The dynamics of yeast telomeres and silencing proteins through the cell cycle.

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.

Functional interaction between the estrogen receptor and CTF1: analysis of the vitellogenin gene B1 promoter in yeast.

Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at -430 to -270 (+/-20 bp) and at -270 to - 100 (+/-20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at - 101 to -114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure.

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3.

The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-beta (TGF-beta) have been analyzed. We show that TGF-beta specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-beta-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-1 was shown to mediate TGF-beta induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-1-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-1-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-beta in vivo. Moreover, point mutations in the TRD that inhibit TGF-beta induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.

Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains.

Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.