Heterochromatin suppresses gross chromosomal rearrangements at centromeres by repressing Tfs1/TFIIS-dependent transcription

Abstract

Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats.

Fig. 1

Clr4 methyltransferase suppresses gross chromosomal rearrangements (GCRs) through H3K9 methylation. a Illustration of an extra-chromosome ChL. Positions of LEU2, ura4⁺, ade6⁺, and centromere 3 (cen3) are indicated. When GCRs associated with the loss of ura4⁺ and ade6⁺ take place, Leu⁺ Ura⁺ Ade⁺ cells become Leu⁺ Ura^– Ade^– cells. b Wild-type and clr4∆ strains (TNF5676 and 5702, respectively) grown in EMM + UA were plated onto YNB + UA (2 × 10² cells) and 5FOA + A (2 × 10⁴ cells) media to count Leu⁺ and Leu⁺ Ura^– colonies, respectively. Plates were incubated at 30 °C for 6–9 days. wt, wild type. c GCR rates of wild-type, clr4∆, mat2-3∆, mat2-3∆ clr4∆, mat2-3∆ rec12∆, and mat2-3∆ rec12∆ clr4∆ strains (TNF3896, 5440, 5676, 5702, 5701, and 5766, respectively). Each dot represents the GCR rate determined using a single colony formed on EMM + UA plates in scatter plots. Lines represent the median. The GCR rate relative to that of the wild-type clr4⁺ strain is indicated on the top of each column. Statistical significance of differences between pairs of strains was determined using the two-tailed Mann–Whitney test. ****P < 0.0001. d GCR rates of wild-type, clr4∆, rik1∆, clr4-set, mlo3KA, mlo3KR, H3K9, H3K9A, and H3K9R strains in the mat2-3∆ background (TNF5676, 5702, 6121, 6958, 6155, 6157, 5738, 6223, and 5802, respectively). The GCR rate relative to that of wild type is indicated on the top of each column. In the cases of H3K9, H3K9A, and H3K9R strains, the GCR rate relative to that of the wild-type H3K9 strain is also shown in parentheses. Statistical significance of differences relative to wild type (the top of each column), and of differences between pairs of strains was determined using the two-tailed Mann–Whitney test

Fig. 2

Clr4 and Rik1 suppress the formation of isochromosomes whose breakpoints are located in centromere repeats. a Repetitive sequences in cen3 of ChL are shown. Units of centromere repeats are indicated as arrows. b Illustration of the gross chromosomal rearrangement (GCR) products that have lost ura4⁺ and ade6⁺ from ChL: translocation, truncation, and isochromosome. The position of probe A used in Southern hybridization is indicated as filled box. c Chromosomal DNAs of wild-type, clr4∆, and rik1∆ strains (TNF5676, 5702, and 6121, respectively) were separated by broad-range pulse field gel electrophoresis (PFGE) and stained with ethidium bromide (EtBr). Positions of chr1, chr2, chr3, and ChL (5.7, 4.6, ~3.5, and 0.5 Mb, respectively) in the parental strain are indicated on the left of the panel. DNAs were transferred onto a nylon membrane and hybridized with probe A. P, Parental. d Chromosomal DNAs were separated by short-range PFGE and stained with EtBr. Sizes of the λ DNA ladder are indicated on the left of the panel. e Pie charts depict proportions of different types of GCRs. f Breakpoints were determined by PCR reactions using GCR products recovered from agarose gel. Both sides of cnt3–imr3 junctions were amplified in the reaction containing im1, cn1, and cn2 primers. irc3L and irc3R were amplified using rc1 and rc2 primers, and the PCR products were digested by ApoI and separated by agarose gel electrophoresis. A, ApoI. Uncropped images of depicted gels and blots are shown in Supplementary Figs. 10 and 11

Fig. 3

Both HP1 homologs, Swi6 and Chp2, and the RNAi component Chp1 are the chromodomain proteins essential for full suppression of gross chromosomal rearrangements (GCRs). The chromodomain proteins Clr4, Swi6, Chp2, and Chp1 that bind to H3K9 methylation marks are illustrated. GCR rates of wild-type, clr4∆, clr4-W31G, swi6∆, chp2∆, swi6∆ chp2∆, chp1∆, and swi6∆ chp2∆ chp1∆ strains (TNF5676, 5702, 6012, 5706, 5685, 5900, 5708, and 6151, respectively) are shown. The two-tailed Mann-Whitney test. ***P < 0.001, ****P < 0.0001; ns, not significant

Fig. 4

RNAi machinery plays an essential role to suppress gross chromosomal rearrangements (GCRs) at the centromeres. Illustrated is the RNAi system that utilizes small RNAs and facilitates H3K9 methylation at the centromeres. GCR rates of wild-type, clr4∆, ago1∆, chp1∆, tas3∆, arb1∆, arb2∆, rdp1∆, and dcr1∆ strains (TNF5676, 5702, 5688, 5708, 7335, 7337, 7331, 7333, and 5687, respectively) are shown. The two-tailed Mann–Whitney test. ****P < 0.0001. ncRNA, noncoding RNA. arb2∆ caused a higher rate of GCRs than arb1∆ (P < 0.05), suggesting that Arb2 has an Arb1-independent function to suppress GCRs

