SNIC
SUPR
SNIC SUPR
K. lactis MNaseSeq
Dnr:

SNIC 2018/8-76

Type:

SNAC Small

Principal Investigator:

Stefan Åström

Affiliation:

Stockholms universitet

Start Date:

2018-02-15

End Date:

2019-03-01

Primary Classification:

10602: Biochemistry and Molecular Biology

Webpage:

Allocation

Abstract

In Baker’s yeast (Saccharomyces cerevisiae) an induced DNA double strand break at the mating type locus (MAT) leads to a programmed genome rearrangement, in which the MAT locus DNA sequence is replaced by the DNA sequence from a cryptic mating type locus. There are two cryptic (not expressed) mating type loci: Hidden MAT Left (HML) and Hidden MAT right (HMR), where the HML locus contains the alpha-mating type information and the HMR locus contains the a-mating type information. In principle, the HMRa and HMLalpha loci should have equal probability being used as donors, as the gene conversion mechanism simply assess sequence homology to a recessed end and both HMRa and HMLalpha share homology to MAT. However, a recombination enhancer (RE) biases which donor locus is used. The RE is active in MATa cells and activates the HMLalpha locus as donor for the gene conversion. In MATalpha cells the RE is inactivated by the alpha-specific transcriptional repressor alpha2, leading to that the HMRa locus is used as donor. This donor preference is efficient, resulting in a switch of mating type in ~90% of the events. The RE resides on the same chromosome arm as HMLalpha, but at a distance of 16kb. The details how the RE promotes the use of HMLalpha as donor are poorly understood, but a DNA-looping model has been proposed. The milk yeast (Kluyveromyces lactis) is similar to S. cerevisiae in that its genome contains MAT, HMLalpha and HMRa loci. Moreover, K. lactis switch mating type using a gene conversion process and in unpublished experiments we have observed donor preference. However, we have been unable to identify the RE in K. lactis. It is not possible to identify the K. lactis RE based on the presence of alpha2-binding sites (because there are too many) or synteny (because of divergence in gene order) so another approach is necessary. To identify the RE, we will analyze a Micrococcal nuclease sequencing (Mnase-seq) experiment of K. lactis chromatin. Mnase digest the chromatin between nucleosomes, but leaves the nucleosomal DNA intact. The samples were subjected to paired-end deep sequencing, which identifies the nucleosomal landscape of the genome. We recently performed Mnase-seq experiments using S. cerevisiae strains that either had an active RE or an inactive RE. The nucleosome positioning/occupancy at the RE was distinctly different between the strains. We hypothesize that the RE in K. lactis also displays distinctive chromatin states in MATa and MATalpha. We will analyze Mnase-seq experiments using isogenic MATa, MATalpha, MATa sir2 and MATa sir2 hml strains. Comparing the MATa and MATalpha strains should identify the RE. Comparing the MATa sir2 and MATa sir2 hml strains will show if the RE-activity is regulated similarly as in S. cerevisiae and also identify other loci that are directly regulated by the conserved Sir2 histone deacetylase. Comparing the RE from K. lactis with that of S. cerevisiae should establish themes and variations in how these RE-elements function.