Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14 -100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170 -220 nm and a mass density of Ϸ110 -150 bp͞nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.higher-order structure ͉ 30-nm fiber ͉ nucleosomes G enetic studies indicate that the spatial positioning of the genome in interphase contributes to the regulation of nuclear functions, yet the principles that govern the organization of interphase chromosomes (Chrs) are largely unknown. At the simplest level, DNA is folded through interaction with histones forming the nucleosome core particle (NCP), which yields a 6:1 or 7:1 compaction ratio depending on linker length. Arrays of nucleosomes are further condensed by Ϸ6-fold into a higherorder structure, the so-called 30-nm fiber, whose in vivo architecture is unresolved. Several models have been proposed for this structure (ref. 1 and reviewed in ref. 2), yet species' specific variation in linker histones and nucleosome repeat lengths may lead to variation in fiber characteristics. How interphase Chrs fold beyond this level of organization is even less well understood, although in addition to fiber compaction, local looping and͞or anchoring to subnuclear elements may influence chromatin conformation (3, 4).To analyze chromatin compaction ratios in interphase nuclei, laboratories have generally applied fluorescence in situ hybridization (FISH), using differentially derivatized probes. These data were interpreted as identifying mega bp-sized loops (averaging 3,000 kb) with the bases of the loops being distributed in a random walk throughout the nucleoplasm (5-7) or as a chain of chromosomal subcompartments each comprising Ϸ10-20 loops of Ϸ120 kb (8). The random distribution of the chain may reflect local chromatin dynamics, which have been recently well documented in living cells by rapi...

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...

Telomeres form the ends of linear chromosomes and protect these ends from being recognized as DNA doublestrand breaks. Telomeric sequences are maintained in most cells by telomerase, a reverse transcriptase that adds TG-rich repeats to chromosome ends. In budding yeast, telomeres are organized in clusters at the nuclear periphery by interactions that depend on components of silent chromatin and the telomerase-binding factor yeast Ku (yKu). In this study, we examined whether the subnuclear localization of telomeres affects end maintenance. A telomere anchoring pathway involving the catalytic yeast telomerase subunits Est2, Est1, and Tlc1 is shown to be necessary for the perinuclear anchoring activity of Yku80 during S phase. Additionally, we identify the conserved Sad1-UNC-84 (SUN) domain protein Mps3 as the principal membrane anchor for this pathway. Impaired interference with Mps3 anchoring through overexpression of the Mps3 N terminus in a tel1 deletion background led to a senescence phenotype and to deleterious levels of subtelomeric Y9 recombination. This suggests that telomere binding to the nuclear envelope helps protect telomeric repeats from recombination. Our results provide an example of a specialized structure that requires proper spatiotemporal localization to fulfill its biological role, and identifies a novel pathway of telomere protection.[Keywords: Telomere protection; telomerase; nuclear envelope; Sad1/UNC-84 (SUN) homology domain; Mps3; nuclear organization; ATM homolog Tel1] Supplemental material is available at http://www.genesdev.org.