The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Eukaryotic cells repair DSBs using both non-homologous end joining (NHEJ) and homologous recombination (HR). How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important questions. In vertebrates, phosphorylation of the histone variant H2A.X (γ-H2A) occurs rapidly after formation of DSBs 1 , spreads over megabase chromatin domains, and is required for stable accumulation of DNA repair proteins at DNA damage foci 2 . In Saccharomyces cerevisiae, phosphorylation of the two major H2A species is also signaled by DSB formation, spreading ∼40 Kb in either direction from a DSB 3 . Here we show that near a DSB γ-H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease. However, γ-H2A and nucleosome loss occur independently of one another. The DNA damage sensor MRX (Mre11-Rad50-Xrs2) 4 is required for histone eviction, which additionally depends on the ATP-dependent nucleosome-remodeling complex, INO80 5 . The repair protein Rad51 6 shows delayed recruitment to a DSB in the absence of histone loss, suggesting that MRX-dependent nucleosome remodeling regulates the accessibility of factors with direct roles in DNA damage repair by HR. We propose that MRX regulates two pathways of chromatin changes, including nucleosome displacement, required for efficient recruitment of HR proteins, and γ-H2A, which modulates checkpoint responses to DNA damage 2 .To elucidate the chromatin pathways leading to DSB repair in Saccharomyces cerevisiae, we employed a MATα haploid strain that lacks HMR and HML donor sequences and carries a galactose-inducible HO gene 7 . In this strain, HO endonuclease introduces a DSB at MAT that can only be repaired by NHEJ, although the major HR proteins are recruited to the break site 6 . We analyzed chromatin structure along 12-20 Kb encompassing the DSB by chromatin immunoprecipitation (ChIP) followed by real-time PCR, which provided sensitive measurement of the kinetics and spatial distribution of chromatin changes and recruitment of repair proteins around the break site.Budding yeast H2A is phosphorylated on serine 129 by the ATM/ATR homologs Tel1/ Mec1 8 . In agreement with a recent report 3 , we found that γ-H2A accumulated rapidly and extensively on either side of the DSB, and that γ-H2A levels were lower close to the DSB relative to 6 Kb distant (Figure 1a; Supplementary Figure 3a). These latter results suggested a loss in nucleosome integrity near the DSB. The nucleosome consists of 146 bp of DNA wrapped ∼two times around a histone octamer comprising an (H3/H4) 2 tetramer and two H2A/H2B dimers. To determine if nucleosome stability was altered at the DSB, we performed ChIP in strains expressing either Flag-H2B or Flag-H3. The levels of both histones decreased 60-90 min after HO induction and were reduced three-fold by 120 min (Figure 1a) loss of both histones suggests that ...

The monoubiquitylation of histone H2B has been associated with transcription initiation and elongation, but its role in these processes is poorly understood. We report that H2B ubiquitylation is required for efficient reassembly of nucleosomes during RNA polymerase II (Pol II)-mediated transcription elongation in yeast. This role is carried out in cooperation with the histone chaperone Spt16, and in the absence of H2B ubiquitylation and functional Spt16, chromatin structure is not properly restored in the wake of elongating Pol II. Moreover, H2B ubiquitylation and Spt16 play a role in each other's regulation. H2B ubiquitylation is required for the stable accumulation of Spt16 at the GAL1 coding region, and Spt16 regulates the formation of ubiquitylated H2B both globally and at the GAL1 gene. These data provide a mechanism linking H2B ubiquitylation to Spt16 in the regulation of nucleosome dynamics during transcription elongation.

Epigenetic control is an important aspect of gene regulation. Despite detailed understanding of protein-coding gene expression, the transcription of non-coding RNA genes by RNA polymerase (pol) III is less well characterized. Here we profile the epigenetic features of pol III target genes throughout the human genome. This reveals that the chromatin landscape of pol III-transcribed genes resembles that of pol II templates in many ways, although there are also clear differences. Our analysis also discovered an entirely unexpected phenomenon, namely that pol II is present at the majority of genomic loci that are bound by pol III.

[1] We employ a numerical surface processes model to study the controls on postbreakup landscape development and denudational history of the southeast African margin. Apatite fission track data, presented in the companion paper, suggest that the Drakensberg Escarpment formed by rapid postbreakup river incision seaward of a preexisting drainage divide, located close to its present position, and subsequently retreated at rates of only $100 m m.y.À1 . Numerical modeling results support such a scenario and show that the prebreakup topography of the margin has exerted a fundamental control on subsequent margin evolution. The rheology of the lithosphere, lithological variations in the eroding upper crust, and inland base level falls provided secondary controls. A relatively low flexural rigidity of the lithosphere (T e % 10 km) is required to explain the observed pattern of denudation as well as the observed geological structure of the southeast African margin. Lithological variations have contributed to the formation of flat-topped ridges buttressing the main escarpment, as well as major fluvial knickpoints. Both these features have previously been interpreted as supporting significant Cenozoic uplift of the margin. An inland base level fall, possibly related to back-cutting of the Orange River drainage system and occurring 40-50 m.y. after breakup, explains the observed denudation inland of the escarpment as well as the development of inland drainage parallel to the escarpment. Our model results suggest that in contrast to widely accepted inferences from classical geomorphic studies, the southeast African margin has remained tectonically stable since breakup and escarpment retreat has been minimal (<25 km).

Novel yeast histone mutations that confer Swi±Snf independence (Sin ± ) were used to investigate the mechanisms by which transcription coactivator complexes relieve chromatin repression in vivo. Derepression of the¯occulation gene FLO1, which is normally repressed by the Tup1±Ssn6 corepressor, leads to its identi®cation as a constitutive Swi±Snf-dependent gene. We demonstrate that Tup1±Ssn6 is a chromatin remodelling complex that rearranges and also orders nucleosomal arrays on the promoter and over 5 kb of upstream intergenic region. Our results con®rm that the Swi±Snf complex disrupts nucleosome positioning on promoters, but reveal that it can also rearrange nucleosomes several kilobases upstream from the transcription start site. The antagonistic chromatin remodelling activities of Swi±Snf and Tup1±Ssn6 detected in an array of 32 nucleosomes upstream of FLO1 extend far beyond the scale of promoter-based models of chromatin-mediated gene regulation. The Swi±Snf coactivator and Tup1±Ssn6 corepressor control an extensive chromatin domain in which regulation of the FLO1 gene takes place.