Our genes are not alone the blueprint for our body. Supplementing the coding capacity of the linear DNA molecule are a series of "epigenetic" factors, which regulate the accessibility of DNA for enzymatic transactions. The compaction of DNA around histones, the covalent modification of histones on cores and tails, the methylation of cytosine residues in DNA, and the folding of chromatin into compact structures, all modulate the accessibility and functionality of the primary coding sequence. Rather than replacing a genetic code, the epigenetic code complements and extends it, allowing for cell-type specific patterns of gene expression and memory of expression states. Epigenetic modifications can respond to both internal and external signals, such as hormones, developmental cues, nutrients, stress and environmental insults. Thus, understanding the modulation of gene expression through epigenetic modification will have significant implications for the reprogramming of gene expression, for tissue engineering and cell fate decisions. Not surprisingly, the enzymes that place or remove epigenetic marks have been directly implicated in many human diseases, including aging and cancer.
Despite its obvious biomedical importance, most of our insights into epigenetic control mechanisms stem from studies in model organisms, including yeast, worms, plants, flies and mice. Model organisms have allowed epigenetic information to be examined over a range of environmental and pharmacological conditions. Genetic manipulation of epigenetic control mechanisms have shown how they impact disease.
The Gasser laboratory pursues two related areas of research: mechanisms that maintain genomic stability through replication, and those that mediate epigenetic inheritance of transcriptional states through cell division and development. We address these questions at both molecular and systemic levels, combining genetics with high resolution live imaging, coupled with biochemical approaches. We have had particular success in extending the approaches optimized in budding yeast to the nematode C. elegans. Recent breakthroughs allow us to identify a role for nuclear organization in cellular differentiation.
The epigenome can be modified in response to both internal and external signals, yet has the capacity to be transmitted through replication into daughter cells. Thus the establishment and modulation of epigenetic marks can significantly impact transcriptional reprogramming and cell fate decisions. This is relevant both for tissue engineering by differentiating stem cells, and for controlling the reprogramming that occurs during oncogenic transformation. To understand epigenetic controls and their inheritance during tissue-specific differentiation events, we have examined how non-histone repressors create inaccessible heterochromatic states. We further explore how the organization of such chromatin in the nucleus contributes to its establishment and/or propagation.
Our area of expertise is the 3D organization of chromatin in the nucleus and the impact of structural considerations on regulatory and hereditary mechanisms. Like nuclei in all organisms, the budding yeast nucleus is divided into open zones of active gene transcription and closed zones of heterochromatin. In many organisms, including yeast, repressive zones are found adjacent to the nuclear envelope. These micro-environments are proposed to facilitate repression by accumulating repressive histone-binding factors, and in yeast they are created by the clustering of telomeric repeats. In contrast to these repressive zones, we found that the association of active genes with nuclear pores can actually enhance transcription (Taddei et al., 2006). Pore association does not seem increase initiation but rather elongation, mRNA processing or its export. Similar mechanisms may fine-tune chromosomal expression in higher eukaryotes, as exemplified by the pore-association of up-regulated genes on the X-chromosome in male flies (Ahktar and Gasser, 2007).
We have identified a novel pathway for the anchoring of telomeric repeats in S-phase cells, which involves the highly conserved SUN domain protein, Mps3 (Schober et al., 2009). This telomere anchoring pathway depends on the catalytic yeast telomerase subunit Est2, Est1 and Tlc1 to which binds Yku80. The conserved Sad1-UNC-84-Nesprin (SUN) domain protein Mps3 is the principal membrane anchor for this pathway, and impaired anchoring to the Mps3 N-terminus in a tel1 deletion background led to deleterious levels of recombination between subtelomeric repeats. Through this we argue that telomere binding to the nuclear envelope helps protect the sequences from recombination events. Our results provide an example of a specialized structure that requires proper spatio-temporal localization to fulfill its biological role, and suggests a novel pathway of telomere protection.
The loss of telomeric anchoring has several other consequences for yeast cells. In a strain lacking both the Yku- anchoring pathway and a second anchoring protein called Enhancer of silent chromatin 1, we find that the Silent Information Regulatory (SIR) proteins 2, 3 and 4 are dispersed throughout the nucleus, rather than being sequestered in peripheral foci. 
