A new member of the Sin3 family of corepressors is essential for cell viability and required for retroelement propagation in fission yeast. (1/2629)

Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1(+), a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1(+) is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1(+) in modulating the nuclear import of Tf1 virus-like particles.  (+info)

Transcriptional regulation of the mouse ferritin H gene. Involvement of p300/CBP adaptor proteins in FER-1 enhancer activity. (2/2629)

We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP.  (+info)

A non-isotopic assay for histone deacetylase activity. (3/2629)

Inhibitors of histone deacetylase (HD) bear great potential as new drugs due to their ability to modulate transcription and to induce apoptosis or differentiation in cancer cells. To study the activity of HD and the effect of potential inhibitors in vitro so far only radio-active assays have existed. For the search of new inhibitors and for the use in HD identification and purification we established a simple, non-radioactive assay that allows screening of large numbers of compounds. The assay is based on an aminocoumarin derivative of an Omega-acetylated lysine as enzyme substrate.  (+info)

A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. (4/2629)

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.  (+info)

Cooperation between phosphorylation and acetylation processes in transcriptional control. (5/2629)

We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118-124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 microM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H1(0) gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148-24153, 1997) showed that the induction of the H1(0) gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.  (+info)

Oxamflatin is a novel antitumor compound that inhibits mammalian histone deacetylase. (6/2629)

Oxamflatin [(2E)-5-[3-[(phenylsufonyl) aminol phenyl]-pent-2-en-4-ynohydroxamic acid] induces transcriptional activation of junD and morphological reversion in various NIH3T3-derived transformed cell lines. We found that oxamflatin showed in vitro antiproliferative activity against various mouse and human tumor cell lines with drastic changes in the cell morphology and in vivo antitumor activity against B16 melanoma. Oxamflatin caused an elongated cell shape with filamentous protrusions as well as arrest of the cell cycle at the G1 phase in HeLa cells. These phenotypic changes of HeLa cells were apparently similar to those by trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC). The effect of oxamflatin on the transcriptional activity of the cytomegalovirus (CMV) promoter was examined and compared with known HDAC inhibitors, TSA, sodium n-butyrate, and FR901228. Oxamflatin as well as all these inhibitors greatly enhanced the transcriptional activity of the CMV promoter in a dose-dependent manner. Oxamflatin, like TSA, inhibited intracellular HDAC activity, as a result of which marked amounts of acetylated histone species accumulated. Finally, effects on expression of several endogenous genes involved in cell morphology and cell cycle control in HeLa cells were analysed. Expression of gelsolin, cyclin E and Cdk inhibitors including p21WAF1/Cip1 was highly augmented, while that of cyclin A and cyclin D1 was decreased by oxamflatin. These results suggest that changes in the expression pattern of the genes regulating cell morphology and the cell cycle due to histone hyperacetylation are responsible for the antitumor activity, the morphological change and the cell cycle arrest induced by oxamflatin.  (+info)

Trichostatin A up-regulates human papillomavirus type 11 upstream regulatory region-E6 promoter activity in undifferentiated primary human keratinocytes. (7/2629)

Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using the lacZ reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the lacZ reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-lacZ and LTR-lacZ in murine retrovirus producer cell lines of fibroblast origin.  (+info)

A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation. (8/2629)

We have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P1. This domain includes the promoter P0, which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation.  (+info)