Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation

Abstract

Replication of nuclear DNA occurs in the context of chromatin and is influenced by histone modifications. In the ciliate Tetrahymena thermophila, we identified TXR1, encoding a histone methyltransferase. TXR1 deletion resulted in severe DNA replication stress, manifested by the accumulation of ssDNA, production of aberrant replication intermediates, and activation of robust DNA damage responses. Paired-end Illumina sequencing of ssDNA revealed intergenic regions, including replication origins, as hot spots for replication stress in ΔTXR1 cells. ΔTXR1 cells showed a deficiency in histone H3 Lys 27 monomethylation (H3K27me1), while ΔEZL2 cells, deleting a Drosophila E(z) homolog, were deficient in H3K27 di- and trimethylation, with no detectable replication stress. A point mutation in histone H3 at Lys 27 (H3 K27Q) mirrored the phenotype of ΔTXR1, corroborating H3K27me1 as a key player in DNA replication. Additionally, we demonstrated interactions between TXR1 and proliferating cell nuclear antigen (PCNA). These findings support a conserved pathway through which H3K27me1 facilitates replication elongation.

Keywords: H3 Lys 27 methylation; histone methyltransferase; replication elongation; replication origin; replication stress; ssDNA.

Figure 1.

Accumulation of ssDNA in ΔTXR1 and HU-treated wild-type (WT) cells. (A) Cell cycle-dependent distribution of ssDNA in ΔTXR1 cells after BrdU pulse-chase. For pulse-labeling, cells were incubated for 30 min with 0.4 mM BrdU in SPP medium and immediately fixed for immunofluorescence staining. For the chase experiment, pulse-labeled cells were washed twice before being resuspended with fresh SPP medium, and time points were taken at half-hour intervals for 4 h. (S) S and early G2 phase; (G2) mid and late G2 phase; (AM) amitosis; (CEB+) BrdU staining in CEBs. (White arrowheads) Micronuclei; (red arrowheads) macronuclear regions poorly stained with DAPI. Note that the cell cycle-dependent redistribution of ssDNA does not reflect the temporal replication program as described in mammalian cells (O'Keefe et al. 1992). (B) Quantification of cell cycle distribution of BrdU-positive ΔTXR1 cells. Details for cell cycle staging are described in the Supplemental Material. (C) Induction of RPA1, RAD51, PARP1, and γ-H2A.X in ΔTXR1 cells. Note the accumulation of RPA1 and γ-H2A.X in micronuclei (white arrowheads) as well as in macronuclei. (D) Accumulation of RPA1 in HU-treated wild-type cells. Cells were incubated for 20 h with 5 mM HU in SPP medium before being fixed for immunofluorescence staining. Note the similarity in abnormal nuclear morphology of ΔTXR1 and HU-treated cells.

Figure 2.

Accumulation of RIs in ΔTXR1 cells. (A) Accumulation of short nascent strands in ΔTXR1 cells, revealed by BrdU pulse-chase and alkaline agarose gel electrophoresis. For detecting incorporated BrdU, DNA was transferred to Nylon N⁺ membrane and probed by α-BrdU antibody. (Numbers) Time (in hours) after BrdU pulse-labeling; (M and M′) DNA markers; (EtBr) ethidium bromide staining; (BrdU) BrdU immunoblotting. (B) Accumulation of RIs in ΔTXR1 cells, revealed by BrdU pulse-chase and neutral agarose gel electrophoresis. (Red arrows) The rDNA minichromosome; (dashed arrows) shift from RIs to finished products. (C) Accumulation of DSBs in ΔTXR1 cells, revealed by BrdU pulse-chase and neutral agarose gel electrophoresis. DNA was in-gel-trapped (Khan and Nawaz 2007) and probed by α-BrdU antibody. (D) Accumulation of aberrant RIs (red arrowheads) in ΔTXR1 cells, revealed by 2D neutral–neutral agarose gel electrophoresis. (+BND) RI enrichment by BND cellulose prior to the 2D gel. Illustrated in the schematics are the interpretations of normal RIs in wild-type (WT) cells and aberrant RI-1 and RI-2 in ΔTXR1 cells.

Figure 3.

Distribution of ssDNA in the rDNA minichromosome in ΔTXR1 cells. (A) Schematic of the palindromic rDNA minichromosome, illustrating the transcribed region, the 5′ NTS, the 3′ NTS, telomeres, and the palindromic HindIII fragment containing replication origins. Details of the 5′ NTS: replication origins (ori1 and ori2), phased nucleosomes, and DNA elements (pausing sites and type I/III elements). (B) Distribution of ssDNA in the rDNA from wild-type (WT) and ΔTXR1 cells in both forward and reverse directions. The Y-axes in B–D represent abundance of the ssDNA reads. Note the symmetry around ori1 and its correspondence to the schematic of nascent strands in a replication bubble. (C,D) Distribution of 5′ (C) and 3′ (D) ends of the ssDNA from ΔTXR1 cells. (E) Illumina sequencing coverage ratio (log₂) of ΔTXR1 and wild-type cells, calculated along the length of the rDNA (details provided in the Supplemental Material). The ratio is based on the abundance of total mappable reads, which are predominantly dsDNA. Note its strong negative correlation with the ssDNA abundance in ΔTXR1 cells. (F) Validation of rDNA replication origins by analysis of short nascent strands (SNSs). Note the enrichment in the rDNA 3′ NTS in ΔTXR1 cells, normalized against SNS levels in the rDNA transcribed region (TS). The difference in fold of enrichment is due to the higher background of ssDNA in ΔTXR1 cells.

