A new member of the Sin3 family of corepressors is essential for cell viability and required for retroelement propagation in fission yeast.
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)
Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor.
The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and forms a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis. (+info)
Transcriptional repression of the cystic fibrosis transmembrane conductance regulator gene, mediated by CCAAT displacement protein/cut homolog, is associated with histone deacetylation.
Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. Our studies of human CFTR transcription focus on identifying transcription factors bound to an inverted CCAAT consensus or "Y-box element." The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant human CFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. We have found 1) that human CDP/cut acts as a repressor of CFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1; 2) that the ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTR Y-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element; 5) that the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence in CFTR; 6) that immunocomplexes of CDP/cut possess an associated histone deacetylase activity; 7) that the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. We propose that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter. (+info)
A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors.
Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2. The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA. We isolated two classes of mutants: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, we did not isolate any genes involved in rRNA transcription. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4. Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3 also restored mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background. (+info)
A non-isotopic assay for histone deacetylase activity.
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 Smad transcriptional corepressor.
Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell. (+info)
Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes.
The Ikaros gene family encodes zinc finger DNA-binding proteins essential for lineage determination and control of proliferation in the lymphoid system. Here, we report that, in the nucleus of a T cell, a major fraction of Ikaros and Aiolos proteins associate with the DNA-dependent ATPase Mi-2 and histone deacetylases, in a 2 MD complex. This Ikaros-NURD complex is active in chromatin remodeling and histone deacetylation. Upon T cell activation, Ikaros recruits Mi-2/HDAC to regions of heterochromatin. These studies reveal that Ikaros proteins are capable of targeting chromatin remodeling and deacetylation complexes in vivo. We propose that the restructuring of chromatin is a key aspect of Ikaros function in lymphocyte differentiation. (+info)
Overexpression of metastasis-associated MTA1 mRNA in invasive oesophageal carcinomas.
The MTA1 gene is a recently identified novel candidate breast cancer metastasis-associated gene which has been implicated in the signal transduction or regulation of gene expression. We examined the mRNA expression levels of the MTA1, the human homologue of the rat mta1 gene in 47 surgically resected oesophageal squamous cell carcinomas by quantitative reverse transcription polymerase chain reaction. The relative overexpression of MTA1 mRNA (tumour/normal ratio > or = 2) was observed in 16 out of 47 (34.0%) oesophageal carcinomas. Oesophageal tumours overexpressing MTA1 mRNA (T/N ratio > or = 2) showed significantly higher frequencies of adventitial invasion (P < 0.05) and lymph node metastasis (P < 0.05), and tended to have a higher rate of lymphatic involvement than the remaining tumours. Thus, the data suggest that the MTA1 gene might play an important role in invasion and metastasis of oesophageal carcinomas. (+info)