Tip60 is a key histone acetyltransferase (HAT) enzyme that plays a central role in diverse biological processes critical for general cell function; however, the chromatin-mediated cell-type specific developmental pathways that are dependent exclusively upon the HAT activity of Tip60 remain to be explored. Here, we investigate the role of Tip60 HAT activity in transcriptional control during multicellular development in vivo by examining genome-wide changes in gene expression in a Drosophila model system specifically depleted for endogenous dTip60 HAT function. We show that amino acid residue E431 in the catalytic HAT domain of dTip60 is critical for the acetylation of endogenous histone H4 in our fly model in vivo, and demonstrate that dTip60 HAT activity is essential for multicellular development. Moreover, our results uncover a novel role for Tip60 HAT activity in controlling neuronal specific gene expression profiles essential for nervous system function as well as a central regulatory role for Tip60 HAT function in general metabolism.

The target of rapamycin (TOR) pathway regulates cell growth and aging in various organisms. In Saccharomyces cerevisiae, silent information regulator 2 (Sir2) modulates cellular senescence. Moreover, Sir2 plays a crucial role in promoting ribosomal DNA (rDNA) stability and longevity under TOR inhibition. Here we review the implication of rDNA stabilizers in longevity, discuss how Sir2 stabilizes rDNA under TOR inhibition and speculate on the link between sumoylation and Sir2-related pro-aging pathways.

Recent studies have established that the highly condensed and transcriptionally silent heterochromatic domains in budding yeast are virtually dynamic structures. The underlying mechanisms for heterochromatin dynamics, however, remain obscure. In this study, we show that histones are dynamically acetylated on H4K12 at telomeric heterochromatin, and this acetylation regulates several of the dynamic telomere properties. Using a de novo heterochromatin formation assay, we surprisingly found that acetylated H4K12 survived the formation of telomeric heterochromatin. Consistently, the histone acetyltransferase complex NuA4 bound to silenced telomeric regions and acetylated H4K12. H4K12 acetylation prevented the over-accumulation of Sir proteins at telomeric heterochromatin and elimination of this acetylation caused defects in multiple telomere-related processes, including transcription, telomere replication, and recombination. Together, these data shed light on a potential histone acetylation mark within telomeric heterochromatin that contributes to telomere plasticity.

GCN5 encodes one of the non-essential Histone Acetyl Transferases in Saccharomyces cerevisiae. Extensive evidence has indicated that GCN5 is a key regulator of gene expression and could also be involved in transcriptional elongation, DNA repair and centromere maintenance. Here we show that the deletion of GCN5 decreases the stability of mini-chromosomes; that the tethering of Gcn5p to a crippled origin of replication stimulates its activity; that high dosage of GCN5 suppresses conditional phenotypes caused by mutant alleles of bona fide replication factors, orc2-1, orc5-1 and mcm5-461. Furthermore, Gcn5p physically associates with origins of DNA replication, while its deletion leads to localized condensation of chromatin at origins. Finally, Deltagcn5 cells display a deficiency in the assembly of pre-replicative complexes. We propose that GCN5 acts as a positive regulator of DNA replication by counteracting the inhibitory effect of Histone Deacetylases.

Acetylation of histones is an essential element regulating chromatin structure and transcription. MYST (Moz, Ybf2/Sas3, Sas2, Tip60) proteins form the largest family of histone acetyltransferases and are present in all eukaryotes. Surprisingly, until recently this protein family was poorly studied. However, in the last few years there has been a substantial increase in interest in the MYST proteins and a number of key studies have shown that these chromatin modifiers are required for a diverse range of cellular processes, both in health and disease. Translocations affecting MYST histone acetyltransferases can lead to leukemia and solid tumors. Some members of the MYST family are required for the development and self-renewal of stem cell populations; other members are essential for the prevention of inappropriate heterochromatin spreading and for the maintenance of adequate levels of gene expression. In this review we discuss the function of MYST proteins in vivo.

