Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors

NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3′ end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3′ end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription.

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title: In search of antisense.
abstract: In recent years, natural antisense transcripts (NATs) have been implicated in many aspects of eukaryotic gene expression including genomic imprinting, RNA interference, translational regulation, alternative splicing, X-inactivation and RNA editing. Moreover, there is growing evidence to suggest that antisense transcription might have a key role in a range of human diseases. Consequently, there have been several recent attempts to identify novel NATs. To date, approximately 2500 mammalian NATs have been found, indicating that antisense transcription might be a common mechanism of regulating gene expression in human cells. There are increasingly diverse ways in which antisense transcription can regulate gene expression and evidence for the involvement of NATs in human disease is emerging. A range of bioinformatic resources could be used to assist future antisense research.

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title: Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA
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title: Mechanisms of Antisense Transcription Initiation from the 3′ End of the GAL10 Coding Sequence In Vivo
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title: A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster.
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title: H3 lysine 4 di‐ and tri‐methylation deposited by cryptic transcription attenuates promoter activation
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title: The 19S proteasome subcomplex promotes the targeting of NuA4 HAT to the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional initiation in vivo
abstract: Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

title: Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo
abstract: (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions.

title: Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase.
abstract: The Esa1-containing NuA4 histone acetylase complex can interact with activation domains in vitro and stimulate transcription on reconstituted chromatin templates. In yeast cells, Esa1 is targeted to a small subset of promoters in an activator-specific manner. Esa1 is specifically recruited to ribosomal protein (RP) promoters, and this recruitment appears to require binding by Rap1 or Abf1. Esa1 is important for RP transcription, and Esa1 recruitment to RP promoters correlates with coordinate regulation of RP genes in response to growth stimuli. However, following Esa1 depletion, H4 acetylation decreases dramatically at many loci, but transcription is not generally affected. Therefore, the transcription-associated targeted recruitment of Esa1 to RP promoters occurs in a background of more global nontargeted acetylation that is itself not required for transcription.

title: Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae
abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one‐step PCR‐mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C‐ or N‐terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N‐ or C‐terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.

title: A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
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title: Esa1p Is an Essential Histone Acetyltransferase Required for Cell Cycle Progression
abstract: Histones are dynamically modified during chromatin assembly, as specific transcriptional patterns are established, and during mitosis and development. Modifications include acetylation, phosphorylation, ubiquitination, methylation, and ADP-ribosylation, but the biological significance of each of these is not well understood. For example, distinct acetylation patterns correlate with nucleosome formation and with transcriptionally activated or silenced chromatin, yet mutations in genes encoding several yeast histone acetyltransferase (HAT) activities result in either no cellular phenotype or only modest growth defects. Here we report characterization of ESA1, an essential gene that is a member of the MYST family that includes two yeast silencing genes, human genes associated with leukemia and with the human immunodeficiency virus type 1 Tat protein, and Drosophila mof, a gene essential for male dosage compensation. Esa1p acetylates histones in a pattern distinct from those of other yeast enzymes, and temperature-sensitive mutant alleles abolish enzymatic activity in vitro and result in partial loss of an acetylated isoform of histone H4 in vivo. Strains carrying these mutations are also blocked in the cell cycle such that at restrictive temperatures,esa1 mutants succeed in replicating their DNA but fail to proceed normally through mitosis and cytokinesis. Recent studies show that Esa1p enhances transcription in vitro and thus may modulate expression of genes important for cell cycle control. These observations therefore link an essential HAT activity to cell cycle progression, potentially through discrete transcriptional regulatory events.

title: The Swi/Snf Complex Is Important for Histone Eviction during Transcriptional Activation and RNA Polymerase II Elongation In Vivo
abstract: The Swi/Snf nucleosome-remodeling complex is recruited by DNA-binding activator proteins, whereupon it alters chromatin structure to increase preinitiation complex formation and transcription. At the SUC2 promoter, the Swi/Snf complex is required for histone eviction in a manner that is independent of transcriptional activity. Swi/Snf travels through coding regions with elongating RNA polymerase (Pol) II, and swi2 mutants exhibit sensitivity to drugs affecting Pol elongation. In FACT-depleted cells, Swi/Snf is important for internal initiation within coding regions, suggesting that Swi/Snf is important for histone eviction that occurs during Pol II elongation. Taken together, these observations suggest that Swi/Snf is important for histone eviction at enhancers and that it also functions as a Pol II elongation factor.

title: Opposing roles for Set2 and yFACT in regulating TBP binding at promoters
abstract: Previous work links histone methylation by Set2 with transcriptional elongation. yFACT (Spt16–Pob3 and Nhp6) reorganizes nucleosomes and functions in both transcriptional initiation and elongation. We show that growth defects caused by spt16 or pob3 mutations can be suppressed by deleting SET2, suggesting that Set2 and yFACT have opposing roles. Set2 methylates K36 of histone H3, and K36 substitutions also suppress yFACT mutations. In contrast, set1 enhances yFACT mutations. Methylation at H3 K4 by Set1 is required for set2 to suppress yFACT defects. We did not detect an elongation defect at an 8 kb ORF in yFACT mutants. Instead, pob3 mutants displayed reduced binding of both pol II and TBP to the GAL1 promoter. Importantly, both GAL1 transcription and promoter binding of pol II and TBP are significantly restored in the pob3 set2 double mutant. Defects caused by an spt16 mutation are enhanced by either TBP or TFIIA mutants. These synthetic defects are suppressed by set2, demonstrating that yFACT and Set2 oppose one another during transcriptional initiation at a step involving DNA binding by TBP and TFIIA.