Under these conditions, we find 60 genes reproducibly misregulated. Among those with higher expression, 22% were near telomeres, confirming that telomere anchoring helps repress subtelomeric genes. On the other hand, loci that were down-regulated by dispersed SIR factors were distributed across all chromosomes. Further controls allowed us to conclude that released SIR factors can repress promiscuously. Thus, the clustering of repeats in heterochromatic foci serves two functions; it both favors subtelomeric repression and prevents promiscuous effects of dispersed repressors. Crucially, these findings argue that subnuclear compartments help regulate gene expression patterns.
Despite vast genetic characterization, it is remains unclear what repressive chromatin actually looks like structurally. To examine this we have set up an in vitro system that allows the reconstitution of nucleosomes with the yeast heterochromatin proteins, Sir2, Sir3 and Sir4. To nucleosomal arrays created from recombinant histones, we add the yeast Sir2-3-4 complex purified from baculoviral-infected insect cells.
The loading of the SIR complex is cooperative and generates a stable structure bearing one SIR complex (Sir2-3-4 in 1:1:1 ratio) between adjacent nucleosomes. The affinity of Sir3 binding in particular is affected by the N-terminal tail of histone H4, and the acetylation of H4K16. Sir3 additionally interacts with the face of the nucleosome, requiring unmodified histone H3K79. Intriguingly we find that the by-product of Sir2-mediated NAD hydrolysis, O-acetyl ADP ribose, greatly stimulates the assembly of SIR proteins onto nucleosomes, by a still-obscure mechanism (Martino et al., 2009).
The impact of histone modification and active histone deacetylation on heterochromatin assembly is being examined by mapping the precise contributions of specific domains of Sir3 and Sir4 as they bind histones and DNA. We propose that O-AADPR binds the AAA+ helicase-like domain of Sir3 to induce a conformation change, that stabilizes the chromatin-SIR complex. Images of these changes are being gathered by electron microscopy.
How nuclear compartments regulated gene expression and repression during metazoan development is a formidable challenge, although we know that heterochromatin accumulates as cells differentiate. Using the worm C. elegans we have explored how nuclear compartments change during development, and whether genes expressed in a tissue-specific manner assume unique positions in nuclei of differentiating cells.
Partial 3D reconstitution of a 120-cell-stage embryo carrying a lacO-tagged transgene and expressing GFPLacI. anti-GFP: Green; anti-LMN-1: Red; and DNA (Hoechst): Blue. see Meister et al., 2010.
A genome-wide RNAi screen for factors that derepress and/or relocalize large heterochromatic arrays reveals a role for lysine methylation in peripheral anchoring. The anchored heterochromatic arrays carry methylation on both histone H3K9 and H3K27 (targets of the Suv3-9 or Polycomb histone methyl transferases respectively). Further studies will identify exactly which marks are involved and what recognizes them.
In contrast to the developmentally regulated promoters, our experiments with the heat-shock promoter hsp-16.2 shows that stress-activated arrays do not relocate upon induction, but remain at the nuclear periphery as they transcribe. Both the high level transcription and the association with the nuclear periphery requires heat-shock transcription factor Hsf-1. Genome-wide mapping of genes associated with nuclear pores and the nuclear lamina suggests that the reporter genes we study bind nuclear pores, but not the lamin-associated protein, lem-2. Taken together our study of C. elegans nuclear organization argues that there are transcriptionally active domains in the nuclear interior which harbour differentiation-induced genes during cell fate acquisition, while the nuclear periphery consists of both inactive zones of heterochromatin, and active sites for stress induced genes at nuclear pores.
