · Vierstra 1
| motif | database | consensus | bits |
|---|---|---|---|
| M02665_2.00 | CIS-BP | CCCCAAAcCACCCcCcccC | 22.3 |
| RREB1 (MA0073.2) | JASPAR | CCCCAAAcCACCCcCcccC | 22.1 |
| AC0602:RREB:C2H2_ZF | Vierstra | CCCCAAACCACCCc | 17.6 |
HOCOMOCO tags read as assay + quality grade: P ChIP-seq, S HT-SELEX, M methyl-HT-SELEX, G GHT-SELEX, I SMiLE-seq, B PBM; grade A means two or more assay types agreed. A P-only matrix is a record of where the protein was crosslinked, which need not be a sequence it binds itself.
The factors, elsewhere
These entries are written from the primary literature and every citation is checked against PubMed, but this one can still be out of date, too confident, or simply wrong — especially where a motif’s published site and its database matrices disagree. Corrections and additions are welcome, and they are the fastest way to make this page better for everyone.