Histone deacetylase inhibitors: a new mode for inhibition of cholesterol metabolism.

BACKGROUND: Eukaryotic gene expression is a complex process involving multiple cis and trans activating molecules to either facilitate or inhibit transcription. In recent years, many studies have focused on the role of acetylation of histone proteins in modulating transcription, whereas deacetylation of these same proteins is associated with inactivation or repression of gene expression. This study explores gene expression in HepG2 and F9 cell lines treated with Trichostatin A (TSA), a potent histone deacetylase inhibitor. RESULTS: These experiments show that TSA treatment results in clear repression of genes involved in the cholesterol biosynthetic pathway as well as other associated pathways including fatty acid biosynthesis and glycolysis. TSA down regulates 9 of 15 genes in this pathway in the F9 embryonal carcinoma model and 11 of 15 pathway genes in the HepG2 cell line. A time course study on the effect of TSA on gene expression of various enzymes and transcription factors involved in these pathways suggests that down regulation of Srebf2 may be the triggering factor for down regulation of the cholesterol biosynthesis pathway. CONCLUSION: Our results provide new insights in the effects of histone deacetylases on genes involved in primary metabolism. This observation suggests that TSA, and other related histone deacetylase inhibitors, may be useful as potential therapeutic entities for the control of cholesterol levels in humans.

Global gene expression profiles associated with retinoic acid-induced differentiation of embryonal carcinoma cells.

We have evaluated the effects of retinoic acid (RA) treatment of F9 embryonal carcinoma (EC) cells, which induces differentiation into primitive endoderm, on gene expression patterns. F9 cells were exposed to RA in culture, and global expression patterns were examined with cDNA-based microarrays at early (8 hr) and later times (24 hr) after exposure. Of the 1,176 known transcripts examined, we identified 57 genes (4.8%) that were responsive to RA at 8 and/or 24 hr: 35 were induced, 20 were repressed, and 2 were differentially regulated at these time points. To determine if our results were dependent on the array technology employed, we also evaluated the response to RA at 24 hr with oligonucleotide-based arrays. With these more dense arrays (12,488 genes), we identified an additional 353 RA-regulated genes (2.8%): 173 were upregulated and 180 were downregulated. Thus, a total of 410 genes regulated by RA were identified with roughly equivalent numbers induced or repressed. Although the expression of many genes found on both array platforms was consistent, the results for some genes were disparate. Quantitative PCR studies on a subset of these genes supported the results obtained with the cDNA arrays. Our results confirmed the regulation of several known RA-responsive genes and we also identified a number of genes not previously known to be RA-responsive. Those novel genes that were induced presumably contribute to the cellular processes required for a shift from proliferation to differentiation, whereas those new genes that were downregulated may possibly contribute to the maintenance of cell proliferation.

The bHLH protein MyoR inhibits the differentiation of early embryonic endoderm.

MyoR is a bHLH protein whose expression was reported to be almost exclusively restricted to the precursors of the skeletal muscle lineage where it was postulated to function as a transcriptional repressor of myogenesis. However, previous studies in our laboratory suggested a much broader role for MyoR in embryonic cell differentiation. We demonstrated that, besides being expressed in several adult tissues of non-muscle lineage, MyoR was expressed at a much earlier stage in mammalian development than had previously been reported, that is, as early as the blastocyst stage, well before skeletal muscle specification. We also found that, as in skeletal muscle precursor cells, MyoR expression is inversely correlated with the cellular differentiative state of ectodermal, non-muscle embryonal carcinoma (EC) cells. Retinoic acid (RA) treatment of ectodermal EC or embryonal stem (ES) cells promotes their differentiation into primitive endoderm. However, in the present study, we show that the RA-induced expression of endodermal markers such as EndoA, collagen IV, and t-PA are inhibited by exogenous MyoR expression and that the level of inhibition of these markers correlates with the level of MyoR expressed. Conversely, knock-down of MyoR expression via RNA interference enhances RA-induced differentiation of EC cells, promoting earlier and much higher expression of the above-mentioned endodermal markers following RA treatment. Finally, we have narrowed the period of exogenous MyoR-induced embryonic lethality to between 3.5 and 5.5 days post-coitum (dpc), the period during which embryonic endoderm differentiates from the embryonic ectoderm. Our results suggest, therefore, that inhibition of endodermal differentiation between 3.5 and 5.5 dpc contributes to the embryonic death of mouse embryos overexpressing exogenous MyoR and consequently that MyoR may serve as a repressor of embryonal endoderm differentiation.

MyoR is expressed in nonmyogenic cells and can inhibit their differentiation.

The development of skeletal muscle in mammals is promoted by the muscle-specific basic helix-loop-helix transcription factors of the MyoD family. Evidence also suggests that there are basic helix-loop-helix proteins that specifically inhibit skeletal myogenesis, including Mtwist, Mist1, and the most recently described, MyoR. It has been suggested that MyoR expression is limited to the precursors of the skeletal muscle lineage and acts as a transcriptional repressor of the muscle differentiation program. However, our results demonstrate that MyoR is expressed in several different, nonmuscle adult tissues. Furthermore, MyoR is expressed in the embryonic ectoderm of blastocyst stage mouse embryos, well before skeletal muscle specification and even before delineation of the mesodermal germ layer. Using embryonic ectoderm analogous stem cells, we demonstrate that in these nonmuscle cells, as in skeletal muscle precursor cells, expression of MyoR is inversely correlated with the extent of cellular differentiation as induced by retinoic acid. Our preliminary results indicate that overexpression of exogenous MyoR inhibits retinoic-acid-induced differentiation in EC cells and is lethal to early mouse embryos. Our results suggest a much broader role for MyoR in the repression and/or determination of embryonic cell differentiation.