Nucleosomes affect local transformation efficiency

Abstract

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today's recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.

Figure 1.

Nucleosome density affects transformation efficiency. (A) The transformation efficiency differs significantly between nucleosome-occupied (Peak: positions with a high nucleosome density) or nucleosome-depleted (Valley: positions with a low nucleosome density) positions. (B) Transformation efficiency anti-correlates with nucleosome density.

Figure 2.

(A) Nucleosome positions do not change during the transformation procedure. We collected cells from three different steps during a typical transformation experiment and determined the nucleosome positions of a region with a well-positioned nucleosome pattern at these different stages of the transformation procedure. (B) Different transformants can grow equally well in different concentrations of hygromycin (HYG). We grew the transformants with HYG cassette inserted in different positions with high nucleosome density (referred to as Peak) (n = 30) or low nucleosome density (referred to as Valley) (n = 30) in different media (YPD containing different concentrations of hygromycin). Results show no variation in growth on selective media between the two groups.

Figure 3.

(A) Transformation efficiency anti-correlates with nucleosome density (in a region with well-positioned nucleosome pattern). Transformation efficiency (black bars) in a 300 bp region of the S. cerevisiae genome containing well-positioned nucleosomes depicts a wave-like pattern which anti-correlates with nucleosome density (orange line). In the nucleosome map (orange line), peaks represent the presence of a well-positioned nucleosome on the DNA sequence and valleys represent the nucleosome-depleted ‘linker’ DNA. (B) Insertion of the marker cassette (every 7 bp) in a 300 bp region of the S. cerevisiae genome lacking well-positioned nucleosomes shows that in this case, transformation efficiency (black bars) does not show a clear wave-like pattern as observed in panel A.

Figure 4.

(A) Transformation efficiency does not correlate with properties of local DNA. The GC content and melting temperature of sequences of homology for each target position do not show correlation with local transformation efficiency. (B) Changes in the nucleosome pattern affect local transformation efficiency. Insertion of a marker cassette (every 7 bp) in a region of ∼100 bp in the same genomic locus in two strains that have a different nucleosome structure in this region reveals that nucleosome positions directly affect transformation efficiency. Left: the S288c strain shows a well-positioned nucleosome centered over the middle of the locus under investigation (seen as a peak in the relative nucleosome density, orange line). The local transformation efficiencies (black bars) show a strong anti-correlation with nucleosome density (orange line). Right: the same genomic region in a mutant derived from the same S288c strain that contains a different sequence upstream of the locus under investigation that disrupts the local nucleosome structure (orange line). In this mutant, the transformation efficiencies (black bars) do not show a clear pattern anymore. Moreover, despite both wild type S288c strain (left) and the mutant (right) have exactly the same DNA sequence at the locus under investigation, the transformation efficiencies differ greatly between these two strains.