Histone modifications that regulate chromatin-dependent processes are catalyzed by multi-subunit complexes. These can function in both targeting activities to specific genes and in regulating genome-wide levels of modifications. In Saccharomyces cerevisiae, Esa1 and Rpd3 have opposing enzymatic activities and are catalytic subunits of multiple chromatin modifying complexes with key roles in processes such as transcriptional regulation and DNA repair. Esa1 is an essential histone acetyltransferase that belongs to the highly conserved MYST family. This study presents evidence that the yeast histone deacetylase gene, RPD3, when deleted, suppressed esa1 conditional mutant phenotypes. Deletion of RPD3 reversed rDNA and telomeric silencing defects and restored global H4 acetylation levels, in addition to rescuing the growth defect of a temperature-sensitive esa1 mutant. This functional genetic interaction between ESA1 and RPD3 was mediated through the Rpd3L complex. The suppression of esa1's growth defect by disruption of Rpd3L was dependent on lysine 12 of histone H4. We propose a model whereby Esa1 and Rpd3L act coordinately to control the acetylation of H4 lysine 12 to regulate transcription, thereby emphasizing the importance of dynamic acetylation and deacetylation of this particular histone residue in maintaining cell viability.

Transcriptional silencing is a crucial process that is mediated through chromatin structure. The histone deacetylase Sir2 silences genomic regions that include telomeres, ribosomal DNA (rDNA) and the cryptic mating-type loci. Here, we report an unsuspected role for the enzyme Gas1 in locus-specific transcriptional silencing. GAS1 encodes a beta-1,3-glucanosyltransferase previously characterized for its role in cell wall biogenesis. In gas1 mutants, telomeric silencing is defective and rDNA silencing is enhanced. We show that the catalytic activity of Gas1 is required for normal silencing, and that Gas1's role in silencing is distinct from its role in cell wall biogenesis. Established hallmarks of silent chromatin, such as Sir2 and Sir3 binding, H4K16 deacetylation, and H3K56 deacetylation, appear unaffected in gas1 mutants. Thus, another event required for telomeric silencing must be influenced by GAS1. Because the catalytic activity of Gas1 is required for telomeric silencing, Gas1 localizes to the nuclear periphery, and Gas1 and Sir2 physically interact, we propose a model in which carbohydrate modification of chromatin components provides a new regulatory element that may be critical for chromatin function but which is virtually unexplored in the current landscape of chromatin analysis.

In the eukaryotic genome, transcriptionally silent chromatin tends to propagate along a chromosome and encroach upon adjacent active chromatin. The silencing machinery can be stopped by chromatin boundary elements. We performed a screen in Saccharomyces cerevisiae for proteins that may contribute to the establishment of a chromatin boundary. We found that disruption of histone deacetylase Rpd3p results in defective boundary activity, leading to a Sir-dependent local propagation of transcriptional repression. In rpd3Delta cells, the amount of Sir2p that was normally found in the nucleolus decreased and the amount of Sir2p found at telomeres and at HM and its adjacent loci increased, leading to an extension of silent chromatin in those areas. In addition, Rpd3p interacted directly with chromatin at boundary regions to deacetylate histone H4 at lysine 5 and at lysine 12. Either the mutation of histone H4 at lysine 5 or a decrease in the histone acetyltransferase (HAT) activity of Esa1p abrogated the silencing phenotype associated with rpd3 mutation, suggesting a novel role for the H4 amino terminus in Rpd3p-mediated heterochromatin boundary regulation. Together, these data provide insight into the molecular mechanisms for the anti-silencing functions of Rpd3p during the formation of heterochromatin boundaries.

In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA (rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.

The nucleolus is an informative model structure for studying how chromatin-regulated transcription relates to nuclear organisation. In this review, we describe how chromatin controls nucleolar structure through both the modulation of rDNA activity by convergently-evolved remodelling complexes and by direct effects upon rDNA packaging. This packaging not only regulates transcription but may also be important for suppressing internal recombination between tandem rDNA repeats. The identification of nucleolar histone chaperones and novel chromatin proteins by mass spectrometry suggests that structure-specific chromatin components remain to be characterised and may regulate the nucleolus in novel ways. However, it also suggests that there is considerable overlap between nucleolar and non-nucleolar-chromatin components. We conclude that a fuller understanding of nucleolar chromatin will be essential for understanding how gene organisation is linked with nuclear architecture.

