Developmentally regulated internal transcription initiation during meiosis in budding yeast.

Sporulation of budding yeast is a developmental process in which cells undergo meiosis to generate stress-resistant progeny. The dynamic nature of the budding yeast meiotic transcriptome has been well established by a number of genome-wide studies. Here we develop an analysis pipeline to systematically identify novel transcription start sites that reside internal to a gene. Application of this pipeline to data from a synchronized meiotic time course reveals over 40 genes that display specific internal initiations in mid-sporulation. Consistent with the time of induction, motif analysis on upstream sequences of these internal transcription start sites reveals a significant enrichment for the binding site of Ndt80, the transcriptional activator of middle sporulation genes. Further examination of one gene, MRK1, demonstrates the Ndt80 binding site is necessary for internal initiation and results in the expression of an N-terminally truncated protein isoform. When the MRK1 paralog RIM11 is downregulated, the MRK1 internal transcript promotes efficient sporulation, indicating functional significance of the internal initiation. Our findings suggest internal transcriptional initiation to be a dynamic, regulated process with potential functional impacts on development.

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80-induced genes, PES4 and MIP6, encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts.

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis.

N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

Sequestration of mRNAs Modulates the Timing of Translation during Meiosis in Budding Yeast.

Starvation of diploid cells of the budding yeast Saccharomyces cerevisiae induces them to enter meiosis and differentiate into haploid spores. During meiosis, the precise timing of gene expression is controlled at the level of transcription, and also translation. If cells are returned to rich medium after they have committed to meiosis, the transcript levels of most meiotically upregulated genes decrease rapidly. However, for a subset of transcripts whose translation is delayed until the end of meiosis II, termed protected transcripts, the transcript levels remain stable even after nutrients are reintroduced. The Ime2-Rim4 regulatory circuit controls both the delayed translation and the stability of protected transcripts. These protected mRNAs localize in discrete foci, which are not seen for transcripts of genes with different translational timing and are regulated by Ime2. These results suggest that Ime2 and Rim4 broadly regulate translational delay but that additional factors, such as mRNA localization, modulate this delay to tune the timing of gene expression to developmental transitions during sporulation.

Biochemical characterization of Hpa2 and Hpa3, two small closely related acetyltransferases from Saccharomyces cerevisiae.

Based on their sequences, the Saccharomyces cerevisiae Hpa2 and Hpa3 proteins are annotated as two closely related members of the Gcn5 acetyltransferase family. Here, we describe the biochemical characterization of Hpa2 and Hpa3 as bona fide acetyltransferases with different substrate specificities. Mutational and MALDI-TOF analyses showed that Hpa3 translation initiates primarily from Met-19 rather than the annotated start site, Met-1, with a minor product starting at Met-27. When expressed in Escherichia coli and assayed in vitro, Hpa2 and Hpa3 (from Met-19) acetylated histones and polyamines. Whereas Hpa2 acetylated histones H3 and H4 (at H3 Lys-14, H4 Lys-5, and H4 Lys-12), Hpa3 acetylated only histone H4 (at Lys-8). Additionally, Hpa2, but not Hpa3, acetylated certain small basic proteins. Hpa3, but not Hpa2, has been reported to acetylate D-amino acids, and we present results consistent with that. Overexpression of Hpa2 or Hpa3 is toxic to yeast cells. However, their deletions do not show any standard phenotypic defects. These results suggest that Hpa2 and Hpa3 are similar but distinct acetyltransferases that might have overlapping roles with other known acetyltransferases in vivo in acetylating histones and other small proteins.

Structural basis for allosteric stimulation of Sir2 activity by Sir4 binding.

The budding yeast Sir2 (silent information regulator 2) protein is the founding member of the sirtuin family of NAD-dependent histone/protein deacetylases. Its function in transcriptional silencing requires both the highly conserved catalytic domain and a poorly understood N-terminal regulatory domain (Sir2N). We determined the structure of Sir2 in complex with a fragment of Sir4, a component of the transcriptional silencing complex in Saccharomyces cerevisiae. The structure shows that Sir4 is anchored to Sir2N and contacts the interface between the Sir2N and the catalytic domains through a long loop. We discovered that the interaction between the Sir4 loop and the interdomain interface in Sir2 is critical for allosteric stimulation of the deacetylase activity of Sir2. These results bring to light the structure and function of the regulatory domain of Sir2, and the knowledge should be useful for understanding allosteric regulation of sirtuins in general.

MYST protein acetyltransferase activity requires active site lysine autoacetylation.

The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

A conserved patch near the C terminus of histone H4 is required for genome stability in budding yeast.

