Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1^kd,3a^−/−,3b^−/−]). Sequencing of 960 RRBS clones from Dnmt[1^kd,3a^−/−,3b^−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1^kd,3a^−/−,3b^−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.     

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1^−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1^−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.     

Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1o^mat−/− mouse embryos born to Dnmt1^Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1^Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.     

Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA^−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA^−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.     

Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1^−/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1^−/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3^−/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1^−/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1^−/− cells are associated with the accumulation of aberrant replication fork structures.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K_m^AdoMet, for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K_m^DNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT ≥ CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.     

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.     

XRCC1 interacts with the p58 subunit of DNA Pol α-primase and may coordinate DNA repair and replication during S phase

Repair of single-stranded DNA breaks before DNA replication is critical in maintaining genomic stability; however, how cells deal with these lesions during S phase is not clear. Using combined approaches of proteomics and in vitro and in vivo protein–protein interaction, we identified the p58 subunit of DNA Pol α-primase as a new binding partner of XRCC1, a key protein of the single strand break repair (SSBR) complex. In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property. Overexpression of the XRCC1-BRCT1 domain in HeLa cells induces poly(ADP-ribose) synthesis, PARP-1 and XRCC1-BRCT1 poly(ADP-ribosyl)ation and a strong S phase delay in the presence of DNA damage. Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks. Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.     

The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells

During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4^Cdt2)–PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2–proteasome pathway to facilitate TLS pathway.     

A Novel Function of the DNA Repair Gene rhp6 in Mating-Type Silencing by Chromatin Remodeling in Fission Yeast

Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1⁻ mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1⁻/rhp6⁻ mutation on silencing are indirect consequences of changes in chromatin structure.     

Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG^+/+ and UNG^−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG^−/− cells accumulated significantly greater uracil and DSBs compared with UNG^+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG^+/+ cells. Furthermore, DSBs in UNG^+/+ and UNG^−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG^−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG^+/+ and UNG^−/− cells treated with IC₅₀-level pemetrexed. However, following pemetrexed exposure, UNG^−/− but not UNG^+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG^−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.