title: SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae
abstract: Mutations selected as suppressors of Ty and solo delta insertion mutations is Saccharomyces cerevisiae have identified a number of genes important for transcription initiation. One of these gens, SPT15, encodes the TATA-binding protein, and three others, SPT3, SPT7, and SPT8, encode proteins functionally related to the TATA-binding protein. To identify additional related functions, we have selected for new spt mutations. This work has identified one new gene, SPT20. Null mutations in SPT20 cause poor growth and a set of severe transcriptional defects very similar to those caused by null mutations in SPT3, SPT7, and SPT8 and also very similar to those caused by certain missense mutations in SPT15. Consistent with its having an important function in transcription in vivo, SPT20 was also recently identified as ADA5 and has been shown to be important for transcriptional activation (G.A. Marcus,   1996.

title: Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B.
abstract: Covalent modifications of the histone N tails play important roles in eukaryotic gene expression. Histone acetylation, in particular, is required for the activation of a subset of eukaryotic genes through the targeted recruitment of histone acetyltransferases. We have reported that a histone C tail modification, ubiquitylation of H2B, is required for optimal expression of several inducible yeast genes, consistent with a role in transcriptional activation. H2B was shown to be ubiquitylated and then deubiquitylated at the GAL1 core promoter following galactose induction. We now show that the Rad6 protein, which catalyzes monoubiquitylation of H2B, is transiently associated with the GAL1 promoter upon gene activation, and that the period of its association temporally overlaps with the period of H2B ubiquitylation. Rad6 promoter association depends on the Gal4 activator and the Rad6-associated E3 ligase, Bre1, but is independent of the histone acetyltransferase, Gcn5. The SAGA complex, which contains a ubiquitin protease that targets H2B for deubiquitylation, is recruited to the GAL1 promoter in the absence of H2B ubiquitylation. The data suggest that Rad6 and SAGA function independently during galactose induction, and that the staged recruitment of these two factors to the GAL1 promoter regulates the ubiquitylation and deubiquitylation of H2B. We additionally show that both Rad6 and ubiquitylated H2B are absent from two regions of transcriptionally silent chromatin but present at genes that are actively transcribed. Thus, like histone H3 lysine 4 and lysine 79 methylation, two modifications that it regulates, Rad6-directed H2B ubiquitylation defines regions of active chromatin.

title: Elongating RNA Polymerase II Is Disassembled through Specific Degradation of Its Largest but Not Other Subunits in Response to DNA Damage in Vivo*
abstract: Although previous biochemical studies have demonstrated global degradation of the largest subunit, Rpb1p, of RNA polymerase II in response to DNA damage, it is still not clear whether the initiating or elongating form of Rpb1p is targeted for degradation in vivo. Further, whether other components of RNA polymerase II are degraded in response to DNA damage remains unknown. Here, we show that the Rpb1p subunit of the elongating, but not initiating, form of RNA polymerase II is degraded at the active genes in response to 4-nitroquinoline-1-oxide-induced DNA damage in Saccharomyces cerevisiae. However, other subunits of RNA polymerase II are not degraded in response to DNA damage. Further, we show that Rpb1p is essential for RNA polymerase II assembly at the active gene, and thus, the degradation of Rpb1p following DNA damage disassembles elongating RNA polymerase II. Taken together, our data demonstrate that Rpb1p but not other subunits of elongating RNA polymerase II is specifically degraded in response to DNA damage, and such a degradation of Rpb1p is critical for the disassembly of elongating RNA polymerase II at the DNA lesion in vivo.

title: Rad26p, a transcription-coupled repair factor, is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner in vivo
abstract: Rad26p, a yeast homologue of human Cockayne syndrome B with an ATPase activity, plays a pivotal role in stimulating DNA repair at the coding sequences of active genes. On the other hand, DNA repair at inactive genes or silent areas of the genome is not regulated by Rad26p. However, how Rad26p recognizes DNA lesions at the actively transcribing genes to facilitate DNA repair is not clearly understood in vivo. Here, we show that Rad26p associates with the coding sequences of genes in a transcription-dependent manner, but independently of DNA lesions induced by 4-nitroquinoline-1-oxide in Saccharomyces cerevisiae. Further, histone H3 lysine 36 methylation that occurs at the active coding sequence stimulates the recruitment of Rad26p. Intriguingly, we find that Rad26p is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner. However, Rad26p does not recognize DNA lesions in the absence of active transcription. Together, these results provide an important insight as to how Rad26p is delivered to the damage sites at the active, but not inactive, genes to stimulate repair in vivo, shedding much light on the early steps of transcription-coupled repair in living eukaryotic cells.