Figure 4.

Global distribution of ssDNA in ΔTXR1 cells. (A) Clustering of ssDNA in ΔTXR1 cells in their distribution in non-rDNA chromosomes. The X-axis represents the inter-ssDNA distances (binned at 25-bp intervals and cut off at 500 bp), while the Y-axis represents their abundance (arbitrary units). The same total number of ssDNA were analyzed in the ΔTXR1 and randomized sample. (B) Distribution of ssDNA and mRNA in a genomic region around ARS1-A and ARS1-B, two known replication origins in non-rDNA chromosomes. (C–G) Average view analyses (binned at 50-bp intervals) of the distribution of ssDNA (C), mRNA (D), AT ratio (E), poly-(dA:dT) (F), and Illumina sequencing coverage ratio (log₂) of ΔTXR1 and wild-type (WT) cells (G), showing features consistent with being replication origins. The Y-axes represent the abundance of the studied features. The same total number of the studied features was analyzed in the ΔTXR1 and randomized sample. Details of calculation are provided in the Supplemental Material.

Figure 5.

Transcriptional responses to replication stress in ΔTXR1 cells. (A) Induction of a cluster of functionally related genes in ΔTXR1 cells, revealed by GO analysis (GO term: biological process) and colored according to the P-value. (B) Induction of many genes in the categories of response to DNA damage, DNA repair, and DNA metabolic process in ΔTXR1 and HU-treated cells, with significant overlap between them. (C) The core of transcriptional responses in ΔTXR1 cells, as revealed by phylogenetic and microarray analysis, is similar to those in HU-treated cells but not ΔEZL2 cells. The left panels are colored as the following: (green) homologs identified by the best hits of BLASTP search in both directions; (dark green) homologs identified by the best hits in one direction; (red) no homolog. (Pro) An assembly of prokaryotes (see the Supplemental Material); (Sce) S. cerevisiae; (Dme) Drosophila melanogaster; (Hsa) Homo sapiens; (Ath) Arabidopsis thaliana; (Pte) Paramecium tetraurelia; (Tth) T. thermophila. The right panels are colored according to the ratio (log₂) of transcript levels of the mutants or treated cells normalized against the wild-type (WT) controls. (NER) Nucleotide excision repair; (MMR) mismatch repair; (HR) homologous recombination; (NHEJ) nonhomologous end joining. (D) Representative genes highly induced in ΔTXR1 but not ΔEZL2 cells, shown by both microarray and qPCR analysis.

Figure 6.

Replication stress in ΔTXR1 cells is mediated by a deficiency in H3K27me1. (A) Abnormal nuclear morphology of ΔTXR1 cells, revealed by DAPI staining. (B) Abnormal aggregation of nucleoli in ΔTXR1 cells, revealed by rDNA FISH (probe corresponding to the 5′ NTS). (White arrow) Aggregated nucleoli at the periphery of the macronucleus. (C) Box plot analysis of PTMs in all four core histones quantified by MS, which covers 53 combinations of PTMs and their unmodified counterparts (see Supplemental Fig. 11 for a complete list). All outliers in the mutant/wild-type (WT) ratio are marked with open ovals. H3K27-related species are denoted with symbols specified in the inset. (D) MS quantification of H3K27me1 levels in wild-type, ΔTXR1, and ΔEZL2 cells. ¹⁵N-labeled histone H3 was used as the internal standard. (E) H3K27me1 deficiency in ΔTXR1 cells, shown by immunofluorescence staining with the modification-specific antibody. (F) H3K27 methylation deficiency in ΔTXR1 and ΔEZL2 cells, shown by immunoblotting with the modification-specific antibodies.(G) Accumulation of RPA1 in H3 K27Q cells. Note its similarity to ΔTXR1 and HU-treated cells.

Figure 7.

A TXR1-dependent, H3K27me1-mediated pathway regulating replication elongation. (A) TXR1 PIP peptide pull-down of PCNA, with recombinant PCNA or cell lysate as the input. (B) Effective competition by the PIP peptide (50–200 μM) but not a similar peptide with mutations in the conserved aromatic residues (PIP-AA). (C) ssDNA and PCNA distribution in TXR1 mutants, including the PIP (Y215A F216A) and SET (Y526N) mutant. (D) Model for origin-proximal replication elongation defects in ΔTXR1 cells. (E) Model for a TXR1-dependent, H3K27me1-mediated pathway regulating replication elongation.