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Eukaryotic genomes typically show a uniform G + C content among chromosomes, but on smaller scales, many species have a G + C density that fluctuates with a characteristic wavelength. This oscillation is evident in many insect species, with wavelengths ranging between 700 bp and 4 kb. Measures of evolutionary conservation oscillate in phase with G + C content, with conserved regions having higher G + C. Loci with large regulatory regions show more regular oscillations; coding sequences and heterochromatic regions show little or no oscillation. There is little oscillation in vertebrate genomes in regions with densely distributed mobile repetitive elements. However, species with few repeats show oscillation in both G + C density and sequence conservation. These oscillations may reflect optimal spacing of cis-regulatory elements.

Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in shorter 3’ mRNA isoforms for most genes. This altered poly(A) pattern is extremely similar to that observed in cells containing Pol II derivatives with slow elongation rates. Conversely, cells containing derivatives with fast elongation rates show a subtle downstream shift in poly(A) sites. Polyadenylation patterns of many genes are sensitive to both fast and slow elongation rates, and a global shift of poly(A) utilization is strongly linked to increased purine content of sequences flanking poly(A) sites. Pol II processivity is impaired in diauxic cells, but strains with reduced processivity and normal Pol II elongation rates have normal polyadenylation profiles. Thus, Pol II elongation speed is important for poly(A) site selection and for regulating poly(A) patterns in response to environmental conditions.

Here we describe CLIP-READS, a technique that combines elements of CLIP (Cross-Linking and ImmunoPrecipitation) and 3’ READS (Jin et al., 2015) to provide a genome-wide map of mRNA 3’ isoform binding by a given messenger ribonucleoprotein (mRNP). In CLIP-READS, cells are grown to logarithmic phase and are irradiated with UV light (254nm) to form RNA-protein adducts. The protein-mRNA complexes are immunoprecipitated from cell extracts with an antibody specific to the protein of interest, after which the protein component is digested away with Pronase. Messenger RNAs are then subjected to 3’ region extraction and deep sequencing (3’ READS) (Jin et al., 2015). An input sample processed by 3’ READS in parallel allows for the relative quantification of isoform-specific binding by the mRNP of interest.

Summary Alternative polyadenylation generates numerous 3′ mRNA isoforms that can vary in biological properties, such as stability and localization. We developed methods to obtain transcriptome-scale structural information and protein binding on individual 3′ mRNA isoforms in vivo. Strikingly, near-identical mRNA isoforms can possess dramatically different structures throughout the 3′ UTR. Analyses of identical mRNAs in different species or refolded in vitro indicate that structural differences in vivo are often due to trans-acting factors. The level of Pab1 binding to poly(A)-containing isoforms is surprisingly variable, and differences in Pab1 binding correlate with the extent of structural variation for closely spaced isoforms. A pattern encompassing single-strandedness near the 3′ terminus, double-strandedness of the poly(A) tail, and low Pab1 binding is associated with mRNA stability. Thus, individual 3′ mRNA isoforms can be remarkably different physical entities in vivo. Sequences responsible for isoform-specific structures, differential Pab1 binding, and mRNA stability are evolutionarily conserved, indicating biological function.

SummaryWe measured half-lives of 21,248 mRNA 3′ isoforms in yeast by rapidly depleting RNA polymerase II from the nucleus and performing direct RNA sequencing throughout the decay process. Interestingly, half-lives of mRNA isoforms from the same gene, including nearly identical isoforms, often vary widely. Based on clusters of isoforms with different half-lives, we identify hundreds of sequences conferring stabilization or destabilization upon mRNAs terminating downstream. One class of stabilizing element is a polyU sequence that can interact with poly(A) tails, inhibit the association of poly(A)-binding protein, and confer increased stability upon introduction into ectopic transcripts. More generally, destabilizing and stabilizing elements are linked to the propensity of the poly(A) tail to engage in double-stranded structures. Isoforms engineered to fold into 3′ stem-loop structures not involving the poly(A) tail exhibit even longer half-lives. We suggest that double-stranded structures at 3′ ends are a major determinant of mRNA stability.

In eukaryotes, RNA polymerase II-driven transcription and processing results in the formation of numerous mRNA 3' isoforms that for any given gene may differ from one another by as little as a single nucleotide. These 3' isoforms can vary in physical properties that may affect their function and stability. Here, we outline a systematic framework to measure individual mRNA 3' isoform half-lives on a genome-wide level in S. cerevisiae. Our approach utilizes the Anchor-Away system to sequester RNA polymerase II (Pol II) in the cytoplasm followed by direct single-molecule RNA sequencing to generate a highly detailed view of 3' isoform stability under most physiological conditions without many of the adverse effects associated with commonly used alternative approaches.