Chromatin modification is important for virtually all aspects of DNA metabolism but little is known about the consequences of such modification in trypanosomatids, early branching protozoa of significant medical and veterinary importance. MYST-family histone acetyltransferases in other species function in transcription regulation, DNA replication, recombination and repair. Trypanosoma brucei HAT3 was recently shown to acetylate histone H4K4 and we now report characterization of all three T. brucei MYST acetyltransferases (HAT1-3). First, GFP-tagged HAT1-3 all localize to the trypanosome nucleus. While HAT3 is dispensable, both HAT1 and HAT2 are essential for growth. Strains with HAT1 knock-down display mitosis without nuclear DNA replication and also specific de-repression of a telomeric reporter gene, a rare example of transcription control in an organism with widespread and constitutive polycistronic transcription. Finally, we show that HAT2 is responsible for H4K10 acetylation. By analogy to the situation in Saccharomyces cerevisiae, we discuss low-level redundancy of acetyltransferase function in T. brucei and suggest that two MYST-family acetyltransferases are essential due to the absence of a Gcn5 homologue. The results are also consistent with the idea that HAT1 contributes to establishing boundaries between transcriptionally active and repressed telomeric domains in T. brucei.

The MYST family of lysine acetyltransferases has been intensely studied because of its broad conservation and biological significance. In humans, there are multiple correlations between the enzymes and development and disease. In model organisms, genetic and biochemical studies have been particularly productive because of mechanistic insights they provide in defining substrate specificity, the complexes through which the enzymes function, and the sites of their activity within the genome. Established and emerging data from yeast reveal roles for the three MYST enzymes in diverse chromosomal functions. In particular, recent studies help explain how MYST complexes coordinate with other modifiers, the histone variant H2A.Z, and remodeling complexes to demarcate silent and active chromosomal domains, facilitate transcription, and enable repair of DNA damage.

S. pombe Mst1 is a member of the MYST family of histone acetyltransferases, and is the likely ortholog of S. cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1) and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.

Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain-RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded beta-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional beta-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.

Normal primary cells have a finite ability to divide in culture and after a number of population doublings enter a state of irreversible cell cycle arrest known as replicative senescence. Several cellular stresses have been shown to induce a senescence-like growth arrest including shortened telomeres, DNA-damaging stresses, and drastic changes in chromatin structure, for example, through histone deacetylase (HDAC) induction. Histones are core components of chromatin which are subject to a number of chemical modifications that influence the dynamic state of chromatin structure. Proper chromatin structure formation is crucial for most DNA-dependent processes including transcription, replication, and repair which have a profound impact on cellular proliferation and senescence. Several genes important for chromatin remodeling such as the tumor suppressors p53 and retinoblastoma (Rb) affect cellular senescence by mediating changes in chromatin structure and gene expression. The Morf4-Related Gene (MRG) family of transcription factors forms stable interactions with chromatin-modifying complexes including histone acetyltransferase (HAT) and HDAC complexes and interact with Rb. Further, the MRG family was founded by a gene, Mortality Factor on Chromosome 4, capable of inducing senescence in immortalized cell lines. In this paper, we review the role of the MRG family of proteins in chromatin dynamics and cellular senescence.

