Construction and characterization of a series of vectors for Schizosaccharomyces pombe.

A set of vectors was created to allow cloning and expression studies in Schizosaccharomyces pombe. These vectors had a uniform backbone with an efficient Sz. pombe ARS, ARS3002, but different selectable markers--his3+, leu1+, ade6+ and ura4+. The vectors functioned efficiently as autonomously replicating plasmids that could also be converted into integrating vectors. The ura4+-containing vector was used to construct a Sz. pombe genomic library.

Sir2p exists in two nucleosome-binding complexes with distinct deacetylase activities.

The absolute requirement for the histone deacetylase activity of Sir2p in silencing coupled with the conservation of Sir2p-like proteins in larger eukaryotes suggests that this molecule plays an important role in gene regulation in all organisms. Here we report the purification and characterization of two Sir2p-containing protein complexes; one of which contains Sir4p and the other Net1p. The Sir4p-containing complex has an NAD-dependent histone deacetylase activity, while the Net1p-containing complex possesses deacetylase activity but only weak NAD-dependent histone deacetylase activity. Finally, we demonstrate that the Sir2p-containing complexes bind nucleosomes efficiently and partially restrict accessibility of the linker DNA to enzymatic probes.

RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. Several DNA elements have been identified that act to separate these chromatin domains. We report a detailed characterization of one of these elements, identifying it as a unique tRNA gene possessing the ability to block the spread of silent chromatin in Saccharomyces cerevisiae efficiently. Transcriptional potential of the tRNA gene is critical for barrier activity, as mutations in the tRNA promoter elements, or in extragenic loci that inhibit RNA polymerase III complex assembly, reduce barrier activity. Also, we have reconstituted the Drosophila gypsy element as a heterochromatin barrier in yeast, and have identified other yeast sequences, including the CHA1 upstream activating sequence, that function as barrier elements. Extragenic mutations in the acetyltransferase genes SAS2 and GCN5 also reduce tRNA barrier activity, and tethering of a GAL4/SAS2 fusion creates a robust barrier. We propose that silencing mediated by the Sir proteins competes with barrier element-associated chromatin remodeling activity.

In vitro transcription.

In vitro analysis of transcription and the factors that play a role in transcription require preparation of an extract that faithfully reproduces in vivo transcription. This unit describes protocols for generating extracts of mammalian cells and Drosophila embryos that are competent for in vitro transcription reactions using naked DNA or chromatin templates and for primer extension analysis of transcripts.

A histone variant, Htz1p, and a Sir1p-like protein, Esc2p, mediate silencing at HMR.

Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.

The RCAF complex mediates chromatin assembly during DNA replication and repair.

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.

A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae.

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed.

Chromatin assembly: biochemical identities and genetic redundancy.

Investigations on chromatin assembly in vitro implicate chromatin assembly factor 1 (CAF1) as a chaperone for histones H3/H4 and nucleosome assembly protein 1 (NAP1) as a chaperone for histones H2A/H2B. Deletion analysis of CAF1 in vivo suggests multiple redundant pathways for deposition of the histones. Histone deposition requires acetylation of the amino-terminal tails and analysis of mutants suggests a specific but redundant role for acetylation of the tails in assembly. Furthermore, studies on the HAT1 acetyltransferase raise the possibility that acetylation of histones occurs following their transport into the nucleus but prior to their deposition onto DNA. Identification of the factors involved in the redundant pathways of assembly is awaited.

The boundaries of the silenced HMR domain in Saccharomyces cerevisiae.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains that provide a mechanism to compact the DNA as well as delineate independent units of gene activity. It is believed that insulator/boundary elements separate these domains. Here we report the identification and characterization of boundary elements that flank the transcriptionally repressed HMR locus in the yeast Saccharomyces cerevisiae. Deletion of these boundary elements led to the spread of silenced chromatin, whereas the ectopic insertion of these elements between a silencer and a promoter blocked the repressive effects of the silencer on that promoter at HMR and at telomeres. Sequence analysis indicated that the boundary element contained a TY1 LTR, and a tRNA gene and mutational analysis has implicated the Smc proteins, which encode structural components of chromosomes, in boundary element function.

Sir- and silencer-independent disruption of silencing in Saccharomyces by Sas10p.

A promoter fusion library of Saccharomyces cerevisiae genes was used to exploit phenotypes associated with altered protein dosage. We identified a novel gene, SAS10, by the ability of Sas10p, when overproduced, to disrupt silencing. The predicted Sas10p was 70,200 kD and strikingly rich in charged amino acids. Sas10p was exclusively nuclear in all stages of the cell cycle. Overproduction of Sas10p caused derepression of mating type genes at both HML and HMR, as well as of URA3, TRP1, and ADE2 when inserted near a telomere or at HMR or the rDNA locus. Repressed genes not associated with silenced chromatin were unaffected. Sas10p was essential for viability, and the termination point following Sas10p depletion was as large budded cells. Remarkably, Sas10p overproduction disrupted silencing even under conditions that bypassed the requirement for Sir proteins, ORC, and Rap1p in silencing. These data implied that Sas10p function was intimately connected with the structure of silenced chromatin.

