The Gasser laboratory focuses on two key lines of research: the mechanisms that spatially organize and silence heterochromatin during development (using C. elegans) and the mechanisms that ensure genomic stability during replication and repair of the genome, primarily pursued in yeast. A third line of research has come to an end with a low resolution structure of the yeast silent chromatin regulatory complex, Sir2-Sir3-Sir4 (collaboration with H Stahlberg, UniBasel) and a model for how the SIR complex spreads along chromatin to repress promoters.
Key findings on the establishment and maintenance of heterochromatin in C. elegans include the following:
1. Step-wise methylation of histone H3K9 is necessary to sequester heterochromatin at the nuclear periphery in C elegans embryos. Two HMTs carrying out step-wise methylation are identified (MET-2, SET-25). A non-HP1 chromodomain protein called CEC-4, is required for peripheral localization, but not repression. Phenotypes that stem from heterochromatin mislocalization are under study. (Towbin et al. 2012; Gonzalez-Sandoval, submitted)
2. A heat-shock promoter, hsp-16.2, leads to gene association with nuclear pores, which is enhanced when the promoter is induced. Anchoring is dependent on RNA pol II and promoter-bound factors, HSF-1 and SAGA. (Rohner et al., 2013)
3. The worm telomeric Shelterin complex component POT-1 and the inner nuclear membrane SUN-1 protein anchor worm telomeres at the nuclear envelope in embryos. ALT telomeres are clustered and less anchored. (Ferreira et al., 2013)
From yeast to man, heterochromatin is sequestered away from enchromatin at the nuclear envelope. The nuclear envelope factors that bind transcriptionally repressed heterochromatin remain uncharacterized. In a genome-wide RNAi screen we found that depletion of S-adenosyl methionine (SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in C. elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms which are enriched for histone H3 lysine 9 methylation. We then showed that these two HMTs target H3K9 in consecutive fashion: MET-2, a SetDB1 homolog, mediates mono- and dimethylation, and SET-25, a Suv39h-like enzyme, deposits me3. SET-25 co-localizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This co-localization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin. Importantly, the methylation status of histone H3K9 (either me1/2 or me3) leads to chromatin positioning in the nucleus, independent of its transcriptional status (Towbin et al., Cell, 2012).
Surprisingly worms lacking all H3K9 methylation as embryos survive and differentiate. At L1 stage they still do not have significant H3K9me, yet tissue-specific repression and its sequestration at the nuclear envelope occurs. This means there are redundant pathways for anchoring and silencing that kick-in later in development. We note that H3K9me-free strains do show delayed or irregular developmental timing and heat-induced (25°C) sterility.
To identify pathways that mediate survival in worms lacking H3K9 HMTs we performed a second genome-wide RNAi screen to identify pathways that mediate this survival. A whole genome RNAi synthetic lethality screen comparing a set-25 met-2 mutant with wild-type worms identified many genes whose down-regulation is specifically and reproducibly lethal in the set-25 met-2 mutant, but not in N2 worms. The identified genes include general chromatin components such as histones, general and specific transcription factors, as well as chromatin remodelers. The aim now is to find the functional connection leading to the synthetic effect.
DNA transposons are a subgroup of repeat sequences that need to be silenced in order to prevent them from jumping into important regulatory regions. These are often repressed due to H3K9 methylation. Our current hypothesis is that transposon instability may be a key "life-preserving" function of H3K9 methylation. Transposons in worms specifically correlate with H3K9me, and we have preliminary data showing enhanced transposon excision in the set-25 met-2 double mutant. We are examining if and how H3K9me silences of transposons and reduces breaks in heterochromatic regions.
Earlier work from our laboratory studying tagged chromatin loci with live time-lapse microscopy, led to the discovery that chromatin is highly mobile in the nucleus. Recent studies show that this mobility increases upon DNA damage. The degree of dynamics at double strand breaks (DSB) depends on the phase of the cell cycle and the type of damage. We have shown that Cohesin holds sisters together and that by attaching sisters, it greatly dampens DNA mobility (Dion et al. 2013). Moreover, checkpoint kinase Mec1 (ATR) regulates mobility at DSBs, as does the remodeler INO80. Intriguingly, an activated Mec1/ATR kinase (checkpoint response) also increases chromatin movement genome-wide (Dion et al, 2013; Seeber et al., 2013).
Besides increasing its mobility, we have shown that some types of damaged DNA are recruited to the nuclear envelope, where damage either binds to nuclear pores or to a conserved SUN domain protein called Mps3 (HsSUN1). This relocation appears to contribute to DSB processing for certain types of nonhomologous repair (e.g. break induced replication). We continue to study what triggers this relocation to NE sites and study the selectivity. We asked two questions: what is the role of chromatin remodelers (mutations in the remodeler SWR1 ablates relocalization) ? Why are there two sites and do telomere sequences influence the differential targeting to one or the other site, also influencing the differential outcome of repair ?