For many years we have studied the critical molecular interactions between RecQ helicases and the S-phase checkpoint kinase ATR/Mec1 at stalled replication forks (Cobb et al., 2003, 2005). During the complex process of DNA replication, forks frequently encounter obstacles, such as tightly bound protein-complexes, or are challenged by DNA damage. As a consequence replication forks stall, forming fragile DNA structures that need to be stabilized in order to prevent DNA double strand break (DSB) formation and aberrant homologous recombination (HR). To coordinate this, a sophisticated surveillance mechanism called the intra-S phase checkpoint is activated to restrain potential fork collapse and to regulate cell cycle progression, DNA repair and late origin firing. The frequency of fork instability increases during oncogenic transformation, making this intra-S phase checkpoint a major gate-keeper that prevents fork-associated rearrangements that aggravate the cell’s oncogenic potential.
Two important proteins in stabilizing arrested replication forks are the checkpoint kinase ATR (called Mec1 in budding yeast) and the Bloom’s Syndrome helicase, which has a single homologue in yeast, the RecQ helicase Sgs1. It has been proposed that both ATR- and BLM-mediated pathways contribute to ensure fork integrity. 
Loss of both leads to a dramatic increase in fork collapse, gross chromosomal rearrangements, even in the absence of fork-stalling lesions. We hypothesized that in budding yeast these two pathways would converge on Replication Protein A, the single-strand binding heterotrimer necessary for replication and repair. Indeed, RPA recruits Mec1-Ddc2 to stalled replication forks and was earlier shown to bind Sgs1 tightly. We have now examined how these factors cooperate to stabilize stalled replication forks, ensuring both checkpoint activation and appropriate fork recovery. We study this under conditions of fork arrest which are induced by the low rNTP levels provoked by the cytostatic drug hydroxyurea (HU).
We mapped the sites of interaction between Sgs1 and RPA to an acidic domain in Sgs1 N-terminus and the first OB fold of the largest RPA subunit, Rpa70. To test the importance of this interaction for fork stability, we created a mutant, sgs1-r1, that lacks the binding interface. Although the mutation leaves Sgs1 active, it completely disrupts Rpa70 binding in two hybrid assays, although residual interactions can be detected between full-length proteins. Consistent with a role for this interaction in DNA polymerase stability, we found that sgs1-r1 partially displaces DNA pol α from stalled replication forks. However, in contrast to sgs1Δ, sgs1-r1 is epistatic to an intra-S phase specific mutation of ATR (Mec1) called mec1-100. Thus for this aspect of replisome stability, Mec1 and Sgs1 act on the same pathway. Combining this with secondary mutations in Sgs1 showed that both the RPA-binding and the helicase activities of Sgs1 are necessary to ensure the association of DNA pol α at stalled replication forks.
Intriguingly, the region of Sgs1 that interacts with RPA is also a target of Mec1-mediated phosphorylation. The phosphorylated form of Sgs1 binds tightly to the downstream checkpoint kinase, Rad53 (Chk2 in mammals) and contributes to the activation of Rad53 by Mec1 at stalled replication forks. Thus a conserved acidic, N-terminal domain of Sgs1 binds RPA and serves a second function in checkpoint activation. The two functions may be mutually exclusive and regulated by Mec1 phosphorylation, thus reflecting the severity of the lesion at the replication fork.
The major binding site for Sgs1 in the N-terminal oligonucleotide binding (OB) fold of the largest RPA subunit, Rpa70, was also carefully mapped and characterized by in vitro binding studies. A basic cleft on one side of the folded OB structure, reported to mediate p53 binding in human cells, is similarly implicated in Sgs1 binding in the yeast protein. Indeed, a charge reversal mutation pointing into the basic cleft called rfa1-t11, is found to disrupt binding to Sgs1 in two-hybrid assays. In addition, rfa1-t11 displays a genome-wide replication defect in response to replication stress, and affects DNA pol α association at HU-stalled replication forks. These phenotypes are stronger for rfa1-t11 than for sgs1Δ, arguing that only a fraction of its activity can be assigned to Sgs1 binding.
Intriguingly, there is an epistatic relationship between rfa1-t11 and proteins involved in homologous recombination (HR). We therefore suspect that impaired HR between newly replicated sisters (also called strand-switching synthesis) may be the cause of fork restart failure in rfa1-t11 cells. This exciting link between RPA and the recombination machinery provides novel insights into how RPA coordinates replication fork stability.