Silencers and locus control regions: opposite sides of the same coin.

Whether or not genes are in an active or a repressed state in a cell depends on the relative effect of gene silencers and locus control regions (LCRs). Here, we suggest that these elements act as binary switches; the state that prevails (activated or repressed) probably depends on a competition between protein complex formation and the stability of the complexes formed at either of the two elements.

The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.

Postreplicative chromatin assembly by Drosophila and human chromatin assembly factor 1.

To study the relationship between DNA replication and chromatin assembly, we have purified a factor termed Drosophila chromatin assembly factor 1 (dCAF-1) to approximately 50% homogeneity from a nuclear extract derived from embryos. dCAF-1 appears to consist of four polypeptides with molecular masses of 180, 105, 75, and 55 kDa. dCAF-1 preferentially mediates chromatin assembly of newly replicated DNA relative to unreplicated DNA during T-antigen-dependent simian virus 40 DNA replication in vitro, as seen with human CAF-1. Analysis of the mechanism of DNA replication-coupled chromatin assembly revealed that both dCAF-1 and human CAF-1 mediate chromatin assembly preferentially with previously yet newly replicated DNA relative to unreplicated DNA. Moreover, the preferential assembly of the postreplicative DNA was observed at 30 min after inhibition of DNA replication by aphidicolin, but this effect slowly diminished until it was no longer apparent at 120 min after inhibition of replication. These findings suggest that the coupling between DNA replication and chromatin assembly may not necessarily involve a direct interaction between the replication and assembly factors at a replication fork.

Assembly of regularly spaced nucleosome arrays by Drosophila chromatin assembly factor 1 and a 56-kDa histone-binding protein.

To ascertain the mechanism by which nucleosomes are assembled by factors derived from Drosophila embryos, two proteins termed Drosophila chromatin assembly factors (CAFs) 1 and 4 (dCAF-1 and dCAF-4) were fractionated and purified from a Drosophila embryo extract. The assembly of chromatin by dCAF-1, dCAF-4, purified histones, ATP, and DNA is a process that generates regularly spaced nucleosomal arrays with a repeat length that resembles that of bulk native Drosophila chromatin and is not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and dCAF-4 is nearly complete within 10 min. The dCAF-1 activity copurified with the Drosophila version of chromatin assembly factor-1 (CAF-1), a factor that has been found to be required for the assembly of chromatin during large tumor (T) antigen-mediated, simian virus 40 (SV40) origin-dependent DNA replication. The dCAF-4 activity copurified with a 56-kDa core-histone-binding protein that was purified to > 90% homogeneity.

ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates.

GAL4-VP16-mediated nucleosome reconfiguration and transcriptional activation were observed with preassembled chromatin templates that contained regular and physiological nucleosome spacing. Both processes were dependent on adenosine triphosphate (ATP), although binding of GAL4-VP16 to the chromatin was ATP-independent. Factor-mediated nucleosome reconfiguration was not, however, sufficient for transcriptional activation. These experiments recreate in vitro the active participation of nucleosomal cores in the regulation of transcription that occurs in vivo, and they suggest a multistep pathway for transcriptional activation in which factor- and ATP-dependent nucleosome reconfiguration is followed by facilitation by the DNA-bound activator of transcription from the repressed chromatin template.

Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.

The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos.

We describe the preparation and use of a nuclear extract derived from Drosophila embryos that is highly active for transcription in vitro by RNA polymerase This extract, which is termed the soluble nuclear fraction (SNF), can support multiple rounds of transcription and generate about 0.45 transcripts per template per 30 min. Furthermore, the SNF is deficient in nonspecific DNA binding factors that inhibit transcription, such as histone H1, and can be used for the analysis of transcriptional regulation by promoter- and enhancer-binding factors with either naked DNA or chromatin templates.

Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly.

Purified, reconstituted chromatin templates containing regular, physiological nucleosome spacing were transcribed in vitro by RNA polymerase II along with the Gal4-VP16 activator. When Gal4-VP16 was prebound to DNA before reconstitution of either H1-deficient or H1-containing chromatin, the resulting templates were transcribed with a similar efficiency. Under such conditions, we observed long-range (1000 bp) activation of transcription in vitro with H1-containing chromatin, but not naked DNA templates. When Gal4-VP16 was added to preassembled chromatin, the H1-deficient chromatin was transcriptionally active, whereas the H1-containing chromatin, which possessed properties similar to native chromatin, was transcriptionally inert. We then mimicked DNA replication and chromatin assembly at a replication fork and found that Gal4-VP16 could potentiate transcription during, but not after, replication and assembly of histone H1-containing chromatin. These experiments provide biochemical data that support a DNA replication-dependent mechanism for reconfiguration of chromatin structure and activation of transcription by Gal4-VP16.