title: Functional Analysis of Bre1p, an E3 Ligase for Histone H2B Ubiquitylation, in Regulation of RNA Polymerase II Association with Active Genes and Transcription in Vivo*
abstract: Bre1p is required for H2B ubiquitylation that promotes RNA polymerase II association with active genes, and hence transcription.  The RING domain of Bre1p facilitates, but a non-RING domain represses, transcription and RNA polymerase II association with active genes.  Bre1p has a dominant-negative role in addition to its well known stimulatory function in transcription.  This study unravels a hidden role of Bre1p in transcriptional regulation. H2B ubiquitylation is carried out by Bre1p, an E3 ligase, along with an E2 conjugase, Rad6p. H2B ubiquitylation has been previously implicated in promoting the association of RNA polymerase II with the coding sequence of the active GAL1 gene, and hence transcriptional elongation. Intriguingly, we find here that the association of RNA polymerase II with the active GAL1 coding sequence is not decreased in Δbre1, although it is required for H2B ubiquitylation. In contrast, the loss of Rad6p significantly impairs the association of RNA polymerase II with GAL1. Likewise, the point mutation of lysine 123 (ubiquitylation site) to arginine of H2B (H2B-K123R) also lowers the association of RNA polymerase II with GAL1, consistent with the role of H2B ubiquitylation in promoting RNA polymerase II association. Surprisingly, unlike the Δrad6 and H2B-K123R strains, complete deletion of BRE1 does not impair the association of RNA polymerase II with GAL1. However, deletion of the RING domain of Bre1p (that is essential for H2B ubiquitylation) impairs RNA polymerase II association with GAL1. These results imply that a non-RING domain of Bre1p counteracts the stimulatory role of the RING domain in regulating the association of RNA polymerase II with GAL1, and hence RNA polymerase II occupancy is not impaired in Δbre1. Consistently, GAL1 transcription is impaired in the absence of the RING domain of Bre1p, but not in Δbre1. Similar results are also obtained at other genes. Collectively, our results implicate both the stimulatory and repressive roles of Bre1p in regulation of RNA polymerase II association with active genes (and hence transcription) in vivo.

title: Evidence That Proteolysis of Gal4 Cannot Explain the Transcriptional Effects of Proteasome ATPase Mutations*
abstract: The Gal system of Saccharomyces cerevisiae is a paradigm for eukaryotic gene regulation. Expression of genes required for growth on galactose is regulated by the transcriptional activator Gal4. The activation function of Gal4 has been localized to 34 amino acids near the C terminus of the protein. The gal4D allele of GAL4 encodes a truncated protein in which only 14 amino acids of the activation domain remain. Expression of GAL genes is dramatically reduced in gal4Dstrains and these strains are unable to grow on galactose as the sole carbon source. Overexpression of gal4D partially relieves the defect in GAL gene expression and allows growth on galactose. A search for extragenic suppressors of gal4D identified recessive mutations in the SUG1 and SUG2 genes, which encode ATPases of the 19S regulatory complex of the proteasome. The proteasome is responsible for the ATP-dependent degradation of proteins marked for destruction by the ubiquitin system. It has been commonly assumed that effects of SUG1 and SUG2mutations on transcription are explained by alterations in the proteolysis of gal4D protein. We have investigated this assumption. Surprisingly, we find that SUG1 and SUG2alleles that are unable to suppress gal4D cause a larger increase in gal4D protein levels than do suppressing alleles. In addition, mutations in genes encoding subunits of the proteolytic 20S sub-complex of the proteasome increase the levels of gal4D protein but do not rescue its transcriptional activity. Therefore, an alteration in the proteolysis of gal4D by the proteasome cannot explain the effects of mutations in SUG1 and SUG2 on expression of GAL genes. These findings suggest that the 19S regulatory complex may play a more direct role in transcription.

title: Ubp8p, a Histone Deubiquitinase Whose Association with SAGA Is Mediated by Sgf11p, Differentially Regulates Lysine 4 Methylation of Histone H3 In Vivo
abstract: Despite recent advances in characterizing the regulation of histone H3 lysine 4 (H3-K4) methylation at the GAL1 gene by the H2B-K123-specific deubiquitinase activity of Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase)-associated Ubp8p, our knowledge on the general role of Ubp8p at the SAGA-dependent genes is lacking. For this study, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay, we have analyzed the role of Ubp8p in the regulation of H3-K4 methylation at three other SAGA-dependent yeast genes, namely, PHO84, ADH1, and CUP1. Like that at GAL1, H3-K4 methylation is increased at the PHO84 core promoter in the UBP8 deletion mutant. We also show that H3-K4 methylation remains invariant at the PHO84 open reading frame in the Δubp8 mutant, demonstrating a highly localized role of Upb8p in regulation of H3-K4 methylation at the promoter in vivo. However, unlike that at PHO84, H3-K4 methylation at the two other SAGA-dependent genes is not controlled by Ubp8p. Interestingly, Ubp8p and H3-K4 methylation are dispensable for preinitiation complex assembly at the core promoters of these genes. Our ChIP assay further demonstrates that the association of Ubp8p with SAGA is mediated by Sgf11p, consistent with recent biochemical data. Collectively, the data show that Ubp8p differentially controls H3-K4 methylation at the SAGA-dependent promoters, revealing a complex regulatory network of histone methylation in vivo.

title: A functional interaction between the C-terminal domain of RNA polymerase II and the negative regulator SIN1
abstract: The C-terminal domain (CTD) of the largest subunit of yeast RNA polymerase II contains 26-27 tandem copies of a conserved heptapeptide of unknown function. Yeast strains whose CTD contains ten heptamers are viable but defective for transcription of the INO1 gene and cold sensitive for growth. Deletion of the SIN1 gene, which codes for a DNA-binding protein that negatively regulates HO transcription, restores INO1 transcription and reduces the cold sensitivity of such strains. A SIN1 deletion suppresses the lethality of a CTD with nine heptamer repeats but not with seven repeats. These observations indicate a functional relationship between SIN1 and the CTD: the CTD might remove SIN1 from DNA, or removal of SIN1 may be a prerequisite for function of the CTD. The SWI1, SWI2, and SWI3 genes, whose products activate HO transcription by antagonizing SIN1, are also required for INO1 transcription and may assist the CTD. In addition, an intact CTD binds nonspecifically to DNA in vitro.

