Percepción de los estudiantes sobre su escuela y las relaciones en la escuela

Interestingly, Eco1 interacts with H2A.Z and PCNA with distinct binding modules. Whereas PCNA-binding is mediated via the Eco1 PIP-box at the N-terminus (Moldovan et al., 2006), an Eco1 construct lacking this motif can still bind H2A.Z in Y2H-assays (Fig. 27B). Moreover, in vitro pull-down assays identified the region encompassing amino acids 33-141 on Eco1 to be necessary and sufficient for H2A.Z interaction (Fig. 27D). As PCNA travels with the replication fork, an alternative Eco1 recruitment pathway would make sense when cohesion is to be established elsewhere than at the replication fork, e.g. at DSBs and outside of S-phase. Because H2A.Z can still bind to Eco1 variants that are defective in the PIP-box, a prediction from this model is, that overexpression of H2A.Z might rescue the lethality of eco1 PIP-box mutants. In fact, this was not the case (Fig. 30A, left panel). However, when overexpressing H2A.Z versions that lacked the SUMOylation site (either by C-terminal truncation or K126R mutation) these now indeed partially rescued the lethality of the PIP-box mutant eco1QKL18-21AAA _{(Fig. 30A, right panel).}

Results Cohesion establishment in response to DSBs

Figure 30. H2A.Z-SUMO-defective mutants can rescue the eco1 PIP-box mutant

(A) Rescuing effect of overexpression of different H2A.Z constructs on lethality of eco1 PIP-box mutants was tested by shuffling out the WT ECO1 upon plating on 5’FOA-containing medium. Equal amounts of cells were spotted and images were taken after 48h of growth at 30°C. The eco1 PIP-box mutants are inviable. Due to their failure to get recruited to DNA via PCNA, cohesion is not established. Only overexpression of SUMOylation-defective H2A.Z mutants but not of wt H2A.Z can rescue this lethality.

(B) Chromatin binding assay. The eco1 PIP-box mutant (QKL18-21AAA) shows a reduced chromatin (CHR) association, which can be rescued by overexpressing the SUMOylation-defective H2A.Z mutant. (SUP, supernatant; WCE, whole cell extract)

Moreover, chromatin binding assays revealed that indeed, when overexpressing htz1K126R _{the eco1 PIP-box mutant relocates to the chromatin fraction (Fig. 30B). In} conclusion, this supports the notion that the H2A.Z - Eco1 interaction could constitute an alternative recruitment pathway for cohesion establishment, with SUMOylation being a negative regulator of this event.

Intriguingly this is reminiscent of the recruitment pathway involving PCNA. Here, overexpression of PCNA-mutants defective in SUMOylation also rescued eco1 cohesion mutants (Moldovan et al., 2006), indicating that SUMOylation of the recruitment factor PCNA is inhibitory for cohesion. To test whether a parallel regulation really exists for H2A.Z as well, C-terminal, linear H2A.Z-SUMO fusions were generated (Fig. 31A). As the SUMOylation sites (K126 and K133) lie almost at the very C-terminus of the protein, these fusions are likely to resemble the naturally modified species. Remarkably, overexpression of these constructs was severely toxic to cells (Fig. 31B). Moreover, this dominant negative effect was not observable

Results Cohesion establishment in response to DSBs

Figure 31. H2A.Z-SUMO represses cohesion establishment (A) Western blot showing expression of H2A.Z linear fusions.

(B) Overexpression of H2A.Z-SUMO-BD linear fusions is toxic, whereas overexpression of H2A.Z-BD or BD alone does not impair cell growth. This dominant negative effect of the SUMO fusion requires the constructs to be incorporated into chromatin as they loose their toxicity in a Δswr1 background. (C) Overexpression of H2A.Z-SUMO fusions leads to cohesion defects. Quantification of cohesion defects in strains GFP-tagged on Chromosome III. Cells were arrested in metaphase and the number of GFP-signal present in each cell was scored, indicating either normal cohesion (one signal) or a cohesion defect (two signals; see examples in micrographs). Data are represented as means of 3 independent experiments (n>100) ± SEM.

when H2A.Z incorporation into chromatin was impaired, e.g. in Δswr1, or when

domains other than SUMO were linearly fused to the C-terminus of H2A.Z, e.g. the Gal4-BD domain (Fig. 31B).

To test whether the toxicity of the H2A.Z-SUMO fusions correlated or was due to impaired Eco1 function, these constructs were assayed for cohesion defects using cohesion tester strains (Bhalla et al., 2002). These harbor an array of Lac

C

A

Results Cohesion establishment in response to DSBs repressor (LacI) binding sites (LacO) on chromosome IV, which can be made visible by expression of a nuclear-targeted GFP_{LacI fusion protein. Is cohesion established}

properly, the two sister chromatids present in G2 cells will be in close proximity to each other and thus only one GFP focus is observable. However, when cohesion is faulty or lacking, sister chromatids will move away from each other, thereby splitting the GFP signal in two. This system provides a straightforward and accurate means for quantification of cohesion efficiency. Strikingly, the expression of the H2A.Z- SUMO fusions led to a drastic increase in cohesion defects, being even further augmented by the addition of a second SUMO moiety to the fusion protein (Fig. 31C). These data correlate well with the effects observed on cell viability (Fig. 31B) and imply that H2A.Z SUMOylation counteracts cohesion establishment.

Figure 32. H2A.Z-SUMO represses Eco1-binding to chromatin.

Chromatin binding assay showing that overexpression of H2A.Z-SUMO-fusions blocks Eco1 binding to chromatin (denoted by arrow), similar as when Eco1 binding capability to PCNA is abrogated (*PIP). In

Δhtz1 or after DNA damage, Eco1 chromatin association remains unchanged compared to WT.

A reasonable explanation for the above observed toxicity and cohesion defects when overexpressing the H2A.Z-SUMO fusions is that overloading chromatin with H2A.Z-SUMO will act as an Eco1-repellant at the DNA. Indeed, when assayed for chromatin association, cells overexpressing H2A.Z-SUMO showed a similar reduction in Eco1 chromatin binding as when the PCNA-recruitment pathway is blocked by mutations in the PIP-box (Fig. 32).

Taken together, these findings suggest that modification of H2A.Z by SUMO is detrimental for sister chromatid cohesion, whereas unmodified or unmodifyable H2A.Z seems to be stimulatory. At first sight, these results may appear paradoxical, as H2A.Z is constitutively SUMOylated throughout the cell cycle. However, only a very minor fraction of H2A.Z is in fact modified by SUMO. As cohesion appears not to be uniformly distributed throughout the genome (Lengronne et al., 2004), H2A.Z- SUMO might in fact be needed to keep certain chromosomal regions free of cohesion.

Results Cohesion establishment in response to DSBs