Nucleosomes are the structural units of eukaryotic chromatin. The vast majority of the genome is tightly associated with nucleosomes, which modulate all genomic processes through their interaction with regulatory complexes and through the information encoded in epigenetic modifications of histones. Consistent with these specialised functions, most nucleosomes in the genome occupy precise positions relative to the DNA sequence (Figure 1).

1. Most promoters colocalise with short nucleosome-free regions (NDRs) where transcription initiates (Figure 1). The aim of this line of research is to determine how NDRs manage to exclude nucleosomes through the combined action of remodelers, transcription factors, the mechanical properties of DNA and the +1 nucleosome.

2. We are studying how chromatin remodelers regulate the dynamics of the nucleosome pattern. We use a degron-based approach that allows the transient and reversible inactivation of remodelers to assess how modifying the position of nucleosomes in vivo affects the expression pattern of genes involved in different physiological conditions (Figure 2).

3. We are also interested in how chromatin organisation and transcriptional patterns are regenerated after their physical disruption during double-strand break repair using an experimental approach based on genomic sequencing that we have developed recently (Figure 3) (Ramos et al. 2022).

Given the universality of DNA-histone interactions and the conserved regulatory role of chromatin in fundamental genomic processes, we use the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae as model systems for biochemical, genetic, genomic, bioinformatic and computational approaches. We exploit the feasibility of genome manipulation in these yeasts to establish cause-effect relationships between genome manipulation, chromatin organisation and gene expression at a level of resolution that is difficult to achieve in animal cells.

Figure 1. Nucleosomal organisation in S. pombe and S. cerevisiae.
Distribution of nucleosomes along two genomic regions at the indicated genomic positions in chromosomes II and IV. Genes and direction of transcription are indicated by arrows. Asteriks indicate the location of nucleosome-free regions (NDR) at the 5’ end of most genes.

Figure 2. Reversible loss of nucleosome positioning upon remodeler inactivation.
Nucleosome occupancy maps of a S. pombe strain carrying a degron-tagged Snf21 catalytic subunit of the RSC remodeler. Vertical dashed lines indicate the dyad position of +1 nucleosomes of the divergent sib3 and rps2402 genes. These nucleosomes are displaced towards the NDR 30 minutes after degron induction and return to their wild-type position 8-12 hours after degron switch-off.

Figure 3. Visualisation of DSB repair of S. cerevisiae chromosome III by genomic sequencing.
The figure shows the coverage along the entire chromosome III of S. cerevisiae at the indicated times after induction of a double-strand break (DSB) at position 200753. The extension of the resection is visualised by the decrease and recovery of coverage in the region flanking the DSB (red line).