title: Homopurine and homopyrimidine strands complementary in parallel orientation form an antiparallel duplex at neutral pH with A-C, G-T, and T-C mismatched base pairs.
abstract: DNA sequences d-TGAGGAAAGAAGGT (a 14-mer) and d-CTCCTTTCTTCC (a 12-mer) are complementary in parallel orientation forming either Donohue (reverse Watson-Crick) base pairing at neutral pH or Hoogsteen base pairing at slightly acidic pH. The structure of the complex formed by dissolving the two strands in equimolar ratio in water has been investigated by nmr. At neutral pH, the system forms an ordered antiparallel duplex with five A : T and four G : C Watson-Crick base pairs and three mismatches, namely G-T, A-C, and T-C. The nuclear Overhauser effect cross-peak pattern suggests an overall B-DNA conformation with major structural perturbations near the mismatches. The duplex has a low melting point and dissociates directly into single strands with a broad melting profile. The hydrogen-bonding schemes in the mismatched base pairs have been investigated. It has been shown earlier that in acidic pH, the system prefers a triple-stranded structure with two pyrimidine strands and one purine strand. One of the pyrimidine strands has protonated cytosines, forms Hoogsteen base pairing, and is aligned parallel to the purine strand; the other has nonprotonated cytosines and has base-pairing scheme similar to the one discussed in this paper. The parallel duplex is therefore less stable than either the antiparallel duplex or the triplex, in spite of its perfect complementarity.

title: Stability of DNA duplexes containing GG, CC, AA, and TT mismatches.
abstract: We employed salt-dependent differential scanning calorimetric measurements to characterize the stability of six oligomeric DNA duplexes (5'-GCCGGAXTGCCGG-3'/5'-CCGGCAYTCCGGC-3') that contain in the central XY position the GC, AT, GG, CC, AA, or TT base pair. The heat-induced helix-to-coil transitions of all the duplexes are associated with positive changes in heat capacity, DeltaC(p), ranging from 0.43 to 0.53 kcal/mol. Positive values of DeltaC(p) result in strong temperature dependences of changes in enthalpy, DeltaH degrees, and entropy, DeltaS degrees , accompanying duplex melting and cause melting free energies, DeltaG degrees, to exhibit characteristically curved shapes. These observations suggest that DeltaC(p) needs to be carefully taken into account when the parameters of duplex stability are extrapolated to temperatures distant from the transition temperature, T(M). Comparison of the calorimetric and van't Hoff enthalpies revealed that none of the duplexes studied in this work exhibits two-state melting. Within the context of the central AXT/TYA triplet, the thermal and thermodynamic stabilities of the duplexes in question change in the following order: GC > AT > GG > AA approximately TT > CC. Our estimates revealed that the thermodynamic impact of the GG, AA, and TT mismatches is confined within the central triplet. In contrast, the thermodynamic impact of the CC mismatch propagates into the adjacent helix domains and may involve 7-9 bp. We discuss implications of our results for understanding the origins of initial recognition of mismatched DNA sites by enzymes of the DNA repair machinery.

title: A rule of seven in Watson-Crick base pairing of mismatched sequences
abstract: Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid–based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

title: Functional Dissection of the NuA4 Histone Acetyltransferase Reveals Its Role as a Genetic Hub and that Eaf1 Is Essential for Complex Integrity
abstract: The Saccharomyces cerevisiae NuA4 histone acetyltransferase complex catalyzes the acetylation of histone H4 and the histone variant Htz1 to regulate key cellular events, including transcription, DNA repair, and faithful chromosome segregation. To further investigate the cellular processes impacted by NuA4, we exploited the nonessential subunits of the complex to build an extensive NuA4 genetic-interaction network map. The map reveals that NuA4 is a genetic hub whose function buffers a diverse range of cellular processes, many not previously linked to the complex, including Golgi complex-to-vacuole vesicle-mediated transport. Further, we probe the role that nonessential subunits play in NuA4 complex integrity. We find that most nonessential subunits have little impact on NuA4 complex integrity and display between 12 and 42 genetic interactions. In contrast, the deletion of EAF1 causes the collapse of the NuA4 complex and displays 148 genetic interactions. Our study indicates that Eaf1 plays a crucial function in NuA4 complex integrity. Further, we determine that Eaf5 and Eaf7 form a subcomplex, which reflects their similar genetic interaction profiles and phenotypes. Our integrative study demonstrates that genetic interaction maps are valuable in dissecting complex structure and provides insight into why the human NuA4 complex, Tip60, has been associated with a diverse range of pathologies.

title: Eaf1 Is the Platform for NuA4 Molecular Assembly That Evolutionarily Links Chromatin Acetylation to ATP-Dependent Exchange of Histone H2A Variants
abstract: Eaf1 (for Esa1-associated factor 1) and Eaf2 have been identified as stable subunits of NuA4, a yeast histone H4/H2A acetyltransferase complex implicated in gene regulation and DNA repair. While both SWI3-ADA2-N-CoR-TF IIIB domain-containing proteins are required for normal cell cycle progression, their depletion does not affect the global Esa1-dependent acetylation of histones. In contrast to all other subunits, Eaf1 is found exclusively associated with the NuA4 complex in vivo. It serves as a platform that coordinates the assembly of functional groups of subunits into the native NuA4 complex. Eaf1 shows structural similarities with human p400/Domino, a subunit of the NuA4-related TIP60 complex. On the other hand, p400 also possesses an SWI2/SNF2 family ATPase domain that is absent from the yeast NuA4 complex. This domain is highly related to the yeast Swr1 protein, which is responsible for the incorporation of histone variant H2AZ in chromatin. Since all of the components of the TIP60 complex are homologous to SWR1 or NuA4 subunits, we proposed that the human complex corresponds to a physical merge of two yeast complexes. p400 function in TIP60 then would be accomplished in yeast by cooperation between SWR1 and NuA4. In agreement with such a model, NuA4 and SWR1 mutants show strong genetic interactions, NuA4 affects histone H2AZ incorporation/acetylation in vivo, and both preset the PHO5 promoter for activation. Interestingly, the expression of a chimeric Eaf1-Swr1 protein recreates a single human-like complex in yeast cells. Our results identified the key central subunit for the structure and functions of the NuA4 histone acetyltransferase complex and functionally linked this activity with the histone variant H2AZ from yeast to human cells.