In genetic hybrids displaying nucleolar dominance, acetylation of lysines 5, 8, 12 and 16 of histone H4 (H4K5, H4K8, H4K12, H4K16) and acetylation of histone H3 on lysines 9 and 14 (H3K9, H3K14) occurs at the promoters of active ribosomal RNA (rRNA) genes, whereas silenced rRNA genes are deacetylated. Likewise, histone hyperacetylation correlates with the active state of transgenes and of endogenous plant genes involved in physiological processes, including cold tolerance, light-responsiveness and flowering. To investigate histone hyperacetylation dynamics we used sodium butyrate, a histone deacetylase inhibitor known to switch silent rRNA genes on, in order to enrich the pool of acetylated histones. Mass spectrometric analyses revealed unique mono-(K16Ac), di-(K12Ac, K16Ac), tri-(K8Ac, K12Ac, K16Ac), and tetra-acetylated (K5Ac, K8Ac, K12Ac, K16Ac) histone H4 isoforms, suggesting that H4 hyperacetylation occurs in a processive fashion, beginning with lysine 16 and ending with lysine 5. Using a combination of molecular and mass spectrometric assays we then determined the specificities of seven of the nine functional co-activator type histone acetyltransferases (HATs) in Arabidopsis thaliana: specifically HATs of the CBP (HAC1, HAC5, HAC12), GNAT (HAG1, HAG2), and MYST families (HAM1, HAM2). Specific HATs acetylate histone H4K5 (HAM1, HAM2), H4K12 (HAG2), and H3K14 (HAG1), suggesting that acetylation of these lysines may have special regulatory significance. Other acetylation events, including histone H3K9 acetylation, are likely to result from the activities of the broad-specificity HAC1, HAC5, and HAC12 histone acetyltransferases.

The MYST family of histone acetyltransferases (HATs) was initially defined by human genes with disease connections and by yeast genes identified for their role in epigenetic transcriptional silencing. Since then, many new MYST genes have been discovered through genetic and genomic approaches. Characterization of the complexes through which MYST proteins act, regions of the genome to which they are targeted and biological consequences when they are disrupted, all deepen the connections of MYST proteins to development, growth control and human cancers. Many of the insights into MYST family function have come from studies in model organisms. Herein, we review functions of two of the founding MYST genes, yeast SAS2 and SAS3, and the essential yeast MYST ESA1. Analysis of these genes in yeast has defined roles for MYST proteins in transcriptional activation and silencing, and chromatin-mediated boundary formation. They have further roles in DNA damage repair and nuclear integrity. The observation that MYST protein complexes share subunits with other HATs, histone deacetylases and other key nuclear proteins, many with connections to human cancers, strengthens the idea that coordinating distinct chromatin modifications is critical for regulation.Oncogene (2007) 26, 5373-5384; doi:10.1038/sj.onc.1210606.

Three heterochromatin-like domains have been identified in Saccharomyces cerevisiae that are refractory to transcription by Pol II, the silent mating-type loci, telomeres and the ribosomal DNA. Previous work has shown that chromatin remodelers can regulate silent chromatin. Here, we report the findings of an investigation into the role of ISW2 in transcriptional silencing at the rDNA. We show that the levels of retrotransposition and mRNA from a genetically marked Ty1 element located in the rDNA were increased significantly in isw2Delta cells, while transcript levels from Ty1 elements outside of the rDNA were not increased in cells lacking ISW2. Additionally, we show that Isw2 is not required for silencing at a telomere. Our findings demonstrate that Isw2 is required for transcriptional silencing at the rDNA and emphasize the differences in the regulation of transcriptional silencing at silent loci in S. cerevisiae.

Transcriptional silencing of Pol II-transcribed genes in Saccharomyces cerevisiae occurs at the HM loci, telomeres and ribosomal DNA (rDNA) locus. Gene silencing at these loci requires histone-modifying enzymes as well as factors that regulate local chromatin structure. Previous work has shown that the ATP-dependent chromatin remodeling protein Isw1 is required for silencing of a marker gene inserted at the HMR locus, but not at telomeres. Here we show that Isw1 is required for transcriptional silencing of Pol II-transcribed genes in the ribosomal DNA locus. Our results indicate that Isw1 associates with the rDNA and that this interaction is not altered in cells lacking other members of the Isw1a and Isw1b chromatin remodeling complexes. Further, the association of Isw1 with the rDNA is not altered in cells lacking the histone deacetylase Sir2 or the histone methyltransferase Set1, two factors that are required for gene silencing at the rDNA. Notably, the loss of transcriptional silencing at the rDNA in cells lacking Isw1 is correlated with a change in rDNA chromatin structure. Together, our data support a model in which Isw1 acts independently of the previously characterized Isw1a and Isw1b complexes to maintain a heterochromatin-like structure at the rDNA that is required for gene silencing.