CENP-B preserves genome integrity at replication forks paused by retrotransposon LTR

Abstract

Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats.

Figure 1

DNA damage in CENP-B mutants is suppressed by sap1 mutation. a, Images of 10³ plated cells of WT, Δabp1 and Δabp1Δcbh1 with Δabp1Δcbh1sap1-c colonies. Microscopy image inserts: Images showing branched phenotype in Δabp1Δcbh1 background (right) and Δabp1Δcbh1sap1-c mutant (left). Scale bar is 10 μm. b, Pulsed Field Gel blot analysis of wild type (WT), CENP-B mutants (Δabp1, Δcbh1, Δabp1Δcbh1) and 5 CENP-B/sap1-c mutant isolates (Δabp1Δcbh1sap1-c). The position of the three chromosomes is indicated on the right. The image is a false-colored composite of hybridizations for all three chromosomes.

Figure 2

Sap1 and CENP-B colocalize at the LTR of retrotransposons in vivo. Average genome-wide enrichment by ChIP-seq of Sap1, Abp1 and Cbh1 on a, all Tf2 elements, b, euchromatic tRNA and c, solo LTR. Error bars represent Standard Error. d, Left panel: Competition EMSA. Right panel: Inactivation by incubation with anti-Sap1 serum. e, Average Sap1, Abp1 and Cbh1 enrichment around Tf1 de novo insertion points. f, ChIP of Sap1 with LTR of Tf2 in CENP-B and sap1-c mutants and g, of Abp1 with LTR of Tf2 in and sap1 ts mutants. Error bars represent standard deviation for triplicates.

Figure 3

CENP-B promotes replication fork progression through the Sap1 dependent barrier present at the LTR and prevents HR. a, 2D gel electrophoresis of a plasmid fragment containing the Tf2 LTR oriented towards (left) and away (right) from the ars1 origin. Arrows indicate paused replication intermediates and open arrows recombination intermediates. The percentage of signal over the LTR is indicated below each panel. b, Quantification of Rad22 -GFP foci (N >400 nuclei for all mutants). Error bars depict standard errors. c, Rad22 -YFP ChIP with LTR in WT, Δabp1 and Δabp1Δcbh1 mutants. Error bars represent standard deviation for triplicates.

Figure 4

CENP-B and Sap1 have opposite effects on Tf2 stability. a, ectopic recombination fluctuation assay. Two potential mechanisms of ura4 loss from the marked Tf2-6::ura4 are indicated, gene conversion and eviction by LTR recombination. Columns represent total median ura4 loss frequency in WT, Δabp1, Δcbh1, Δabp1sap1-c and Δcbh1sap1-c mutants, error bars represent 95% confidence intervals. Colors indicate distribution of mode of ectopic recombination events in the ura4- colonies obtained from WT (n=93), Δabp1 (n=88), Δcbh1 (n=94), Δabp1sap1-c (n=91), and Δcbh1sap1-c (n=89) mutants. b, Model for the interactions between Abp1, Cbh1, Sap1 and the replication fork at the LTR.