title: A glycolytic burst drives glucose induction of global histone acetylation by picNuA4 and SAGA
abstract: Little is known about what enzyme complexes or mechanisms control global lysine acetylation in the amino-terminal tails of the histones. Here, we show that glucose induces overall acetylation of H3 K9, 18, 27 and H4 K5, 8, 12 in quiescent yeast cells mainly by stimulating two KATs, Gcn5 and Esa1. Genetic and pharmacological perturbation of carbon metabolism, combined with 1H-NMR metabolic profiling, revealed that glucose induction of KAT activity directly depends on increased glucose catabolism. Glucose-inducible Esa1 and Gcn5 activities predominantly reside in the picNuA4 and SAGA complexes, respectively, and act on chromatin by an untargeted mechanism. We conclude that direct metabolic regulation of globally acting KATs can be a potent driving force for reconfiguration of overall histone acetylation in response to a physiological cue.

title: The Saccharomyces cerevisiae Piccolo NuA4 Histone Acetyltransferase Complex Requires the Enhancer of Polycomb A Domain and Chromodomain To Acetylate Nucleosomes
abstract: Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

title: The Tor Pathway Regulates Gene Expression by Linking Nutrient Sensing to Histone Acetylation
abstract: The Tor pathway mediates cell growth in response to nutrient availability, in part by inducing ribosomal protein (RP) gene expression via an unknown mechanism. Expression of RP genes coincides with recruitment of the Esa1 histone acetylase to RP gene promoters. We show that inhibition of Tor with rapamycin releases Esa1 from RP gene promoters and leads to histone H4 deacetylation without affecting promoter occupancy by Rap1 and Abf1. Genetic and biochemical evidence identifies Rpd3 as the major histone deacetylase responsible for reversing histone H4 acetylation at RP gene promoters in response to Tor inhibition by rapamycin or nutrient limitation. Our results illustrate that the Tor pathway links nutrient sensing with histone acetylation to control RP gene expression and cell growth.

title: Evidence that Set1, a Factor Required for Methylation of Histone H3, Regulates rDNA Silencing in S. cerevisiae by a Sir2-Independent Mechanism
abstract: Several types of histone modifications have been shown to control transcription. Recent evidence suggests that specific combinations of these modifications determine particular transcription patterns. The histone modifications most recently shown to play critical roles in transcription are arginine-specific and lysine-specific methylation. Lysine-specific histone methyltransferases all contain a SET domain, a conserved 130 amino acid motif originally identified in polycomb- and trithorax-group proteins from Drosophila. Members of the SU(VAR)3-9 family of SET-domain proteins methylate K9 of histone H3. Methylation of H3 has also been shown to occur at K4. Several studies have suggested a correlation between K4-methylated H3 and active transcription. In this paper, we provide evidence that K4-methylated H3 is required in a negative role, rDNA silencing in Saccharomyces cerevisiae. In a screen for rDNA silencing mutants, we identified a mutation in SET1, previously shown to regulate silencing at telomeres and HML. Recent work has shown that Set1 is a member of a complex and is required for methylation of K4 of H3 at several genomic locations. In addition, we demonstrate that a K4R change in H3, which prevents K4 methylation, impairs rDNA silencing, indicating that Set1 regulates rDNA silencing, directly or indirectly, via H3 methylation. Furthermore, we present several lines of evidence that the role of Set1 in rDNA silencing is distinct from that of the histone deacetylase Sir2. Together, these results suggest that Set1-dependent H3 methylation is required for rDNA silencing in a Sir2-independent fashion.

title: Histone Crosstalk between H2B Monoubiquitination and H3 Methylation Mediated by COMPASS
abstract: COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.

title: Active genes are tri-methylated at K4 of histone H3
abstract: Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.

title: H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A
abstract: Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug  A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.

title: Set3 HDAC Mediates Effects of Overlapping Noncoding Transcription on Gene Induction Kinetics
abstract: The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.

title: Cotranscriptional Set2 Methylation of Histone H3 Lysine 36 Recruits a Repressive Rpd3 Complex
abstract: The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.

title: Histone H3 Methylation by Set2 Directs Deacetylation of Coding Regions by Rpd3S to Suppress Spurious Intragenic Transcription
abstract: Yeast Rpd3 histone deacetylase plays an important role at actively transcribed genes. We characterized two distinct Rpd3 complexes, Rpd3L and Rpd3S, by MudPIT analysis. Both complexes shared a three subunit core and Rpd3L contains unique subunits consistent with being a promoter targeted corepressor. Rco1 and Eaf3 were subunits specific to Rpd3S. Mutants of RCO1 and EAF3 exhibited increased acetylation in the FLO8 and STE11 open reading frames (ORFs) and the appearance of aberrant transcripts initiating within the body of these ORFs. Mutants in the RNA polymerase II-associated SET2 histone methyltransferase also displayed these defects. Set2 functioned upstream of Rpd3S and the Eaf3 methyl-histone binding chromodomain was important for recruitment of Rpd3S and for deacetylation within the STE11 ORF. These data indicate that Pol II-associated Set2 methylates H3 providing a transcriptional memory which signals for deacetylation of ORFs by Rpd3S. This erases transcription elongation-associated acetylation to suppress intragenic transcription initiation.