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

In Saccharomyces cerevisiae, previous measurements of mRNA stabilities have been determined on a per-gene basis. We and others have recently shown that yeast genes give rise to a highly heterogeneous population of mRNAs thanks to extensive alternative 3' end formation. Typical genes can have fifty or more distinct mRNA isoforms with 3' endpoints differing by as little as one and as many as hundreds of nucleotides. In our recent paper [Geisberg et al. Cell (2014) 156: 812-824] we measured half-lives of individual mRNA isoforms in Saccharomyces cerevisiae by using the anchor away method for the rapid removal of Rpb1, the largest subunit of RNA Polymerase II, from the nucleus, followed by direct RNA sequencing of the cellular mRNA population over time. Combining these two methods allowed us to determine half-lives for more than 20,000 individual mRNA isoforms originating from nearly 5000 yeast genes. We discovered that different 3' mRNA isoforms arising from the same gene can have widely different stabilities, and that such half-life variability across mRNA isoforms from a single gene is highly prevalent in yeast cells. Determining half-lives for many different mRNA isoforms from the same genes allowed us to identify hundreds of RNA sequence elements involved in the stabilization and destabilization of individual isoforms. In many cases, the poly(A) tail is likely to participate in the formation of stability-enhancing secondary structures at mRNA 3' ends. Our results point to an important role for mRNA structure at 3' termini in governing transcript stability, likely by reducing the interaction of the mRNA with the degradation apparatus.

Genome-wide mapping of nucleosomes generated by micrococcal nuclease (MNase) suggests that yeast promoter and terminator regions are very depleted of nucleosomes, predominantly because their DNA sequences intrinsically disfavor nucleosome formation. However, MNase has strong DNA sequence specificity that favors cleavage at promoters and terminators and accounts for some of the correlation between occupancy patterns of nucleosomes assembled in vivo and in vitro. Using an improved method for measuring nucleosome occupancy in vivo that does not involve MNase, we confirm that promoter regions are strongly depleted of nucleosomes, but find that terminator regions are much less depleted than expected. Unlike at promoter regions, nucleosome occupancy at terminators is strongly correlated with the orientation of and distance to adjacent genes. In addition, nucleosome occupancy at terminators is strongly affected by growth conditions, indicating that it is not primarily determined by intrinsic histone–DNA interactions. Rapid removal of RNA polymerase II (pol II) causes increased nucleosome occupancy at terminators, strongly suggesting a transcription-based mechanism of nucleosome depletion. However, the distinct behavior of terminator regions and their corresponding coding regions suggests that nucleosome depletion at terminators is not simply associated with passage of pol II, but rather involves a distinct mechanism linked to 3’-end formation.

Evidence against a genomic code for nucleosome positioning Reply to “Nucleosome sequence preferences influence in vivo nucleosome organization”

We assess the role of intrinsic histone-DNA interactions by mapping nucleosomes assembled in vitro on genomic DNA. Nucleosomes strongly prefer yeast DNA over Escherichia coli DNA, indicating that the yeast genome evolved to favor nucleosome formation. Many yeast promoter and terminator regions intrinsically disfavor nucleosome formation, and nucleosomes assembled in vitro show strong rotational positioning. Nucleosome arrays generated by the ACF assembly factor have fewer nucleosome-free regions, reduced rotational positioning and less translational positioning than obtained by intrinsic histone-DNA interactions. Notably, nucleosomes assembled in vitro have only a limited preference for specific translational positions and do not show the pattern observed in vivo. Our results argue against a genomic code for nucleosome positioning, and they suggest that the nucleosomal pattern in coding regions arises primarily from statistical positioning from a barrier near the promoter that involves some aspect of transcriptional initiation by RNA polymerase II. © 2009 Nature America, Inc. All rights reserved.

