A dynamic role for HDAC7 in MEF2-mediated muscle differentiation.

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7. MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Promoter specific sensitivity to inhibition of histone deacetylases: implications for hormonal gene control, cellular differentiation and cancer.

Alterations in histone acetylation status appear to play a central role in the regulation of neoplasia, tumor suppression, cell cycle control, hormone responsiveness and senescence. These alterations of chromatin control gene transcription. The histone acetylation status is regulated by the equilibrium of histone acetyl-transferase activity (HAT) and the histone deacetylase activity (HDAC). Commonly, DNA-transfection assays are used to measure the effect of histone acetylation and deacetylation on gene transcription. Here we have analyzed the response of various viral long terminal repeats and vertebrate promoters to the specific histone deacetylase inhibitor trichostatin A (TSA). We show that the activity of many, but not all, promoters is increased upon TSA treatment. Interestingly, the lysozyme promoter exhibited TSA resistance, while the activity of metallothionine, the human growth hormone, and the thymidine kinase promoters was increased. Furthermore, we found that all tested viral promoters are induced by TSA. Analysis of the transcriptional behaviour of the thyroid hormone receptor (TR), the cellular homologue of the v-erbA oncogene, revealed that TSA reduced the gene silencing function but had no influence on the hormone-induced gene activation function of the receptor. These results on gene specific effects, together with the HDAC structural data (1), may be a basis for the development of HDAC inhibitors as antitumor agents.

Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita.

DAX-1 is an unusual member of the nuclear hormone receptor (NHR) superfamily. Lack of DAX-1-mediated silencing leads to adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Gene silencing through NHRs such as the thyroid hormone receptor (TR) is mediated by corepressors. We have previously characterized a novel corepressor, termed Alien, which interacts with TR and the ecdysone receptor but not with the retinoic acid receptors RAR or RXR. Here, we show that DAX-1 interacts with the corepressor Alien but not with the corepressor SMRT. This interaction is mediated by the DAX-1-silencing domain. Naturally occurring mutants of the DAX-1 gene fail to interact with Alien and have lost silencing function. Because the silencing domain of DAX-1 is unusual for NHRs, we mapped the interaction of Alien with DAX-1 and with TR. We show that Alien exhibits different binding characteristics to DAX-1 and TR. Furthermore, Northern experiments demonstrate that Alien is expressed in the adrenal gland and testis in tissues where DAX-1 is specifically expressed. Interestingly, a novel adrenal gland-specific mRNA of Alien was discovered. Thus, the impairment of Alien binding seems to play an important role in the pathogenesis mediated by DAX-1 mutants.

Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily.

Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor's silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.

Cell-specific inhibition of retinoic acid receptor-alpha silencing by the AF2/tau c activation domain can be overcome by the corepressor SMRT, but not by N-CoR.

The human retinoic acid receptor alpha (hRAR alpha) exhibits cell-specific transcriptional activity. Previously, it was shown that in the absence of hormone the wild-type receptor is a transcriptional silencer in L cells, whereas it lacks silencing function and is a weak activator in CV1 cells. Addition of hormone leads to a further increase in transactivation in CV1 cells. Thus, the retinoic acid response mediated by RAR alpha is weak in these cells. It was shown that the CV1-specific effect is due to the receptor C terminus. We show, that the failure of silencing by RAR is not due to a general lack of corepressors in CV1 cells, since the silencing domain of RAR is functionally active and exhibits active repression in these cells. Furthermore, we show that the conserved AF2/tau c activation function of RAR is responsible for the cell-specific inhibition of silencing. Thereby, the CV1 cell specificity was abolished by replacing AF2/tau c of RAR with the corresponding sequence of the thyroid hormone receptor. Thus, we find a new role of the C-terminal conserved activation function AF2/tau c in that, specifically, the RAR AF2/tau c-sequence is able to prevent silencing of RAR in a cell-specific manner. In addition, we show that the inhibitory effect of AF2/tau c in CV1 cells can be overcome by expression of the corepressor SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), but not by that of N-CoR (nuclear receptor corepressor). The expression of these two corepressors, however, had no measurable effect on RAR-mediated silencing in L cells. Thus, the expression of a corepressor can lead to a dramatic increase of hormonal response in a cell-specific manner.

Diurnal variation of cytochrome P-450 dependent parameters after administration of the phenoxyacid herbicide dichlorprop to rats.

The circadian rhythm of ethylmorphine N-demethylase (ND) and hexobarbital sleeping time (HBS) was studied in noninduced and dichlorophenoxy propionic acid (DP) induced adult male rats. DP was gavaged (40 mg/kg bw) at 1.00, 7.00, 13.00 or 19.00 hr. After 21 days the above mentioned parameters were determined corresponding to the time point of DP administration. ND showed a biphasic oscillation (peak activities at 1.00 and 13.00 hr), whereas HBS was significantly shortened only at 1.00 hr. The effects of DP--ND-induction and HBS-shortening--were significant only, when the circadian phase of these parameters was low (i.e. at 7.00 and 19.00 hr for ND and at 7.00 hr for HBS). Consequently, attention should be paid not only to circadian rhythms of the cytochrome P-450 system per se but also to circadian alterations of the inducibility of this system.