Ann L. Kirchmaier

HST3/HST4-dependent Deacetylation of Lysine 56 of Histone H3 in Silent Chromatin

The composition of posttranslational modifications on newly synthesized histones must be altered upon their incorporation into chromatin. These changes are necessary to maintain the same gene expression state at individual chromosomal loci before and after DNA replication. We have examined how one modification that occurs on newly synthesized histone H3, acetylation of K56, influences gene expression at epigenetically regulated loci in Saccharomyces cerevisiae. H3 K56 is acetylated by Rtt109p before its incorporation into chromatin during S phase, and this modification is then removed by the NAD(+)-dependent deacetylases Hst3p and Hst4p during G2/M phase. We found silenced loci maintain H3 K56 in a hypoacetylated state, and the absence of this modification in rtt109 mutants was compatible with HM and telomeric silencing. In contrast, loss of HST3 and HST4 resulted in hyperacetylation of H3 K56 within silent loci and telomeric silencing defects, despite the continued presence of Sir2p throughout these loci. These silencing defects in hst3Delta hst4Delta mutants could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p could not restore silencing in hst3Delta hst4Delta mutants. Together, our findings argue that HST3 HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.

Cell cycle requirements in assembling silent chromatin in Saccharomyces cerevisiae.

ABSTRACT The establishment of silencing at the silent mating-type locus, HMR, in Saccharomyces cerevisiae requires that yeast pass through S phase of the cell cycle, yet requires neither the initiation of DNA replication at the locus destined to become silenced nor the passage of a replication fork through that locus. We tested whether this S-phase requirement reflects a window within the cell cycle permissive for recruitment of Sir proteins to HMR. The S-phase-restricted event necessary for silencing occurred after recruitment of Sir proteins to HMR. Moreover, cells arrested in early S phase formed silent chromatin at HMR, provided HMR was on a nonreplicating template. Replicating templates required a later step for silencing. These results provide temporal resolution of discrete steps in the formation of silent chromatin and suggest that more than one cell cycle-regulated event may be necessary for the establishment of silencing.

Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin in Saccharomyces cerevisiae via Lysine 56 on Histone H3

The formation and stability of epigenetically regulated chromatin is influenced by DNA replication and factors that modulate post-translational modifications on histones. Here we describe evidence that PCNA can affect silencing in Saccharomyces cerevisiae by facilitating deposition of H3 K56ac onto chromosomes. We propose that PCNA participates in this process through a pathway that includes replication factor C, the chromatin assembly factor Asf1p, and the K56-specific acetyltransferase Rtt109p. We show that mutation of POL30 or loss of K56-acetylation in rtt109 and histone H3 mutants enhances silencing at the crippled HMR locus HMRae** via restoring Sir binding and that pol30 mutants with silencing phenotypes have reduced levels of H3 K56ac. Although loss of acetylation on H3 K56 was generally compatible with silencing, mutations at this residue also led to defects in silencing an ADE2 reporter at HMR and abolished silencing when combined with cac1 or pol30-8. These silencing phenotypes are analogous to those in asf1 mutants or pol30-6 and pol30-79 mutants with defects in ASF1-dependent pathways. On the basis of these findings, we propose that mutations in DNA replication factors alter acetylation of H3 K56. We show that this defect, in turn, contributes to misregulation of epigenetic processes as well as of cellular responses to DNA damage.

Non-replicating Epstein-Barr Virus-based Plasmids Extend Gene Expression and Can Improve Gene Therapy in Vivo

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family ofrepeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.

