Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

The organization of the nucleus into subcompartments creates microenvironments that are thought to facilitate distinct nuclear functions. In budding yeast, regions of silent chromatin, such as those at telomeres and mating-type loci, cluster at the nuclear envelope creating zones that favour gene repression. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently owing to association of the gene with nuclear pore complexes. Here we report that transcriptional activation of a subtelomeric gene, HXK1 (hexokinase isoenzyme 1), by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3' untranslated region (UTR), which is essential for efficient messenger RNA processing and export, consistent with an accompanying report. However, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Notably, when we interfered with HXK1 localization by either antagonizing or promoting association with the pore, transcript levels were reduced or enhanced, respectively. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.

In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP-tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312-aa domain of Sir4 (Sir4 PAD ) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4 PAD -mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku-and Esc1-dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.

Chromatin in the interphase nucleus moves in a constrained random walk. Despite extensive study, the molecular causes of such movement and its impact on DNA-based reactions are unclear. Using high-precision live fluorescence microscopy in budding yeast, we quantified the movement of tagged chromosomal loci to which transcriptional activators or nucleosome remodeling complexes were targeted. We found that local binding of the transcriptional activator VP16, but not of the Gal4 acidic domain, enhances chromatin mobility. The increase in movement did not correlate strictly with RNA polymerase II (PolII) elongation, but could be phenocopied by targeting the INO80 remodeler to the locus. Enhanced chromatin mobility required Ino80's ATPase activity. Consistently, the INO80-dependent remodeling of nucleosomes upon transcriptional activation of the endogenous PHO5 promoter enhanced chromatin movement locally. Finally, increased mobility at a double-strand break was also shown to depend in part on the INO80 complex. This correlated with increased rates of spontaneous gene conversion. We propose that local chromatin remodeling and nucleosome eviction increase large-scale chromatin movements by enhancing the flexibility of the chromatin fiber.[Keywords: chromatin remodeling; nuclear organization; transcription; VP16; Ino80; fluorescence microscopy; homologous recombination] Supplemental material is available for this article.Received August 15, 2011; revised version accepted January 13, 2012.DNA-based transactions such as transcription, replication, and repair take place in distinct nuclear subcompartments. Transcriptional silencing is frequently associated with the nuclear envelope or occurs near nucleoli (Towbin et al. 2009), whereas the activation of tissue-specific genes correlates with a shift of the relevant genes away from the nuclear periphery (Egecioglu and Brickner 2011). In contrast, genes activated under conditions of nutrient or temperature stress move to nuclear pores when they are induced (Taddei 2007). Finally, some types of damagenamely, irreparable DNA double-strand breaks (DSBs), collapsed replication forks, and eroded telomeres-relocate to nuclear pores to be processed for repair, unlike DSBs that can be repaired by recombination with a homologous template (for review, see Nagai et al. 2010). For these relocalization events to occur, whether at a promoter, a replication fork, or a DSB, chromatin must be mobile.Rapid time-lapse fluorescence microscopy of GFP-LacItagged genomic loci has shown that individual chromosomal domains move constantly in a near-random walk within a restrained volume of the nucleus (Hubner and Spector 2010). The measured radius of constraint for the movement of an average chromosomal locus (;0.6 mm) was roughly similar in every species investigated, although mobility was also shown to be influenced by local chromatin context (Marshall et al. 1997;Heun et al. 2001;Vazquez et al. 2001;Chubb et al. 2002;Gartenberg et al. 2004). For instance, lacO arrays inserted near budding yeast ce...

Budding yeast, like other eukaryotes, carries its genetic information on chromosomes that are sequestered from other cellular constituents by a double membrane, which forms the nucleus. An elaborate molecular machinery forms large pores that span the double membrane and regulate the traffic of macromolecules into and out of the nucleus. In multicellular eukaryotes, an intermediate filament meshwork formed of lamin proteins bridges from pore to pore and helps the nucleus reform after mitosis. Yeast, however, lacks lamins, and the nuclear envelope is not disrupted during yeast mitosis. The mitotic spindle nucleates from the nucleoplasmic face of the spindle pole body, which is embedded in the nuclear envelope. Surprisingly, the kinetochores remain attached to short microtubules throughout interphase, influencing the position of centromeres in the interphase nucleus, and telomeres are found clustered in foci at the nuclear periphery. In addition to this chromosomal organization, the yeast nucleus is functionally compartmentalized to allow efficient gene expression, repression, RNA processing, genomic replication, and repair. The formation of functional subcompartments is achieved in the nucleus without intranuclear membranes and depends instead on sequence elements, protein-protein interactions, specific anchorage sites at the nuclear envelope or at pores, and long-range contacts between specific chromosomal loci, such as telomeres. Here we review the spatial organization of the budding yeast nucleus, the proteins involved in forming nuclear subcompartments, and evidence suggesting that the spatial organization of the nucleus is important for nuclear function. TABLE OF CONTENTS Abstract 107Introduction 108

To investigate the mechanisms that assure the maintenance of heterochromatin regions, we took advantage of the fact that clusters of heterochromatin DNA replicate late in S phase and are processed in discrete foci with a characteristic nuclear distribution. At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1α and -β), and chromatin assembly factor 1 (CAF-1). During replication, Hp1α and -β domains of concentration are stably maintained, whereas heterochromatin regions are enriched in both CAF-1 and replication-specific acetylated isoforms of histone H4 (H4Ac 5 and 12). We defined a time window of 20 min for the maintenance of this state. Furthermore, treatment with Trichostatin A (TSA), during and after replication, sustains the H4Ac 5 and 12 state in heterochromatin excluding H4Ac 8 and 16. In comparison, early replication foci, at the same level, did not display any specific enrichment in H4Ac 5 and 12. These data emphasize the specific importance for heterochromatin of the replication-associated H4 isoforms. We propose that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1α and -β, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.

Budding yeast telomeres and cryptic mating-type loci are enriched at the nuclear envelope, forming foci that sequester silent information regulators (SIR factors), much as heterochromatic chromocenters in higher eukaryotes sequester HP1. Here we examine the impact of such subcompartments for regulating transcription genome-wide. We show that the efficiency of subtelomeric reporter gene repression depends not only on the strength of SIR factor recruitment by cisacting elements, but also on the accumulation of SIRs in such perinuclear foci. To monitor the effects of disrupting this subnuclear compartment, we performed microarray analyses under conditions that eliminate telomere anchoring, while preserving SIR complex integrity. We found 60 genes reproducibly misregulated. Among those with increased expression, 22% were within 20 kb of a telomere, confirming that the nuclear envelope (NE) association of telomeres helps repress natural subtelomeric genes. In contrast, loci that were down-regulated were distributed over all chromosomes. Half of this ectopic repression was SIR complex dependent. We conclude that released SIR factors can promiscuously repress transcription at nontelomeric genes despite the presence of ''anti-silencing'' mechanisms. Bioinformatic analysis revealed that promoters bearing the PAC (RNA Polymerase A and C promoters) or Abf1 binding consenses are consistently downregulated by mislocalization of SIR factors. Thus, the normal telomeric sequestration of SIRs both favors subtelomeric repression and prevents promiscuous effects at a distinct subset of promoters. This demonstrates that patterns of gene expression can be regulated by changing the spatial distribution of repetitive DNA sequences that bind repressive factors.