title: Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange
abstract: Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, but it has a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions. In this way, Isw1b and Chd1 are important in maintaining chromatin integrity during transcription elongation by RNAPII.

title: Histone methylation and ubiquitination with their cross-talk and roles in gene expression and stability
abstract: Methylation of lysine residues of histones is associated with functionally distinct regions of chromatin, and, therefore, is an important epigenetic mark. Over the past few years, several enzymes that catalyze this covalent modification on different lysine residues of histones have been discovered. Intriguingly, histone lysine methylation has also been shown to be cross-regulated by histone ubiquitination or the enzymes that catalyze this modification. These covalent modifications and their cross-talks play important roles in regulation of gene expression, heterochromatin formation, genome stability, and cancer. Thus, there has been a very rapid progress within past several years towards elucidating the molecular basis of histone lysine methylation and ubiquitination, and their aberrations in human diseases. Here, we discuss these covalent modifications with their cross-regulation and roles in controlling gene expression and stability.

title: H2B-K123 ubiquitination stimulates RNAPII elongation independent of H3-K4 methylation.
abstract: Eukaryotic gene regulation is closely correlated with histone covalent modifications. Recently, histone H2B lysine-123 (H2B-K123) ubiquitination has been implicated in regulation of transcription as well as histone H3 lysine-4 (H3-K4) methylation which is further associated with active transcription. However, whether H2B-K123 ubiquitination controls transcription through regulation of H3-K4 methylation remains unknown under physiological conditions. Here, we show that H2B-K123 ubiquitination enhances the rate of elongating RNA polymerase II (RNAPII) recruitment to the coding sequence of an inducible yeast gene, GAL1. Consistently, GAL1 transcription is significantly impaired in absence of H2B-K123 ubiquitination. On the other hand, H3-K4 methylation does not alter the rate of elongating RNAPII recruitment at GAL1. Further, these covalent modifications do not regulate pre-initiation complex formation at GAL1. Collectively, our data demonstrate the function of H2B-K123 ubiquitination in regulation of transcriptional elongation independently of H3-K4 methylation in vivo, providing a new insight on epigenetic regulation of gene expression.

title: H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation.
abstract: The monoubiquitylation of histone H2B has been associated with transcription initiation and elongation, but its role in these processes is poorly understood. We report that H2B ubiquitylation is required for efficient reassembly of nucleosomes during RNA polymerase II (Pol II)-mediated transcription elongation in yeast. This role is carried out in cooperation with the histone chaperone Spt16, and in the absence of H2B ubiquitylation and functional Spt16, chromatin structure is not properly restored in the wake of elongating Pol II. Moreover, H2B ubiquitylation and Spt16 play a role in each other's regulation. H2B ubiquitylation is required for the stable accumulation of Spt16 at the GAL1 coding region, and Spt16 regulates the formation of ubiquitylated H2B both globally and at the GAL1 gene. These data provide a mechanism linking H2B ubiquitylation to Spt16 in the regulation of nucleosome dynamics during transcription elongation.

title: Activator-specific recruitment of Mediator in vivo
abstract: The Mediator complex associates with eukaryotic RNA polymerase (Pol) II and is recruited to transcriptional enhancers by activator proteins. It is believed that Mediator is a general component of the Pol II machinery that is crucial to connect enhancer-bound activators to basic transcription factors. However, we show that Mediator does not detectably associate with many highly active Pol II promoters in yeast cells. Furthermore, in response to stress conditions, Mediator association is not directly related to Pol II association and in some cases is not detectable at highly activated promoters. Thus, Mediator is recruited to enhancers in an activator-specific manner, and it does not seem to be a stoichiometric component of the basic Pol II machinery in vivo. Mediator is recruited by many activators involved in stress responses, but not by the major activators that function under optimal conditions.

title: Direct Bre1-Paf1 Complex Interactions and RING Finger-independent Bre1-Rad6 Interactions Mediate Histone H2B Ubiquitylation in Yeast*
abstract: Recent yeast genetic studies have implicated the ubiquitin-conjugating enzyme and ubiquitin ligase functions of yRad6 and yBre1, respectively, in H2B ubiquitylation. However, there have been no corresponding biochemical analyses demonstrating intrinsic enzyme activities of yRad6 and yBre1 or related mechanistic details. Here, we describe a robust in vitro chromatin ubiquitylation assay that involves purified H2B ubiquitylation factors and natural nucleosomes. Our results indicate that yRad6 has an in vitro ability to nonspecifically ubiquitylate all core histones in the absence of an ubiquitin ligase but that yBre1 functions, through direct interactions with yRad6, to direct the ubiquitin conjugating activity of yRad6 toward the physiological H2B ubiquitylation site. Moreover, a yRad6 domain mapping analysis shows that an intact UBC domain is required for binding to yBre1, whereas the C-terminal acidic tail domain that is not required for a stable yBre1-yRad6 interaction is necessary for full enzyme activity of yRad6. We also find that, analogous to heteromeric complex formation by BRE1 paralogues in other organisms, yBre1 forms a homo-multimeric complex. Of special significance, our detailed biochemical analyses further show that the yBre1 RING finger domain is essential for H2B ubiquitylation but, surprisingly, dispensable for interaction of yBre1 with yRad6. In further support of the genetically identified requirement of the RNA polymerase II-associated yPaf1 complex for H2B ubiquitylation, protein interaction studies reveal that a purified yPaf1 complex directly and selectively interacts with yBre1 and thus serves to link the H2B ubiquitylation and general transcription machineries. These studies provide a more detailed mechanistic basis for H2B ubiquitylation in yeast.

