Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot

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.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0233-x) contains supplementary material, which is available to authorized users.

Molecular mechanisms for environmentally induced plasticity in the positioning of meiotic recombination at hotspots

“…The parental imprinting that we discovered using reciprocally configured, but genetically identical, heterozygous meioses (Figure 2) revealed that sequence-specific, hotspot-activating protein-DNA complexes can establish a recombinationally poised epigenetic state prior to meiosis. Recent, temporal analyses of hotspot-recruited chromatin remodeling enzymes and histone PTMs (29), and of chromatin structure at singlenucleosome resolution (30), each support independently this conclusion for the hotspots examined in this study. Some hotspot-specific chromatin remodeling occurs before premeiotic S-phase, further remodeling occurs later in meiosis, leading subsequently to the induction of DSBs.…”

“…Analyses of DNA binding in vitro and in vivo (47,(51)(52)(53)(54)(55), as well as chromatin mapping experiments (29,46,47,51), revealed that the hotspot binding/activating proteins occupy their own DNA binding sites before meiosis. Moreover, the cis-acting regulatory modules (sequencespecific protein-DNA complexes) each promote recombination via the modification of chromatin structure before and during meiosis (30).…”

“…This protein-DNA complex can help to seed both euchromatin and heterochromatin (51,83,84). In addition, complex-dependent hotspot activation is regulated both agonistically and antagonistically by multiple different signaling pathways, including those that respond to environmental and metabolic cues (29,46,47,81,(85)(86)(87)(88)(89). Furthermore, distinct domains of chromosome-bound Atf1-Pcr1 heterodimer promote and repress recombination (42,46), demonstrating that both positive and negative regulation can be Figure 4.…”

“…If factor "A" controls chromatin remodelers that help to activate "B"-type hotspots, this would contribute to the regulation in trans. A recent, proteomics-based approach revealed that many different chromatin remodeling enzymes and histone PTMs function in concert to activate individual hotspots (29), so there are many potential targets for the modulation of hotspot activity at this level.…”

“…Such imprinting can also be inferred from the structure and composition of chromatin at hotspots (28)(29)(30). Differences in temperature during meiosis affect patterns of recombination across the genomes of diverse species (31), and in two of these species the effects of temperature on the global distribution of recombination-initiating DSBs have been examined.…”