A screen of Saccharomyces cerevisiae histone alanine substitution mutants revealed that mutations in any of three adjacent residues, L97, Y98, or G99, near the C terminus of H4 led to a unique phenotype. The mutants grew slowly, became polyploid or aneuploid rapidly, and also lost chromosomes at a high rate, most likely because their kinetochores were not assembled properly. There was lower histone occupancy, not only in the centromeric region, but also throughout the genome for the H4 mutants. The mutants displayed genetic interactions with the genes encoding two different histone chaperones, Rtt106 and CAF-I. Affinity purification of Rtt106 and CAF-I from yeast showed that much more H4 and H3 were bound to these histone chaperones in the case of the H4 mutants than in the wild type. However, in vitro binding experiments showed that the H4 mutant proteins bound somewhat more weakly to Rtt106 than did wild-type H4. These data suggest that the H4 mutant proteins, along with H3, accumulate on Rtt106 and CAF-I in vivo because they cannot be deposited efficiently on DNA or passed on to the next step in the histone deposition pathway, and this contributes to the observed genome instability and growth defects.

The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A.

The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase-like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase-Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.

The JmjC domain of Gis1 is dispensable for transcriptional activation.

Yeast Gis1 protein functions as a transcription factor after nutrient limitation and oxidative stress. In this report, we show that Gis1 also regulates the induction of several genes involved in spore wall synthesis during sporulation. Gis1 contains a JmjC domain near its N-terminus. In many proteins, JmjC domains provide histone demethylase activity. Whether the JmjC domain of Gis1 contributes to its transcriptional activation is still unknown. Here, we show that gis1 point mutations that abolish Fe (II) and α-ketoglutarate binding, known cofactors in other JmjC proteins, are still able to induce transcription normally during glucose starvation and sporulation. Even the deletion of the entire JmjC domain does not affect transcriptional activation by Gis1. Moreover, the JmjC domain is not required for the toxicity associated with Gis1 overexpression. The data demonstrate that the JmjC domain is dispensable for transcriptional activation by Gis1 during nutrient stress and sporulation.

Promoter strength influences the S phase requirement for establishment of silencing at the Saccharomyces cerevisiae silent mating type Loci.

In Saccharomyces cerevisiae, the two cryptic mating type loci, HML and HMR, are transcriptionally silent. Previous studies on the establishment of silencing at HMR identified a requirement for passage through S phase. However, the underlying mechanism for this requirement is still unknown. In contrast to HMR, we found that substantial silencing of HML could be established without passage through S phase. To understand this difference, we analyzed several chimeric HM loci and found that promoter strength determined the S phase requirement. To silence a locus with a strong promoter such as the a1/a2 promoter required passage through S phase while HM loci with weaker promoters such as the α1/α2 or TRP1 promoter did not show this requirement. Thus, transcriptional activity counteracts the establishment of silencing but can be overcome by passage through S phase.

Mutational analysis of the Sir3 BAH domain reveals multiple points of interaction with nucleosomes.

Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.

A yeast sir2 mutant temperature sensitive for silencing.

A screen for Saccharomyces cerevisiae temperature-sensitive silencing mutants identified a strain with a point mutation in the SIR2 gene. The mutation changed Ser276 to Cys. This amino acid is in the highly conserved NAD(+) binding pocket of the Sir2 family of proteins. Haploid strains of either mating type carrying the mutation were severely defective at mating at 37 degrees but normal at 25 degrees . Measurements of RNA from the HMR locus demonstrated that silencing was lost rapidly upon shifting the mutant from the low to the high temperature, but it took >8 hours to reestablish silencing after a shift back to 25 degrees . Silencing at the rDNA locus was also temperature sensitive. On the other hand, telomeric silencing was totally defective at both temperatures. Enzymatic activity of the recombinant wild-type and mutant Sir2 protein was compared by three different assays. The mutant exhibited less deacetylase activity than the wild-type protein at both 37 degrees and 25 degrees . Interestingly, the mutant had much more NAD(+)-nicotinamide exchange activity than wild type, as did a mutation in the same region of the protein in the Sir2 homolog, Hst2. Thus, mutations in this region of the NAD(+) binding pocket of the protein are able to carry out cleavage of NAD(+) to nicotinamide but are defective at the subsequent deacetylation step of the reaction.

NADP regulates the yeast GAL induction system.

Transcriptional regulation of the galactose-metabolizing genes in Saccharomyces cerevisiae depends on three core proteins: Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UAS(GAL)); Gal80p, a repressor that binds to the carboxyl terminus of Gal4p and inhibits transcription; and Gal3p, a cytoplasmic transducer that, upon binding galactose and adenosine 5'-triphosphate, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm. However, the rapid induction of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the carboxyl-terminal activation domain of Gal4p reveals the existence of a dinucleotide that mediates the interaction between the two. Biochemical and in vivo experiments suggests that nicotinamide adenine dinucleotide phosphate (NADP) plays a key role in the initial induction event.

Structural basis of inhibition of the human NAD+-dependent deacetylase SIRT5 by suramin.

Sirtuins are NAD(+)-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD(+)-dependent deacetylase activity with an IC(50) value of 22 microM. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD(+), the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.