title: The Rtf1 Component of the Paf1 Transcriptional Elongation Complex Is Required for Ubiquitination of Histone H2B*
abstract: In yeast cells, the Rtf1 and Paf1 components of the Paf1 transcriptional elongation complex are important for recruitment of Set1, the histone H3-lysine 4 (H3-Lys4) methylase, to a highly localized domain at the 5′ portion of active mRNA coding regions. Here, we show that Rtf1 is essential for global methylation of H3-Lys4 and H3-Lys79, but not H3-Lys36. This role of Rtf1 resembles that of Rad6, which mediates ubiquitination of histone H2B at lysine 123. Indeed, Rtf1 is required for H2B ubiquitination, suggesting that its effects on H3-Lys4 and H3-Lys79 methylation are an indirect consequence of its effect on H2B ubiquitination. Rtf1 is important for telomeric silencing, with loss of H3-Lys4 and H3-Lys79 methylation synergistically reducing Sir2 association with telomeric DNA. Dot1, the H3-Lys79 methylase, associates with transcriptionally active genes, but unlike the association of Set1 and Set2 (the H3-Lys36 methylase), this association is largely independent of Rtf1. We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor.

title: The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4.
abstract: Previous studies demonstrated that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex facilitates the binding of TATA-binding protein (TBP) during transcriptional activation of the GAL1 gene of Saccharomyces cerevisiae. TBP binding was shown to require the SAGA components Spt3 and Spt20/Ada5, but not the SAGA component Gcn5. We have now examined whether SAGA is directly required as a coactivator in vivo by using chromatin immunoprecipitation analysis. Our results demonstrate that SAGA is physically recruited in vivo to the upstream activation sequence (UAS) regions of the galactose-inducible GAL genes. This recruitment is dependent on both induction by galactose and the Gal4 activation domain. Furthermore, we demonstrate that another well-characterized activator, Gal4-VP16, also recruits SAGA in vivo. Finally, we provide evidence that a specific interaction between Spt3 and TBP in vivo is important for Gal4 transcriptional activation at a step after SAGA recruitment. These results, taken together with previous studies, demonstrate a dependent pathway for the recruitment of TBP to GAL gene promoters consisting of the recruitment of SAGA by Gal4 and the subsequent recruitment of TBP by SAGA.

title: SAGA is an essential in vivo target of the yeast acidic activator Gal4p.
abstract: Despite major advances in characterizing the eukaryotic transcriptional machinery, the function of promoter-specific transcriptional activators (activators) is still not understood. For example, in no case have the direct in vivo targets of a transcriptional activator been unambiguously identified, nor has it been resolved whether activators have a single essential target or multiple redundant targets. Here we address these issues for the prototype acidic activator yeast Gal4p. Gal4p binds to the upstream activating sequence (UAS) of GAL1 and several other GAL genes and stimulates transcription in the presence of galactose. Previous studies have shown that GAL1 transcription is dependent on the yeast SAGA ( Using formaldehyde-based in vivo cross-linking, we show that the Gal4p activation domain recruits SAGA to the GAL1 UAS. If SAGA is not recruited to the UAS, the preinitiation complex (PIC) fails to assemble at the GAL1 core promoter, and transcription does not occur. SAGA, but not other transcription components, is also recruited by the Gal4p activation domain to a plasmid containing minimal Gal4p-binding sites. Recruitment of SAGA by Gal4p and stimulation of PIC assembly is dependent on several SAGA subunits but not the SAGA histone acetyl-transferase (HAT) GCN5. Based on these and other results, we conclude that SAGA is an essential target of Gal4p that, following recruitment to the UAS, facilitates PIC assembly and transcription.

title: Continuous and widespread roles for the Swi–Snf complex in transcription
abstract: Chromatin presents a significant obstacle to transcription, but two means of overcoming its repressive effects, histone acetylation and the activities of the Swi–Snf complex, have been proposed. Histone acetylation and Swi–Snf activity have been shown to be crucial for transcriptional induction and to facilitate binding of transcription factors to DNA. By regulating the activity of the Swi–Snf complex in vivo, we found that active transcription requires continuous Swi–Snf function, demonstrating a role for this complex beyond the induction of transcription. Despite the presumably generalized packaging of genes into chromatin, previous studies have indicated that the transcriptional requirements for the histone acetyltransferase, Gcn5, and the Swi–Snf complex are limited to a handful of genes. However, inactivating Swi–Snf function in cells also lacking GCN5 revealed defects in transcription of several genes previously thought to be SWI–SNF‐ and GCN5‐independent. These findings suggest that chromatin remodeling plays a widespread role in gene expression and that these two chromatin remodeling activities perform independent and overlapping functions during transcriptional activation.

title: Rad26p regulates the occupancy of histone H2A–H2B dimer at the active genes in vivo
abstract: Recently, we have demonstrated a predominant association of Rad26p with the coding sequences but not promoters of several GAL genes following transcriptional induction. Here, we show that the occupancy of histone H2A–H2B dimer at the coding sequences of these genes is not altered following transcriptional induction in the absence of Rad26p. A histone H2A–H2B dimer-enriched chromatin in Δrad26 is correlated to decreased association of RNA polymerase II with the active coding sequences (and hence transcription). However, the reduced association of RNA polymerase II with the active coding sequence in the absence of Rad26p is not due to the defect in formation of transcription complex at the promoter. Thus, Rad26p regulates the occupancy of histone H2A–H2B dimer, which is correlated to the association of elongating RNA polymerase II with active GAL genes. Similar results are also found at other inducible non-GAL genes. Collectively, our results define a new role of Rad26p in orchestrating chromatin structure and hence transcription in vivo.