Fig. 5

mlo3∆ but not cid14∆ reduces RNAPII chromatin binding and suppresses gross chromosomal rearrangements (GCRs) at the centromeres in ago1∆ cells. a GCR rates of wild-type, cid14∆, mlo3∆, ago1∆, cid14∆ ago1∆, and mlo3∆ ago1∆ strains (TNF5676, 6153, 5764, 5688, 6411, and 6188, respectively). The two-tailed Mann–Whitney test. ****P < 0.0001; ns, not significant. b Chromatin immunoprecipitation (ChIP) analysis was performed to determine H3K9me2, H3 and RNAPII (Rpb1) levels at centromere repeats (dg, dh, and imr3) and at a non-centromeric region of chr2 (adl1) in wild-type, cid14∆, mlo3∆, ago1∆, cid14∆ ago1∆, and mlo3∆ ago1∆ strains (TNF5921, 6276, 5923, 5922, 6550, and 6210, respectively). DNA levels were quantified by real-time PCR, and percentages of input DNA were obtained. Data are presented as the mean ± s.e.m. from three biologically independent experiments. Dots represent individual measurements from distinct samples. Statistical significance of differences relative to wild type (top of bars), and of differences between pairs of mutant strains was determined using the two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Repression of RNAPII suppresses centromeric gross chromosomal rearrangements (GCRs) in the absence of H3K9 methylation. a GCR rates of wild-type, mlo3∆, clr4∆, mlo3∆ clr4∆, rik1∆, mlo3∆ rik1∆, rad51∆, and mlo3∆ rad51∆ strains (TNF5676, 5764, 5702, 5824, 6121, 6378, 6244, and 6383, respectively). The two-tailed Mann-Whitney test. ***P < 0.001, ****P < 0.0001. b Chromatin immunoprecipitation (ChIP) analysis of RNAPII (Rpb1) and H3 in wild-type, mlo3∆, clr4∆, and mlo3∆ clr4∆ strains (TNF5921, 5923, 5948, and 5925, respectively). The two-tailed Student’s t-test. *P < 0.05, **P < 0.01. c ChIP analysis of H3K9me2, H3K9me3, H3K9ac, and H3K14ac in wild-type, mlo3∆, clr4∆, and mlo3∆ clr4∆ strains. mlo3∆ reduced the level of H3K9me3 but not that of H3K9me2, suggesting that Mlo3 is required for the transition from H3K9me2 to H3K9me3 state

Fig. 7

Clr4 suppresses centromeric gross chromosomal rearrangements (GCRs) by repressing transcription that is dependent on RNAPII CTD Ser7 and Tfs1/TFIIS. a GCR rates of wild-type, rpb1-S7A, tfs1∆, ell1∆, leo1∆, spt4∆, clr4∆, rpb1-S7A clr4∆, tfs1∆ clr4∆, ell1∆ clr4∆, leo1∆ clr4∆, and spt4∆ clr4∆ strains (TNF5676, 6848, 6688, 7042, 7130, 7055, 5702, 6850, 6726, 7063, 7154, and 7057, respectively). The two-tailed Mann-Whitney test. **P < 0.01, ****P < 0.0001; ns, not significant. b Chromatin immunoprecipitation (ChIP) analysis of RNAPII (Rpb1) and H3 in wild-type, rpb1-S7A, tfs1∆, clr4∆, rpb1-S7A clr4∆, and tfs1∆ clr4∆ strains (TNF5921, 6862, 6722, 5948, 6864, and 6799, respectively). The two-tailed Student’s t-test. *P < 0.05, ***P < 0.001. c Northern blotting using total RNAs prepared from log phase cultures of rpb1-S7A, wild-type, tfs1∆, rpb1-S7A clr4∆, clr4∆, and tfs1∆ clr4∆ strains. Illustrated are the positions of DNA probes used in Northern blotting (magenta bars) and the readthrough transcript of adl1 (a green arrow). RNAs were separated by 1.0% agarose gel under denatured condition, stained with ethidium bromide (EtBr) (the bottom panel), transferred onto a nylon membrane, and hybridized with specific probes (the top panel). Uncropped images of depicted gels and blots are shown in Supplementary Fig. 12. d A model that explains how heterochromatin suppresses GCRs at centromeres. With the aid of the RNAi system, Clr4 catalyzes H3K9 methylation at centromeres. H3K9 methylation marks are recognized by the chromodomain proteins including Clr4, Swi6, Chp2, and Chp1. Both HP1 homologs, Swi6 and Chp2, and an RNAi component Chp1 are required for full suppression of GCRs. In addition to the Clr4 recruitment, RNAi machinery may prevent transcription of noncoding RNAs from centromere repeats to suppress GCRs. RNAPII transcription that depends on CTD Ser7, Mlo3, and Tfs1/TFIIS causes centromeric GCRs possibly by removing DNA binding proteins, such as replication factors, from DNA. e Tfs1/TFIIS-dependent transcription might remove the roadblock that binds to DNA and produce R-loops, which facilitate interaction between centromere repeats at non-allelic positions and cause crossover and/or break-induced replication (BIR) that leads to GCRs