Most eukaryotic genes are transcribed into mRNAs with alternative poly(A) sites. Emerging evidence suggests that mRNA isoforms with alternative poly(A) sites can perform critical regulatory functions in numerous biological processes. In recent years, a number of strategies utilizing high-throughput sequencing technologies have been developed to aid in the identification of genome-wide poly(A) sites. This unit describes a modified protocol for a recently published 3'READS (3' region extraction and deep sequencing) method that accurately identifies genome-wide poly(A) sites and that can be used to quantify the relative abundance of the resulting 3' mRNA isoforms. This approach minimizes nonspecific sequence reads due to internal priming and typically yields a high percentage of sequence reads that are ideally suited for accurate poly(A) identification.

In yeast, TFIID strongly associates with nearly all ribosomal protein (RP) promoters, but a TAF-independent form of TBP preferentially associates with other active promoters. RP promoters are regulated in response to growth stimuli, in most cases by a Rap1-containing activator. This Rap1-dependent activator is necessary and sufficient for TFIID recruitment, whereas other activators do not efficiently recruit TFIID. TAFs are recruited to RP promoters even when TBP and other general transcription factors are not associated, suggesting that TFIID recruitment involves a direct activator-TAF interaction. Most RP promoters lack canonical TATA elements, and they are preferentially activated by the Rap1-containing activator. These results demonstrate activator-specific recruitment of TFIID in vivo, and they suggest that TFIID recruitment is important for coordinate expression of RP genes.

Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene-replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site-specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR-based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR- and oligonucleotide-based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene.

The amount of sequence obtained by modern sequencing machines greatly exceeds the sequencing depth requirements of many experiments, especially those involving organisms with small genomes. In the interest of economy and efficiency, various strategies have been developed for multiplexing, in which samples are uniquely tagged with short identifying sequences known as barcodes, pooled, and then sequenced together in a single lane. The resulting combined sequence data are subsequently sorted by barcode before bioinformatic analysis. This unit contains a barcoding protocol for the preparation of up to 96 ChIP samples for multiplex sequencing in a single flow cell lane on the Illumina platform. This strategy may be extended to even larger numbers of samples and may also be generalized to other sequencing applications or sequencing platforms.

The transcription factor TFIID, a central component of the eukaryotic RNA polymerase II (Pol II) transcription apparatus, comprises the TATA-binding protein (TBP) and approximately ten TBP-associated factors (TAFs). Although the essential role of TBP in all eukaryotic transcription has been extensively analysed in vivo and in vitro, the function of the TAFs is less clear. In vitro, TAFs are dispensable for basal transcription but are required for the response to activators. In addition, specific TAFs may act as molecular bridges between particular activators and the general transcription machinery. In vivo, TAFS are required for yeast and mammalian cell growth, but little is known about their specific transcriptional functions. Using conditional alleles created by a new double-shutoff method, we show here that TAF depletion in yeast cells can reduce transcription from some promoters lacking conventional TATA elements. However, TAF depletion has surprisingly little effect on transcriptional enhancement by several activators, indicating that TAFs are not generally required for transcriptional activation in yeast.

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

We use nucleosome maps obtained by high-throughput sequencing to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a secondary role. Furthermore, similar sequence signatures are exhibited by control experiments in which genomic DNA is either sonicated or digested with micrococcal nuclease in the absence of nucleosomes, making it possible that current predictions based on high-throughput nucleosome positioning maps are biased by experimental artifacts., Comment: 36 pages, 13 figures

Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

Disruptions in local chromatin structure often indicate features of biological interest such as regulatory regions. We find that sonication of cross-linked chromatin, when combined with a size-selection step and massively parallel short-read sequencing, can be used as a method (Sono-Seq) to map locations of high chromatin accessibility in promoter regions. Sono-Seq sites frequently correspond to actively transcribed promoter regions, as evidenced by their co-association with RNA Polymerase II ChIP regions, transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3) marks, and CpG islands; signals over other sites, such as those bound by the CTCF insulator, are also observed. The pattern of breakage by Sono-Seq overlaps with, but is distinct from, that observed for FAIRE and DNase I hypersensitive sites. Our results demonstrate that Sono-Seq can be a useful and simple method by which to map many local alterations in chromatin structure. Furthermore, our results provide insights into the mapping of binding sites by using ChIP–Seq experiments and the value of reference samples that should be used in such experiments.

Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-tag context to include the FLAG, HSV, V5 and VSV-G epitopes. The cloning strategy used would be easily applicable to the construction of a similar tag-URA3-tag molecule for essentially any desired epitope. Oligonucleotides designed for PCR from one plasmid may be used interchangeably with any of the other template molecules to allow tagging with different epitopes without the need for new primer synthesis. We have tagged Tfc6 with each of the triple epitope tags and assessed the efficiency of these epitopes for chromatin immunoprecipitation (ChIP). For all the tagged alleles, ChIP occupancy signals are easily detectable at known Tfc6 target genes. These new tags provide additional options in experimental schemes requiring multiple tagged proteins.