title: The multifunctional transcription factors Abf1p, Rap1p and Reb1p are required for full transcriptional activation of the chromosomalPGK gene inSaccharomyces cerevisiae
abstract: We have identified two new transcription factor binding sites upstream of the previously defined UAS within the phosphoglycerate kinase (PGK) gene promoter inSaccharomyces cerevisiae. These sites are bound in vitro by the multifunctional factors Cpf1p and Reb1p. We have generated targeted deletions of Rap1p, Abf1p and Reb1p binding sites in the promoter of the chromosomal copy of thePGK gene. Northern blot analysis confirmed that mostPGK promoter activity is mediated through the Rap1p binding site. However, significant effects are also mediated through both the Reb1p and Abf1p sites. In contrast, when the promoter is present on a high-copy-number plasmid, both the Abf1p and Reb1p sites play no role in transcriptional activation. The role of Cpf1p was examined using acfp1 null strain. Cpf1p was found to have little if any, effect on activation of either the chromosomal or plasmid-bornePGK gene.

title: Importance of general regulatory factors Rap1p, Abf1p and Reb1p for the activation of yeast fatty acid synthase genes FAS1 and FAS2.
abstract: The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are under transcriptional control of pathway-specific regulators of phospholipid biosynthesis. However, site-directed mutagenesis of the respective cis-acting elements upstream of FAS1 and FAS2 revealed that additional sequences activating both genes must exist. A deletion analysis of the FAS1 promoter lacking the previously characterized inositol/choline-responsive-element motif defined a region (nucleotides -760 to -850) responsible for most of the remaining activation potency. Gel-retardation experiments and in-vitro DNase footprint studies proved the binding of the general regulatory factors Rap1p, Abf1p and Reb1p to this FAS1 upstream region. Mutation of the respective binding sites led to a drop of gene activation to 8% of the wild-type level. Similarly, we also demonstrated the presence of a Reb1p-binding site upstream of FAS2 and its importance for gene activation. Thus, in addition to the previously characterized FAS-binding factor 1 interacting with the inositol/choline-responsive-element motif, a second motif common to the promoter regions of both FAS genes could be identified. Transcription of yeast fatty acid synthase genes is therefore subjected to both the pathway-specific control affecting genes of phospholipid biosynthesis and to the activation by general transcription factors allowing a sufficiently high level of constitutive gene expression.

title: A REB1-binding site is required for GCN4-independent ILV1 basal level transcription and can be functionally replaced by an ABF1-binding site.
abstract: The ILV1 gene of Saccharomyces cerevisiae encodes the first committed step in isoleucine biosynthesis and is regulated by general control of amino acid biosynthesis. Deletion analysis of the ILV1 promoter revealed a GC-rich element important for the basal level expression. This cis-acting element, called ILV1BAS, is functional independently of whether GCN4 protein is present. Furthermore, unlike the situation at HIS4, the magnitude of GCN4-mediated derepression is independent of ILV1BAS. The element has homology to the consensus REB1-binding sequence CGGGTARNNR. Gel retardation assays showed that REB1 binds specifically to this element. We show that REB1-binding sites normally situated in the SIN3 promoter and in the 35S rRNA promoter can substitute for the ILV1 REB1 site. Furthermore, a SIN3 REB1 site containing a point mutation that abolishes REB1 binding does not support ILV1 basal level expression, suggesting that binding of REB1 is important for the control of ILV1 basal level expression. Interestingly, an ABF1-binding site can also functionally replace the ILV1 REB1-binding site. A mutated ABF1 site that displays a very low affinity for ABF1 does not functionally replace the ILV1 REB1 site. This suggests that ABF1 and REB1 may have related functions within the cell. Although the REB1-binding site is required for the ILV1 basal level expression, the site on its own stimulates transcription only slightly when combined with the CYC1 downstream promoter elements, indicating that another ILV1 promoter element functions in combination with the REB1 site to control high basal level expression.

title: Transcriptional regulatory code of a eukaryotic genome
abstract: DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.

title: Regulation of Chromatin Assembly/Disassembly by Rtt109p, a Histone H3 Lys56-specific Acetyltransferase, in Vivo*
abstract: Rtt109p, a histone acetyltransferase, associates with active genes and acetylates lysine 56 on histone H3 in Saccharomyces cerevisiae. However, the functional role of Rtt109p or H3 Lys56 acetylation in chromatin assembly/disassembly (and hence gene expression) immediately switching transcription on or off has not been clearly elucidated in vivo. Here, we show that Rtt109p promotes the eviction of histone H3 from a fast inducible yeast gene, GAL1, following transcriptional initiation via histone H3 Lys56 acetylation. Conversely, the deposition of histone H3 to GAL1 is significantly decreased in the presence of Rtt109p following transcriptional termination. Intriguingly, we also find that the deposition of histone H2B on preexisting non-acetylated histone H3 Lys56 at GAL1 in Δrtt109 is significantly increased independently of histone H3 deposition immediately following transcriptional termination subsequent to a short induction. Consistently, histone H2B is not efficiently evicted from GAL1 in the absence of Rtt109p immediately following transcriptional induction. Furthermore, we show that the stimulated eviction or reduced deposition of histones by Rtt109p promotes the association of RNA polymerase II with GAL1 and hence the synthesis of GAL1 mRNA. These results, taken together, support the fact that Rtt109p regulates the deposition/eviction of histone H2B in addition to its role in stimulating histone H3 eviction, thus providing insight into chromatin assembly/disassembly and hence gene expression in vivo.