Histone acetylation influences thyroid hormone and retinoic acid-mediated gene expression.

Thyroid hormone (T3) and retinoic acid (RA) receptors regulate transcription of the rat growth hormone (GH) gene through binding to a common hormone response element (HRE) in the promoter. We have investigated the effect of histone acetylation on hormone-dependent expression of the rat GH gene. We examined the effect of butyrate, which induces histone hyperacetylation, and trichostatin A (TSA), a highly specific inhibitor of histone deacetylases. GH-mRNA levels were significantly increased in pituitary GH4C1 cells incubated with T3 and RA, and this response was further stimulated in the presence of 1 mM butyrate. The effect of butyrate was mimicked by TSA. Butyrate and TSA also enhanced the activity of recombinant constructs containing the GH promoter directing chloramphenicol acetyl transferase (CAT) reporter gene expression. CAT activity increased by 4- to 8-fold after incubation with 1 nM T3 and 1 microM RA, and this response was stimulated 2- to 4-fold further in the presence of 0.25 mM butyrate. This concentration of butyrate did not influence basal expression of CAT. TSA produced a dose-dependent increase of CAT activity in the absence of ligands, and between 5 and 200 nM potentiated the effect of T3 and RA. These compounds also increased the hormonal response of constructs in which the HRE was linked to heterologous [mouse mammary tumor virus (MMTV) and thymidine kinase (TK)] promoters. With butyrate >1 mM, basal activity of the GH promoter increased by more than 10-fold and the effect of T3 and RA was no longer observed. Overexpression of T3 receptors was able to counteract the stimulation of basal CAT levels caused by butyrate. Thus, in the absence of ligand, the T3 receptor acts as a constitutive repressor of gene expression. Upon binding of the hormone, the T3 receptor is converted into an activator. Our findings suggest that histone acetylation, which alters chromatin structure, may play an important role in hormone-mediated transcriptional regulation.

one-eyed pinhead is required for development of the ventral midline of the zebrafish (Danio rerio) neural tube.

Axial midline structures play a central role as signalling centres during the development of the vertebrate embryo. We have isolated mutant alleles of a new locus, one-eyed pinhead (oep), in the zebrafish that are characterized by cyclopia and impaired development of the floor plate. oep mutants fail to establish expression of axial (zebrafish HNF3beta) and sonic hedgehog in the midline of the neural plate but form a notochord that expresses both genes. In the spinal cord of the 1-day-old embryo, mutation of oep impairs floor-plate but not motor-neuron development. Floor-plate development is absolutely dependent on oep only at early stages, since partial recovery of the floor plate can be detected at 48 h in the spinal cord, suggesting compensatory pathways. Ectopic expression of sonic hedgehog and a dominant-negative protein kinase A regulatory subunit induces expression of floor-plate marker genes in the oep mutant neural tube in a manner indistinguishable from wild-type embryos. Our data suggest that the oep mutation does not impair Sonic hedgehog signalling and thus implicate a second process that acts synergistically with Sonic hedgehog signalling in the specification of the midline of the neuroectoderm and that can partially be compensated for during later development.

Comparison of the effects of retinoic acid and nerve growth factor on PC12 cell proliferation, differentiation, and gene expression.

Retinoic acid (RA) produced a dose-dependent inhibition of PC12 cell growth and the appearance of cell clusters without neurite extension. RA-induced cell clumping was similar to that caused by dexamethasone (Dx). Nerve growth factor (NGF) induced neurite extension, and the combination of RA plus NGF produced a maximal decrease in cell proliferation with a mixed morphology in which part of the cell population had neurites and part formed clumps. Transcriptional effects of RA were demonstrated by the increase in the activity of reporter constructs that contain an RA response element. RA also regulated expression of endogenous genes in PC12 cells. The retinoid produced a two- to threefold increase in level of p75LNGFR mRNA (the low-affinity NGF receptor), without altering expression of the trk protoon-cogene (the high-affinity NGF receptor carrying tyrosine kinase activity). RA also caused a transient increase in level of tyrosine hydroxylase (TH) mRNA (twofold after 16 h), which returned to basal levels and then decreased relative to basal levels at 48 h. The effect of NGF on the expression of these genes was identical to that produced by RA. However, incubation with Dx did not induce p75LNGFR mRNA and produced a strong and sustained increase of TH mRNA level (three- to fivefold after 48 h). These results show that, despite the common morphological changes produced by RA and glucocorticoids in PC12 cells, the biochemical changes caused by RA are similar to those produced by NGF. Therefore, RA could initiate a biochemical program of neuronal differentiation in PC12 cells, although a fully differentiated phenotype with neurite extension is not obtained.

Vitamin D interferes with transactivation of the growth hormone gene by thyroid hormone and retinoic acid.

The thyroid hormone, retinoic acid (RA), and vitamin D regulate gene expression by binding to similar receptors which act as ligand-inducible transcription factors. Incubation of pituitary GH4C1 cells with nanomolar concentrations of vitamin D markedly reduces the response of the rat growth hormone mRNA to thyroid hormone triiodothyronine (T3) and RA. The stimulation of growth hormone gene expression by both ligands is mediated by a common hormone response element (TREGH) present in the 5'-flanking region of the gene, and the inhibition caused by vitamin D is due to transcriptional interference of the vitamin D receptor on this DNA element. No inhibition of the basal promoter activity by the vitamin was observed. The response to T3 and RA of a heterologous promoter containing this element, the palindromic T3- and RA-responsive sequence TREPAL, or a direct repeat of the same motif is also inhibited by vitamin D. In contrast, vitamin D strongly induces the activity of constructs containing a vitamin D response element, and neither T3 nor RA reduces vitamin D-mediated transactivation. Transfection with an expression vector for the retinoid X receptor alpha (RXR alpha) increases transactivation by T3 and RA but does not abolish the inhibition caused by the vitamin. Gel retardation experiments show that the vitamin D receptor (VDR) as a heterodimer with RXR weakly binds to the T3- and RA-responsive elements. Additionally, VDR displaces binding of T3 and RA receptors in a dose-dependent manner. Our data suggest the formation of TR-VDR and RAR-VDR heterodimers with RXR. The fact that the same response element mediates opposite effects of at least four different nuclear receptors provides a greater complexity and flexibility of the transcriptional responses to their ligands.

Interaction of thyroid hormone and retinoic acid receptors on the regulation of the rat growth hormone gene promoter.

Retinoic acid transcriptionally regulate growth hormone (GH) gene expression through sequences located in the 5'-flanking region of the gene. A partial induction by retinoic acid was obtained with the -181 bp of the rat GH promoter, and sequences up to -209 were required for a full response. These sequences contain the previously identified thyroid hormone responsive element. The retinoid X receptor RXR increased transactivation by T3 and RA. The retinoid was relatively more effective in stimulating the native GH promoter than an heterologous promoter which contains the response element, thus showing the importance of the promoter context on transactivation by the nuclear receptors.