The most widely used method for detecting genome-wide protein–DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and “spike-ins” comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated.

Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein-DNA cross-links between molecules that are in close proximity on the chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein-DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome-wide basis. in this unit describes the ChIP procedure for Saccharomyces cerevisiae; describes the corresponding steps for mammalian cells.

This unit describes the combination of chromatin immunoprecipitation (ChIP) with microarray hybridization to determine the genome-wide occupancy profile of a DNA-associated protein. After conventional ChIP, the immunoprecipitated material is amplified by a two-step process involving primer extension followed by PCR in the presence of a modified nucleotide. The amplified DNA is fluorescently labeled in a reaction that couples dye to the modified nucleotide, and the labeled sample is hybridized to a microarray representing a complete genome. This method allows the study of a protein's pattern of DNA association across an entire genome with no need for prior knowledge of potential DNA targets. Keywords: Chromatin immunoprecipitation; ChIP; microarray; amplification; PCR; dye coupling; protein-DNA interactions; ChIP-chip; ChIP-on-chip; genome-wide location; hybridization; whole-genome analysis

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

In yeast cells, preferential accessibility of the HIS3-PET56 promoter region is determined by a general property of the DNA sequence, not by defined sequence elements. In vivo, this region is largely devoid of nucleosomes, and accessibility is directly related to reduced histone density. The HIS3-PET56 and DED1 promoter regions associate poorly with histones in vitro, indicating that intrinsic nucleosome stability is a major determinant of preferential accessibility. Specific and genome-wide analyses indicate that low nucleosome density is a very common feature of yeast promoter regions that correlates poorly with transcriptional activation. Thus, the yeast genome is organized into structurally distinct promoter and nonpromoter regions whose DNA sequences inherently differ with respect to nucleosome formation. This organization ensures that transcription factors bind preferentially to appropriate sites in promoters, rather than to the excess of irrelevant sites in nonpromoter regions.

Genes were originally defined more than a century ago by variants or mutations that alter the phenotype of an organism, and this method of identification remains useful today. Genes have also been defined by virtue of the RNAs or proteins they encode, and in many such cases, the phenotypes of mutations have not been assessed. With the advent of complete genomic sequences, genes are defined primarily by computational analysis. Although powerful, computational methods are necessarily biased by the specific properties used to define the genes. As a consequence, computational analyses often fail to identify real genes or, conversely, identify genes that are biologically meaningless. For example, as defined primarily by open reading frames (ORFs) greater than 99 amino acids, the yeast Saccharomyces cerevisiae was originally found to have 6,275 possible ORFs, 5,885 of which were predicted to be translated into proteins (9). Subsequent identification of transcribed regions of the yeast genome revealed 160 ORFs that had escaped annotation based on the original sequence-derived criteria (39). Recent computational studies based on sequence homologies among closely related yeast species suggest that approximately 500 of the originally defined genes are invalid, and they identify more than 40 smaller genes that were initially unrecognized (2, 19). Thus, in order to define the complete collection of genes in an organism, it is essential to use large-scale experimental approaches to complement the computational analysis. In eukaryotic organisms, RNA polymerase (Pol) III is responsible for the transcription of tRNAs and a few other nontranslated RNAs. Although greatly outnumbered by Pol II-transcribed, mRNA-encoding genes, Pol III genes are transcribed at very high frequencies and in fact constitute a much larger fraction of the total cellular RNA. In the yeast Saccharomyces cerevisiae, the non-tRNA genes transcribed by Pol III include the RNA component of RNase P (RPR1), the U6 small nuclear RNA (SNR6), the cytoplasmic RNA of the signal recognition particle (SCR1), and the 5S rRNA. In mammalian cells, the RNA component of RNase MRP is also transcribed by Pol III; its yeast counterpart NME1 has not been identified as a Pol III gene. Several human genes, including the 7SK gene, the RNase MRP RNA gene, and the U6 RNA gene, have extragenic promoter elements and use a specialized Pol recruitment complex known as SNAPc (36), which is not found in yeast. tRNAs and other Pol III genes share specific sequence and structural properties, including conserved A and B block sequences typically found within the coding region (7), thereby making it possible to search an entire genome for Pol III genes by computer-based methods. Such approaches revealed a total of 275 tRNA genes in the complete S. cerevisiae genome (10, 25, 32), as well as Pol III-transcribed RNA170 (31). Northern analysis of long intergenic regions (31) and recent computational analysis based on conservation of secondary structure across species (26) have revealed a number of previously unpredicted noncoding RNAs. In addition, at least one as-yet-unidentified Pol III gene has been implicated in the processing of the major ribosomal (Pol I-dependent) transcript (13). Thus, it is unclear how many additional Pol III genes exist in the yeast genome. The Pol III transcription apparatus consists of the three multisubunit general transcription factors TFIIIB, TFIIIC, and RNA Pol III, with an additional factor, TFIIIA, playing a role in the transcription of the 5S rRNA (8). The assembly factor TFIIIC consists of six subunits, and it recognizes the conserved A and B blocks. These DNA sequence elements are usually found embedded within the RNA-coding sequence, though occasionally, as with SNR6, the B block may be located outside the transcribed region. TFIIIB, which is recruited by TFIIIC to a region upstream of the transcriptional start site, comprises the TATA-binding protein, TBP, the TFIIB-related factor Brf1, and the SANT domain protein Bdp1. RNA Pol III consists of 17 subunits, 10 of which are unique to Pol III, the others being common to two or all three of the yeast RNA Pols (8, 36). We use chromatin immunoprecipitation, followed by microarray hybridization, to determine the genome-wide distribution of the Pol III transcription apparatus. The Pol III transcriptome in yeast includes essentially all tRNA genes, the previously identified non-tRNA genes, and SNR52, which encodes a small nucleolar RNA. These results are in agreement with recent reports that appeared after the work here was completed (11, 34). Unexpectedly, we identify ZOD1, a locus of unknown function associated with all components of the Pol III machinery, as well as eight ETC loci that are occupied by TFIIIC but not by TFIIIB or Pol III. The B-block sequences and association of the Pol III factors at these loci are conserved across Saccharomyces species, raising the possibilities of regulated Pol III recruitment and novel functions of the Pol III machinery.

