Distinct chromatin modulators regulate the formation of accessible and repressive chromatin at the fission yeast recombination hotspot ade6-M26

Abstract

Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) regulate transcription and recombination via alteration of local chromatin configuration. The ade6-M26 allele of Schizosaccharomyces pombe creates a meiotic recombination hotspot that requires a cAMP-responsive element (CRE)-like sequence M26, the Atf1/Pcr1 heterodimeric ATF/CREB transcription factor, the Gcn5 HAT, and the Snf22 SWI2/SNF2 family ADCR. Chromatin alteration occurs meiotically around M26, leading to the activation of meiotic recombination. We newly report the roles of other chromatin remodeling factors that function positively and negatively in chromatin alteration at M26: two CHD-1 family ADCRs (Hrp1 and Hrp3), a Spt-Ada-Gcn5 acetyltransferase component (Ada2), and a member of Moz-Ybf2/Sas3-Sas2-Tip60 family (Mst2). Ada2, Mst2, and Hrp3 are required for the full activation of chromatin changes around M26 and meiotic recombination. Acetylation of histone H3 around M26 is remarkably reduced in gcn5Delta, ada2Delta and snf22Delta, suggesting cooperative functions of these HAT complexes and Snf22. Conversely, Hrp1, another CHD-1 family ADCR, maintains repressive chromatin configuration at ade6-M26. Interestingly, transcriptional initiation site is shifted to a site around M26 from the original initiation sites, in couple with the histone acetylation and meiotic chromatin alteration induced around 3' region of M26, suggesting a collaboration between these chromatin modulators and the transcriptional machinery to form accessible chromatin. These HATs and ADCRs are also required for the regulation of transcription and chromatin structure around M26 in response to osmotic stress. Thus, we propose that multiple chromatin modulators regulate chromatin structure reversibly and participate in the regulation of both meiotic recombination and stress-induced transcription around CRE-like sequences.

Figure 1.

Hrp3 and Ada2 are required for meiotic chromatin remodeling around ade6-M26, whereas Hrp1 functions to suppress the alteration of chromatin structure. Diploid strains D20 (ade6-M26), D51 (ade6-M26, hrp1Δ), D53 (ade6-M26, hrp3Δ), D52 (ade6-M26, ada2Δ), and D72 (ade6-M26, mst2Δ) were cultured in MM + N medium (lanes 0 h). Cells were transferred to MM − N medium and cultured further for 4 h (lanes 4 h) or 6 h (lanes 6 h). Chromatin isolated from the cells was digested with MNase and analyzed as described previously (Mizuno et al., 1997). The vertical and horizontal arrows indicate the ade6 ORF and position of the M26 mutation, respectively. The arrowheads represent altered or unaltered chromatin configuration around M26 in the wild-type (filled), hrp1Δ (open), hrp3Δ, ada2Δ, and mst2Δ (gray) strains, respectively. Lane N indicates MNase digestion of naked DNA.

Figure 2.

The impact of hrp1⁺, hrp3⁺, ada2⁺, and mst2⁺ deletions on meiotic recombination frequency and meiotic DSB formation and genetic interactions between these proteins and Gcn5 or Snf22. (A) Recombination rates at M26 or M375 control allele (indicated as Ade⁺/10⁴ spores) were examined as described in Materials and Methods. All crosses were repeated independently at least three times. Error bars represent SD. (B) Recombination between leu1-32 and his5-303 (indicated as leu+/his+ [percentage]) was measured as described in Materials and Methods. All crosses were repeated independently two or three times. Error bars represent SD.

Figure 3.

