The role of a retinoic acid response element in establishing the anterior neural expression border of Hoxd4 transgenes

The zebrafish hoxd4a locus was compared to its murine ortholog, Hoxd4. The sequence of regulatory elements, including a DR5 type retinoic acid response element (RARE) required for Hoxd4 neural enhancer activity, are highly conserved. Additionally, zebrafish and mouse neural enhancers function identically in transgenic mouse embryos. We tested whether sequence conservation reflects functional importance by altering the spacing and sequence of the RARE in the Hoxd4 neural enhancer. Stabilizing receptor-DNA interactions did not anteriorize transgene expression. By contrast, conversion of the RARE from a DR5 to a DR2 type element decreased receptor-DNA stability and posteriorized expression. Hence, the setting of the Hox anterior expression border is not a simple function of the affinity of retinoid receptors for their cognate element.     

Identification of a retinoic acid‐responsive neural enhancer in the Ciona intestinalis Hox1 gene

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci‐Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci‐Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid‐response element (RARE). Mutation of the RARE in the Ci‐Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci‐Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA‐dependent and one RA‐independent.     

Retinoic acid response element in HOXA-7 regulatory region affects the rate,not the formation of anterior boundary expression

Since it is known that a 307 bp fragment of the position specific regulatory element of human HOXA-7 contains two (DR3 and DR5) retinoic acid response elements (RAREs) at its 3' end, we constructed several deletion constructs containing different numbers of RAREs and examined their effects in vitro and in vivo. The 5' deletion constructs, BM112 and OM213, retaining both RAREs, were highly responsible (about 8 fold induction) for RA in F9 teratocarcinoma cells, versus NM307 (4-5 fold). The construct NS218, with both RAREs deleted but retaining the 5' sequences lost RA responsibility completely, whereas NR271, with one RARE(DR5) deleted retained a 50% inducibility (2.5 fold). In vivo transgenic analysis revealed that the constructs NM307 and NR271, but not OM213 nor BM112, directed the position specific expression pattern. Sequence analysis revealed that HOXA-7 enhancer sequences, including the RARE repeat sequences, were well conserved in human, mouse and chick. Part of the RAREs overlaps with the CDX1 binding site, and sequences of the DR3 RAREs were identical in this species. Two GAGA binding sites were also found to be strictly conserved. Because OM213, which had one GAGA site disrupted but retaining both RAREs, did not direct anterior boundary formation in transgenic mice, these results suggest the importance of the 5' 94 bp region, including the GAGA binding site, in anterior boundary formation and the involvement of the RARE in the rate of expression not in anterior boundary formation.     

Overexpression of the cellular retinoic acid binding protein-I (CRABP- I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells

Treatment of F9 teratocarcinoma stem cells with retinoic acid (RA) causes their irreversible differentiation into extraembryonic endoderm. To elucidate the role of the cellular retinoic acid binding protein-I (CRABP-I) in this differentiation process, we have generated several different stably transfected F9 stem cell lines expressing either elevated or reduced levels of functional CRABP-I protein. Stably transfected lines expressing elevated levels of CRABP-I exhibit an 80-90% reduction in the RA induced expression of retinoic acid receptor (RAR) beta, laminin B1, and collagen type IV (alpha 1) mRNAs at low exogenous RA concentrations, but this reduction is eliminated at higher RA concentrations. Thus, greater expression of CRABP-I reduces the potency of RA in this differentiation system. Moreover, transfection of a CRABP-I expression vector into F9 cells resulted in five- and threefold decreases in the activation of the laminin B1 RARE (retinoic acid response element) and the RAR beta RARE, respectively, as measured from RARE/CAT expression vectors in transient transfection assays. These results support the idea that CRABP-I sequesters RA within the cell and thereby prevents RA from acting to regulate differentiation specific gene expression. Our data suggest a mechanism whereby the level of CRABP-I can regulate responsiveness to RA during development.     

Convergence of cAMP, TGF-beta and retinoic acid signaling pathways in cells of the embryonic palate.

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-beta) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-beta1, cyclic adenosine 3', 5'-monophosphate (cAMP) and RA signaling systems. TGF-beta1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-beta mRNA in MEPM cells, though only TGF-beta1 inhibited RA-induced RAR-beta protein expression. Forskolin, but not TGF-beta1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-beta gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-beta-responsive, element. Thus, a putative TGF-beta Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-beta promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.     

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RARalpha/RXRalpha heterodimer to a novel retinoic acid response element in the promoter

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RARalpha/RXRalpha heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RARalpha/RXRalpha directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RARalpha/RXRalpha) in the induction of hiNOS by RA.     

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.

The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.