Abstract
BackgroundMeiotic recombination hotspots control the frequency and distribution of Spo11 (Rec12)-initiated recombination in the genome. Recombination occurs within and is regulated in part by chromatin structure, but relatively few of the many chromatin remodeling factors and histone posttranslational modifications (PTMs) have been interrogated for a role in the process.ResultsWe developed a chromatin affinity purification and mass spectrometry-based approach to identify proteins and histone PTMs that regulate recombination hotspots. Small (4.2 kbp) minichromosomes (MiniCs) bearing the fission yeast ade6-M26 hotspot or a basal recombination control were purified approximately 100,000-fold under native conditions from meiosis; then, associated proteins and histone PTMs were identified by mass spectrometry. Proteins and PTMs enriched at the hotspot included known regulators (Atf1, Pcr1, Mst2, Snf22, H3K14ac), validating the approach. The abundance of individual histones varied dynamically during meiotic progression in hotspot versus basal control MiniCs, as did a subset of 34 different histone PTMs, implicating these as potential regulators. Measurements of basal and hotspot recombination in null mutants confirmed that additional, hotspot-enriched proteins are bona fide regulators of hotspot activation within the genome. These chromatin-mediated regulators include histone H2A-H2B and H3-H4 chaperones (Nap1, Hip1/Hir1), subunits of the Ino80 complex (Arp5, Arp8), a DNA helicase/E3 ubiquitin ligase (Rrp2), components of a Swi2/Snf2 family remodeling complex (Swr1, Swc2), and a nucleosome evictor (Fft3/Fun30).ConclusionsOverall, our findings indicate that a remarkably diverse collection of chromatin remodeling factors and histone PTMs participate in designating where meiotic recombination occurs in the genome, and they provide new insight into molecular mechanisms of the process.
Highlights
Meiotic recombination hotspots control the frequency and distribution of Spo11 (Rec12)-initiated recombination in the genome
If a chemically cross-linked, 12,500-kbp genome is sheared into chromatin fragments approximately 1 kbp in length and if the target fragment is enriched 1000-fold, greater than 90% of proteins in the purifications would come from genomic regions other than the target locus of interest
The omission of shearing would obviate the need for prior chemical cross-linking of proteins to DNA and would streamline the process, allowing us to purify chromatin rapidly under native conditions
Summary
Meiotic recombination hotspots control the frequency and distribution of Spo (Rec12)-initiated recombination in the genome. Sequence-specific DNA binding proteins, such as the Atf-Pcr heterodimer, Bas, and Prdm, regulate hotspots [3, 5,6,7]. They trigger directly or indirectly posttranslational modifications (PTMs) of histones that. The removal of PTM acceptor residues or the enzymes that place these marks, such as the histone acetyltransferase Gcn, the E3 ubiquitin ligase Bre1/ Brl, and Set methyltransferases (of which Prdm is a member), and the removal of ATP-dependent chromatin remodeling enzymes such as Snf, affect the distribution of recombination at hotspots (e.g., [17,18,19,20,21]). There are differences in dependence on Set methyltransferase activity (e.g., [19,20,21]), and in some species, a DNA binding domain targets the enzyme to the chromosome, whereas in other species there is no DNA binding domain, so the enzymatic activity must be recruited indirectly by other factors (e.g., [19, 22])
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