Hyperacetylation of histone H3 around the M26 mutation site was affected in the gcn5Δ, snf22Δ, and ada2Δ strains. (A) The diploid wild-type strain was cultured to induce meiosis, as described in Figure 1. DNA of the input and ChIP samples were quantified by a slot blotting followed by hybridization with the M26 probe. The time after meiotic induction is indicated on the left side of the panel. (B) Quantified data of acetylated histones H3 and H4 in wild-type, gcn5Δ, snf22Δ, hrp1Δ, hrp3Δ, ada2Δ, and mst2Δ strains. Vertical and horizontal axes represent ChIP efficiency (percentage) (left, histone H3ac; right, histone H4ac) and hours after meiotic induction. Squares and filled circles indicate data for acetylated histones H3 and H4, respectively. All experiments except for mst2Δ were repeated independently two times. Error bars represent SD.

Figure 4.

M26 specific hyperacetylation of histones H3 and H4, and transcriptional activation. (A) Schematics of the probes used to quantify the acetylation of histones H3 and H4 around the ade6 locus. The scale represents 200 bp. Arrows represent the initiation sites of long and short transcript. (B) Quantified data for acetylated histones H3 and H4 in M26 (bold line) and M375 (dotted line) strains. Vertical and horizontal axes represent ChIP efficiency (percentage) and hours after meiotic induction. (C) Meiotic chromatin remodeling is induced around 3′ region of M26 mutation point. Wild-type (D20) cells were cultured in MM + N (lanes; mitosis) and transferred to MM − N and cultured further for 4 h (lanes; meiosis). Open and hatched ovals represent phase and randomly positioned nucleosomes, respectively. (D) The initiation site of the ade6 transcript is shifted to the 3′ region in the M26 strain. The diploid M26 and M375 strains were cultured to induce meiosis, as described in Figure 1. RNA was isolated and analyzed by Northern blotting using the ade6-ORF probe, probe 3 in Figure 4A, and cam1⁺ as a loading control (Takeda and Yamamoto, 1987). (E) The intensity of the band corresponding to the short transcript and whole transcript was quantified, and relative band intensity (short transcript/whole transcript percentage) was calculated. (F) The increase of short transcript of ade6 during meiosis requires Snf22, Gcn5, Hrp3, and Ada2. The diploid wild-type, snf22Δ, gcn5Δ, hrp3Δ, and ada2Δ strains were cultured to induce meiosis, as described in Figure 1, and transcript of ade6 was detected as Figure 4D. The relative intensity (short transcript/whole transcript percentage) was calculated as Figure 4E.

Figure 5.

The chromatin regulation of ade6-M26 by the HATs and ADCRs is coupled to transcriptional activation in response to osmotic stress. (A) The cells of haploid strains (wild type, gcn5Δ, snf22Δ, gcn5Δ/snf22Δ, hrp1Δ, hrp3Δ, and ada2Δ) were cultured in YE to mid-log phase, transferred to YE containing 1.2 M sorbitol, and cultured further for 90 min. Total RNA was isolated from each culture and analyzed by Northern analysis. Arrowheads represent the shorter ade6-M26 transcript. (B) Quantification of the results in A. Vertical axis indicates -fold induction compared with the mRNA levels in the wild-type strain without the sorbitol treatment. (C) The crude nuclei were isolated from the simultaneous cultures indicated in Figure 5A. The chromatin structure was analyzed as described in Figure 1. The vertical and horizontal arrows indicate the ade6 ORF and position of the M26 mutation, respectively. Arrowheads represent the altered or partially altered chromatin configuration around M26 in the wild-type (filled), hrp1Δ (open), hrp3Δ, ada2Δ, and mst2Δ (gray) strains, respectively.

Figure 6.

(A) Cooperative behavior of HATs-ADCRs for the modification and remodeling of chromatin structure around M26. (B) Reversible regulation of M26/CRE-dependent chromatin remodeling by chromatin modifying machineries. The model postulates that distinct remodeling complexes are required for nucleosome disruption to recruit either recombination complexes or transcription complexes. In addition, Hrp1 is involved in the suppression of chromatin remodeling at M26, thereby providing reversible regulation of the M26/CRE-dependent chromatin configuration.