Saccharomyces cerevisiae has a global pattern of histone acetylation in which histone H3 and H4 acetylation levels are lower at protein-coding sequences than at promoter regions. The loss of Eaf3, a subunit of the NuA4 histone acetylase and Rpd3 histone deacetylase complexes, greatly alters the genomic profile of histone acetylation, with the effects on H4 appearing to be more pronounced than those on H3. Specifically, the loss of Eaf3 causes increases in H3 and H4 acetylation at coding sequences and decreases at promoters, such that histone acetylation levels become evenly distributed across the genome. Eaf3 does not affect the overall level of H4 acetylation, the recruitment of the NuA4 catalytic subunit Esa1 to target promoters, or the level of transcription of the genes analyzed for histone acetylation. Whole-genome transcriptional profiling indicates that Eaf3 plays a positive, but quantitatively modest, role in the transcription of a small subset of genes, whereas it has a negative effect on very few genes. We suggest that Eaf3 regulates the genomic profile of histone H3 and H4 acetylation in a manner that does not involve targeted recruitment and is independent of transcriptional activity.

The physiological role of TFIIA was investigated by analyzing transcription in a yeast strain that contains a TATA-binding protein (TBP) mutant (N2-1) defective for interacting with TFIIA. In cells containing N2-1, transcription from a set of artificial his3 promoters dependent on different activators is generally reduced by a similar extent, indicating that TFIIA function is largely nonselective for activators. In addition, TATA element utilization, a core promoter function, is altered at his3 promoters dependent on weak activators. Genomic expression analysis reveals that 3% of the genes are preferentially affected by a factor of 4 or more. Chimeras of affected promoters indicate that the sensitivity to the TFIIA-TBP interaction can map either to the upstream or core promoter region. Unlike wild-type TBP or TFIIA, the N2-1 derivative does not activate transcription when artificially recruited to the promoter via a heterologous DNA binding domain, indicating that TFIIA is important for transcription even in the absence of an activation domain. Taken together, these results suggest that TFIIA plays an important role in both activator-dependent and core promoter functions in vivo. Further, they suggest that TFIIA function may not be strictly related to the recruitment of TBP to promoters but may also involve a step after TBP recruitment.