One goal of this project is to be able to provoke fork collapse selectively in cancer cells, which are hypersensitive to perturbations in replication. Elaboration of this potential chemotherapeutic is pursued in collaboration with NIBR, using a chemicogenetic approach. Compounds that selectively kill cells on low concentrations of HU, and in the presence of sgs1Δ or mec1-100 mutations, now identify novel pathways of intervention for intervention at the replication fork.
We have described the mobility of internal loci on yeast interphase chromosomes by live fluorescence microscopy with high precision. Yeast chromatin moves constantly in a random diffuse manner (radius of constraint = 0.6 ± 0.1 µm), except when it is anchored by protein-protein interactions.
Based on single particle tracking and mathematical simulations of random walks in a confined volume, we characterized the constraints exerted by the chromatin fiber. Intriguingly, local recruitment of the transcriptional activator VP16 and components of the Ino80 chromatin remodeling complex increase diffusion rate, large rapid steps and the radius of constraint of a given locus. Whereas the Ino80 induced mobility increase is dependent on its ATPase activity, the inhibition of transcription did not alter chromatin mobility. this suggests that chromatin remodeling associated with transcriptional activation and repair, rather than transcription per se, alters the mobility of the chromatin fiber.
If DSBs are repaired by homologous recombination (HR), the template must contact the site of damage. We are developing a system to monitor microscopically and genetically the homology search in living yeast cells. We have shown that Rad52-GFP foci, which mark sites of DSB repair, localize preferentially in the nuclear lumen, suggesting that HR occurs away from the nuclear periphery (Bystricky et al. 2009). In addition, we measured the movement of Rad52-GFP foci and found that they move slower and are more constrained than intact genomic loci. The meaning of this is under study and will be correlated with modeling of homology search based on theoretical considerations and nuclear constraints.
The Gasser laboratory was among the first to use Chromatin immunoprecipitation (ChIP) to examine the recruitment of factors to stalled forks and double strand breaks (DSBs) and pioneered the study of chromatin remodelers at these sites (Van Attikum et al., 2007; Dubrana et al., 2008). Using genome-wide approaches, we have found that the INO80 chromatin remodeling complex is present at origins of replication and tRNA genes throughout the yeast genome, but is particularly enriched at stalled replication forks near early firing origins (Shimada et al., 2008). We found that the INO80 complex is dispensible for origin firing and the maintenance of the replication bubble upon HU-induced arrest, yet the resumption of DNA replication was impaired in ino80, arp5 or arp8 mutants (Shimada et al., 2008). These mutants stimulated the HR repair response as they attempted to resume replication after fork arrest. Whether INO80 removes nucleosomes at stalled forks, as it does at breaks (van Attikum et al., 2007) is under study.
Extending our genome-wide mapping activities we recently mapped the binding sites of a closely related complex, the SWR1 remodeling complex (SWR-C or SRCAP in mammals). Despite the fact that both SWR-C and INO80 are recruited to DSBs, their patterns of binding in unperturbed yeast cells show no overlap. The SWR-C complex is found at promoters where it deposits the histone H2A.Z variant. Surprisingly, although the Ino80 actin-related subunit Arp5 and Arp8 always colocalize with the catalytic subunit Ino80, the Swr1 actin-related component, Arp6, has a life of its own. In budding yeast, Arp6 has both SWR-C-dependent and -independent functions in gene expression. The latter is achieved by its contribution to the anchorage of chromatin domains to the nuclear pore complex (Yoshida et al., 2010). Intriguingly, among the genes anchored to pores by Arp6 (but not by SWR-C) are a subset of highly expressed ribosomal protein genes. The loss of ribosomal protein gene positioning at pores in the arp6 mutant correlates surprisingly with an increase in expression (Yoshida et al., 2010). These observations provide first evidence for the involvement of an actin-related protein in long-range chromatin organization in the interphase nucleus, and shows that the modulation of gene expression at nuclear pores holds more mysteries to unveil.
GFP-tagged ARS607 (red) and a CFP-nuclear pore fusion (green) were expressed in a strain carrying a mutant form of Nup133 that allows nuclear pores to cluster. In cells exposed to HU and MMS, the collapsed replication fork moves to nuclear pores.