C2H2 zinc finger-SET histone methyltransferase is a plant-specific chromatin modifier.

Histone modification represents a universal mechanism for regulation of eukaryotic gene expression underlying diverse biological processes from neuronal gene expression in mammals to control of flowering in plants. In animal cells, these chromatin modifications are effected by well-defined multiprotein complexes containing specific histone-modifying activities. In plants, information about the composition of such co-repressor complexes is just beginning to emerge. Here, we report that two Arabidopsis thaliana factors, a SWIRM domain polyamine oxidase protein, AtSWP1, and a plant-specific C2H2 zinc finger-SET domain protein, AtCZS, interact with each other in plant cells and repress expression of a negative regulator of flowering, FLOWERING LOCUS C (FLC) via an autonomous, vernalization-independent pathway. Loss-of-function of either AtSWP1 or AtCZS results in reduced dimethylation of lysine 9 and lysine 27 of histone H3 and hyperacetylation of histone H4 within the FLC locus, in elevated FLC mRNA levels, and in moderately delayed flowering. Thus, AtSWP1 and AtCZS represent two main components of a co-repressor complex that fine tunes flowering and is unique to plants.

Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs.

BACKGROUND: By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions. RESULTS: Targeted silencing by Rtt107/Esc4 was dependent on the SIR genes, which encode obligatory structural and enzymatic components of yeast silent chromatin. Based on its sequence, Rtt107/Esc4 was predicted to contain six BRCT motifs. This motif, originally identified in the human breast tumor suppressor gene BRCA1, is a protein interaction domain. The targeted silencing activity of Rtt107/Esc4 resided within the C-terminal two BRCT motifs, and this region of the protein bound to Sir3 in two-hybrid tests. Deletion of RTT107/ESC4 caused sensitivity to the DNA damaging agent MMS as well as to hydroxyurea. A two-hybrid screen showed that the N-terminal BRCT motifs of Rtt107/Esc4 bound to Slx4, a protein previously shown to be involved in DNA repair and required for viability in a strain lacking the DNA helicase Sgs1. Like SLX genes, RTT107ESC4 interacted genetically with SGS1; esc4Delta sgs1Delta mutants were viable, but exhibited a slow-growth phenotype and also a synergistic DNA repair defect. CONCLUSION: Rtt107/Esc4 binds to the silencing protein Sir3 and the DNA repair protein Slx4 via different BRCT motifs, thus providing a bridge linking silent chromatin to DNA repair enzymes.

SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis.

The mammalian cytoplasmic protein SirT2 is a member of the Sir2 family of NAD+-dependent protein deacetylases involved in caloric restriction-dependent life span extension. We found that SirT2 and its yeast counterpart Hst2 have a strong preference for histone H4K16Ac in their deacetylation activity in vitro and in vivo. We have pinpointed the decrease in global levels of H4K16Ac during the mammalian cell cycle to the G2/M transition that coincides with SirT2 localization on chromatin. Mouse embryonic fibroblasts (MEFs) deficient for SirT2 show higher levels of H4K16Ac in mitosis, in contrast to the normal levels exhibited by SirT1-deficient MEFs. The enzymatic conversion of H4K16Ac to its deacetylated form may be pivotal to the formation of condensed chromatin. Thus, SirT2 is a major contributor to this enzymatic conversion at the time in the cell's life cycle when condensed chromatin must be generated anew.

The BUR1 cyclin-dependent protein kinase is required for the normal pattern of histone methylation by SET2.

BUR1 and BUR2 encode the catalytic and regulatory subunits of a cyclin-dependent protein kinase complex that is essential for normal growth and has a general role in transcription elongation. To gain insight into its specific role in vivo, we identified mutations that reverse the severe growth defect of bur1Delta cells. This selection identified mutations in SET2, which encodes a histone methylase that targets lysine 36 of histone H3 and, like BUR1, has a poorly characterized role during transcription elongation. This genetic relationship indicates that SET2 activity is required for the growth defect observed in bur1Delta strains. This SET2-dependent growth inhibition occurs via methylation of histone H3 on lysine 36, since a methylation-defective allele of SET2 or a histone H3 K36R mutation also suppressed bur1Delta. We have explored the relationship between BUR1 and SET2 at the biochemical level and find that histone H3 is monomethylated, dimethylated, and trimethylated on lysine 36 in wild-type cells, but trimethylation is significantly reduced in bur1 and bur2 mutant strains. A similar methylation pattern is observed in RNA polymerase II C-terminal domain truncation mutants and in an spt16 mutant strain. Chromatin immunoprecipitation assays reveal that the transcription-dependent increase in trimethylated K36 over open reading frames is significantly reduced in bur2Delta strains. These results establish links between a regulatory protein kinase and histone methylation and lead to a model in which the Bur1-Bur2 complex counteracts an inhibitory effect of Set2-dependent histone methylation.

Structure and function of the Saccharomyces cerevisiae Sir3 BAH domain.

Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.