The striking phenomenon of histone acetylases with histone-like substructures that play critical (though distinct) roles in eukaryotic gene regulation raises evolutionary questions. We propose the following speculative scenario that begins with an Archea-like organism containing TBP and histones lacking N-terminal tails. Following the split between Archea and eukaryotes, we imagine a eukaryotic ancestor with histone tails and a histone acetylase that might conceivably be associated with the histones; such an organism would have the ability to modify nucleosome structure. Subsequently, gene duplications and evolutionary divergence results in two sets of histones; the standard nucleosomal histones and the histone-like TAFs that associate with the histone acetylase while losing the ability to form nucleosomes. Next, the primitive histone acetylase complexes diverge into two types, which are distinguished by the catalytic subunit (Gcn5 or TAF-HAT). The TAF-HAT type acquired the ability to interact with TBP, additional TAF subunits, and promoter DNA, whereas the Gcn5 type acquired Ada and Spt subunits to facilitate the interaction with (and hence modification of) nucleosomal histones. Further divergence after the yeast–human split generated novel TAF-like proteins (e.g., PAF65α and PAF65β) that are specific to the human PCAF and hGcn5 complexes. In considering these ideas, it would be of interest to examine very primitive eukaryotes for the presence of histones, TAFs, Spt and Ada proteins, and Gcn5-like histone acetylases.

A major advantage of working with yeast is the ability to replace the wild-type chromosomal copy of a gene with a mutant derivative that is constructed in vitro using a cloned copy of the gene. This technique unavailable in most other eukaryotes allows the phenotype of the mutation to be studied under accurate in vivo conditions, with the mutation present in single copy at its normal chromosomal location. In the first protocol, a plasmid harboring both a selectable marker and a cloned gene of interest is integrated at the chromosomal location of the cloned gene via homologous recombination (integrative transformation). Four methods are described for constructing a mutation in vitro in a cloned gene and reintroducing this mutation at the correct chromosomal site. This allows assessment of the genetic consequences of a mutation, and is often used to determine whether or not a gene is essential (by determining if a complete gene deletion is viable). Two of these techniques integrative disruption and one-step gene disruption generate either insertion or deletion mutations. The third technique transplacement is more generally applicable: it can be used to introduce insertion or deletion mutations containing a selectable marker, but it can also be used to introduce nonselectable mutations, such as conditional lethal mutations in an essential gene. Protocols are also provided to allow creation of modified genes by one-step integrative replacement, and also conditional alleles by a copper-inducible double-shutoff procedure.

In eukaryotic cells the TATA-binding protein (TBP) associates with other proteins known as TBP-associated factors (TAFs) to form multisubunit transcription factors important for gene expression by all three nuclear RNA polymerases. Computer searching of the complete Saccharomyces cerevisiae genome revealed five previously unidentified yeast genes with significant sequence similarity to known human and Drosophila RNA polymerase II TAFs. Each of these genes is essential for viability. A sixth essential gene ( FUN81 ) has previously been noted to be similar to human TAF II 18. Coimmunoprecipitation experiments show that all six proteins are associated with TBP, demonstrating that they are true TAFs. Furthermore, these proteins are present in complexes containing the TAF II 130 subunit, indicating that they are components of TFIID. Based on their predicted molecular weights, these genes have been designated TAF67 , TAF61(68) , TAF40 , TAF23(25) , TAF19 ( FUN81 ), and TAF17 . Yeast TAF61 is significantly larger than its higher eukaryotic homologues, and deletion analysis demonstrates that the evolutionarily conserved, histone-like domain is sufficient and necessary to support viability.

BACKGROUND: Iwr1, a protein conserved throughout eukaryotes, was originally identified by its physical interaction with RNA polymerase (Pol) II. PRINCIPAL FINDINGS: Here, we identify Iwr1 in a genetic screen designed to uncover proteins involved in Pol III transcription in S. cerevisiae. Iwr1 is important for Pol III transcription, because an iwr1 mutant strain shows reduced association of TBP and Pol III at Pol III promoters, a decreased rate of Pol III transcription, and lower steady-state levels of Pol III transcripts. Interestingly, an iwr1 mutant strain also displays reduced association of TBP to Pol I-transcribed genes and of both TBP and Pol II to Pol II-transcribed promoters. Despite this, rRNA and mRNA levels are virtually unaffected, suggesting a post-transcriptional mechanism compensating for the occupancy defect. CONCLUSIONS: Thus, Iwr1 plays an important role in preinitiation complex formation by all three nuclear RNA polymerases.