Confirming previous evidence implicating Mec1/Tel1 kinase and the histone variant, Htz1, we found that the incorporation of Htz1 by the SWR1 remodeler, appears to be necessary for the relocation of DSB to the nuclear envelope. To test whether Htz1 itself can position chromatin, we targeted a lexA-Htz1 fusion, and we confirm that it is sufficient to shift an undamaged locus to Mps3 without the assistance of SWR1 complex. Consistently, the increased, locus-specific mobility of a DSB in yeast was abolished in SWR1-deficient mutants and in cells lacking Htz1. These relocalization assays were confirmed by ChIP for Mps3 or Nup84. The INO80 complex appears necessary only for the binding of damage to Mps3, while Swr1 affects relocation to either pores or Mps3. While Rad52, Rad51 and telomerase machinery were reported to be necessary for anchoring DSBs to Mps3, we found that these are not required for the DSB relocation to the nuclear periphery due to the second binding sites at nuclear pores. This suggests that the recruitment of SWR1 complex and deposition of Htz1 near the DSB increase its mobility and affinity for pores; a second event controls a shift to Mps3, and this appears to require INO80. Short telomeres may be sequestered from promiscuous recombination at Mps3, thus we are systematically studying the relocalization of DSB induced near TG repeat sequences.
We have also more recently studied the role of SUMOylation and Slx5/Slx8 (HsRNF4; SUMO directed Ub ligase) in damage localization ? We see a role for Sumo dependent Ub ligation in relocation, which triggers the recruitment of Slx5/Slx8, which in turn most likely leads to the degradation of the ubiquitinated target. We continue to search for the relevant target to determine how this step is involved in repair pathway choice.
An intriguing result stemmed from a chemicogenetic screen for compounds (and their targeted pathway) that synergize with impaired replication fork stability (Shimada et al., 2013).The screen surprisingly yielded a yeast TOR kinase inhibitor that causes cell death due to massive genomic fragmentation. The chromosome breakage is due to an inability to repair the oxidative damage induced by ionizing radiation or Zeocin. It turns out that inhibition of the cytoplasmic TORC2 complex, or misregulation of the actin polymerization which is controlled by TORC2, renders cells hypersensitive to low level DNA damage. This has led us to study how nonpolymerized actin, like that found in remodelers affects various repair pathways.
Cells deal with a continuous onslaught of endogenous and exogenous DNA damage. Stalled or collapsed forks and double strand breaks trigger a highly conserved DNA damage checkpoint response that requires activation of the PI3K-related kinase ATR (Mec1). Particularly in S-phase, Mec1/ATR is implicated in the maintenance of fork stability and restart potential when replication forks encounter damage or stalling conditions. We have an S-phase deficient allele of MEC1, mec1-100, that carries point mutations near the HEAT repeats and within the FAT domain. This mutant supports the G2 DNA damage checkpoint, but is compromised in G1-S and intra-S-phase checkpoints. Since the mutant kinase has robust enzymatic activity in vitro we hypothesize that Mec1 co-factor or targets are unique in S phase and are sensitive to these mutations. We have approached this with high-throughput epistasis EMAP approaches, with genetics, and with biochemistry and phosphoproteomics. All technique show that the key S phase regulators of Mec1 are PPH3 and PSY2, which form an evolutionary conserved phosphatase complex. We confirmed that PPH3 deletion indeed suppresses HU sensitivity of mec1-100. Mec/Ddc2 and the Pph3 phosphatase form a complex) and comparative phosphoproteomics reveals key targets of Mec1 and the phosphatase in the S-phase checkpoint. One attractive hypothesis is that Pph3 regulates the activating auto-phosphorylation of Mec1. This may shed light on the cell cycle regulation of ATR/Mec1 (Hustedt et al., 2015).
The laboratory spent many years studying the yeast silent chromatin complex Sir2-3-4, leading us towards the final goal of solving the structure of the SIR complex bound to nucleosomes. While we tried valiantly, the isolated heterotrimer was not conducive to crystallization, and the presence of homodimerization domains in Sir3 and Sir4 (as well as Sir3-Sir4 and Sir4-Sir2 interactions) generated a diversity of complexes, rather than a uniform single complex. We find that both Sir3 and Sir4 homodimerization events are essential for efficient repression and spreading (Oppikofer et al., EMBO J 2013, and work in preparation). Below is a model for SIR assembly, based on work of many, but focusing on our key contributions.