Insights into the impact of histone acetylation and methylation on Sir protein recruitment, spreading and silencing in Saccharomyces cerevisiae

Silent chromatin formation in Saccharomyces cerevisiae begins with the recruitment of silent information regulator (Sir) proteins to silencers at the silent mating-type loci and to telomere ends. Next, Sir2/3/4 proteins propagate across these loci as histones are deacetylated by the NAD(+)-dependent histone deacetylase Sir2p, ultimately resulting in the cessation of transcription and in the loss of SET1- and DOT1-dependent methylation of histone H3 within silent chromatin. We analyzed the effects of modifiable lysine residues on histones H3 and H4 on experimentally defined steps in silencing: recruitment of Sir proteins to silencers, Sir protein spreading, and transcriptional repression. Loss of acetylation, but not methylation, facilitated both Sir recruitment and spreading, and Sir spreading across hypoacetylated chromatin could disrupt SET1- and DOT1-dependent histone methylation without silencing underlying genes. Our data indicate that loss of methylation of K4 and K79 on histone H3 reflects intermediate events during the formation of silent chromatin, and that retention of a positive charge at a single residue on histone H4 (K16) was both necessary and sufficient to permit Sir spreading beyond sites of their recruitment.

Ub-family modifications at the replication fork: Regulating PCNA-interacting components

A vast array of proteins is recruited to the replication fork in a dynamic and coordinated manner through physical interactions with Proliferating Cell Nuclear Antigen, PCNA. How this complex exchange of PCNA binding partners is choreographed to ensure proper replication origin licensing, DNA synthesis during normal replication or repair of DNA damage, chromatin assembly, DNA methylation, histone modification, and sister chromatid cohesion is only beginning to be appreciated. In this review, several roles of ubiquitin‐related modifications in the recruitment and turnover of PCNA‐interacting proteins at the replication fork are considered.

Single Molecule Tools Elucidate H2A.Z Nucleosome Composition

Summary Although distinct epigenetic marks correlate with different chromatin states, how they are integrated within single nucleosomes to generate combinatorial signals remains largely unknown. We report the successful implementation of single molecule tools constituting fluorescence correlation spectroscopy (FCS), pulse interleave excitation-based Förster resonance energy transfer (PIE-FRET) and fluorescence lifetime imaging-based FRET (FLIM-FRET) to elucidate the composition of single nucleosomes containing histone variant H2A.Z (Htz1p in yeast) in vitro and in vivo. We demonstrate that yeast nucleosomes containing Htz1p are primarily composed of H4 K12ac and H3 K4me3 but not H3 K36me3 and that these patterns are conserved in mammalian cells. Quantification of epigenetic modifications in nucleosomes will provide a new dimension to epigenetics research and lead to a better understanding of how these patterns contribute to the targeting of chromatin-binding proteins and chromatin structure during gene regulation.

Proliferating cell nuclear antigen (PCNA) is required for cell cycle-regulated silent chromatin on replicated and nonreplicated genes

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793–809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.

Cell cycle regulation of silent chromatin formation.

Identical genes in two different cells can stably exist in alternate transcriptional states despite the dynamic changes that will occur to chromatin at that locus throughout the cell cycle. In mammals, this is achieved through epigenetic processes that regulate key developmental transitions and ensure stable patterns of gene expression during growth and differentiation. The budding yeast Saccharomyces cerevisiae utilizes silencing to control the expression state of genes encoding key regulatory factors for determining cell-type, ribosomal RNA levels and proper telomere function. Here, we review the composition of silent chromatin in S. cerevisiae, how silent chromatin is influenced by chromatin assembly and histone modifications and highlight several observations that have contributed to our understanding of the interplay between silent chromatin formation and stability and the cell cycle. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

[5] Epstein-barr viral plasmid vectors and their amplifiable derivatives

Summary EBV replicates its DNA via two modes: During the latent phase of the EBV life cycle, the virus replicates its DNA as a multicopy plasmid once per cell cycle in S phase (1, 2); during the lytic phase of EBVs life cycle the virus amplifies its DNA 100- to 1000- fold within one or two cell generations (6). These two modes of DNA replication require distinct cis - and trans -acting viral genetic elements, which have been identified and used to construct different vectors that mimic the two modes of the DNA replication of EBV.