CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Analysis of Global and Local DNA Methylation Patterns in Blood Samples of Patients With Autism Spectrum Disorder.

The goal of this investigation was to determine whether there are alterations in DNA methylation patterns in children with autism spectrum disorder (ASD). Material and Methods: Controlled prospective observational case-control study. Within the ASD group, children were sub-classified based on the presence (AMR subgroup) or absence (ANMR subgroup) of neurodevelopmental regression during the first 2 years of life. We analyzed the global levels of DNA methylation, reflected in LINE-1, and the local DNA methylation pattern in two candidate genes, Neural Cell Adhesion Molecule (NCAM1) and Nerve Growth Factor (NGF) that, according to our previous studies, might be associated to an increased risk for ASD. For this purpose, we utilized blood samples from pediatric patients with ASD (n = 53) and their corresponding controls (n = 45). Results: We observed a slight decrease in methylation levels of LINE-1 in the ASD group, compared to the control group. One of the CpG in LINE-1 (GenBank accession no.X58075, nucleotide position 329) was the main responsible for such reduction, highly significant in the ASD subgroup of children with AMR (p < 0.05). Furthermore, we detected higher NCAM1 methylation levels in ASD children, compared to healthy children (p < 0.001). The data, moreover, showed higher NGF methylation levels in the AMR subgroup, compared to the control group and the ANMR subgroup. These results are consistent with our prior study, in which lower plasma levels of NCAM1 and higher levels of NGF were found in the ANMR subgroup, compared to the subgroup that comprised neurotypically developing children. Conclusions: We have provided new clues about the epigenetic changes that occur in ASD, and suggest two potential epigenetic biomarkers that would facilitate the diagnosis of the disorder. We similarly present with evidence of a clear differentiation in DNA methylation between the ASD subgroups, with or without mental regression.

Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs.

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.

DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

DNA Base Excision Repair in Plants: An Unfolding Story With Familiar and Novel Characters.

Base excision repair (BER) is a critical genome defense pathway that deals with a broad range of non-voluminous DNA lesions induced by endogenous or exogenous genotoxic agents. BER is a complex process initiated by the excision of the damaged base, proceeds through a sequence of reactions that generate various DNA intermediates, and culminates with restoration of the original DNA structure. BER has been extensively studied in microbial and animal systems, but knowledge in plants has lagged behind until recently. Results obtained so far indicate that plants share many BER factors with other organisms, but also possess some unique features and combinations. Plant BER plays an important role in preserving genome integrity through removal of damaged bases. However, it performs additional important functions, such as the replacement of the naturally modified base 5-methylcytosine with cytosine in a plant-specific pathway for active DNA demethylation.

Active DNA Demethylation in Plants.

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

Characterization of an AP endonuclease from sugarcane - ScARP1.

Plants are sessile organisms that need to cope with different conditions. The Base Excision Repair (BER) pathway is an important mechanism protecting the genome from DNA lesions. Apurinic/apyrimidinic (AP) endonucleases are key BER enzymes that process AP sites arising either spontaneously or as BER intermediates. In Arabidopsis there are three AP endonucleases: AtARP1, AtAPE1L, and AtAPE2, and in sugarcane two AtARP1 homologues have been identified: ScARP1 and ScARP3. ScARP1 shares 59% sequence identity with Arabidopsis AtARP. Protein modeling of ScARP1 and AtARP1 revealed conserved active sites and metal binding sites. For biochemical characterisation, recombinant ScARP1 protein displayed AP endonuclease activity both in the presence of MnCl2 or MgCl2 and the optimal temperature for its activity was 37 °C. Under these conditions, 3'-exonuclease, 3'-phosphatase, and 3'-phosphodiesteterase activities were not detectable. We also show that ScARP1 protein is able to complement mutant atarp-/- cell extracts deficient in AP endonuclease activity. These results suggest that AP endonucleases from different plant species preserve AP endonuclease activity. The biochemical characterisation of ScARP1 extends our knowledge of the BER pathway to a monocot crop plant group.

Monitoring base excision repair in Chlamydomonas reinhardtii cell extracts.

Base excision repair (BER) is a major defense pathway against spontaneous DNA damage. This multistep process is initiated by DNA glycosylases that recognise and excise the damaged base, and proceeds by the concerted action of additional proteins that perform incision of the abasic site, gap filling and ligation. BER has been extensively studied in bacteria, yeasts and animals. Although knowledge of this pathway in land plants is increasing, there are no reports detecting BER in algae. We describe here an experimental in vitro system allowing the specific analysis of BER in the model alga Chlamydomonas reinhardtii. We show that C. reinhardtii cell-free extracts contain the enzymatic machinery required to perform BER of ubiquitous DNA lesions, such as uracil and abasic sites. Our results also reveal that repair can occur by both single-nucleotide insertion and long-patch DNA synthesis. The experimental system described here should prove useful in the biochemical and genetic dissection of BER in algae, and may contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways in photosynthetic eukaryotes.

Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis.

Abasic (apurinic/apyrimidinic, AP) sites in DNA arise from spontaneous base loss or by enzymatic removal during base excision repair. It is commonly accepted that both classes of AP site have analogous biochemical properties and are equivalent substrates for AP endonucleases and AP lyases, although the relative roles of these two types of enzymes are not well understood. We provide here genetic and biochemical evidence that, in Arabidopsis, AP sites generated by spontaneous loss of N7-methylguanine (N7-meG) are exclusively repaired through an AP endonuclease-independent pathway initiated by FPG, a bifunctional DNA glycosylase with AP lyase activity. Abasic site incision catalyzed by FPG generates a single-nucleotide gap with a 3'-phosphate terminus that is processed by the DNA 3'-phosphatase ZDP before repair is completed. We further show that the major AP endonuclease in Arabidopsis (ARP) incises AP sites generated by enzymatic N7-meG excision but, unexpectedly, not those resulting from spontaneous N7-meG loss. These findings, which reveal previously undetected differences between products of enzymatic and nonenzymatic base release, may shed light on the evolution and biological roles of AP endonucleases and AP lyases.

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase.

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.

Dual control of ROS1-mediated active DNA demethylation by DNA damage-binding protein 2 (DDB2).

By controlling gene expression, DNA methylation contributes to key regulatory processes during plant development. Genomic methylation patterns are dynamic and must be properly maintained and/or re-established upon DNA replication and active removal, and therefore require sophisticated control mechanisms. Here we identify direct interplay between the DNA repair factor DNA damage-binding protein 2 (DDB2) and the ROS1-mediated active DNA demethylation pathway in Arabidopsis thaliana. We show that DDB2 forms a complex with ROS1 and AGO4 and that they act at the ROS1 locus to modulate levels of DNA methylation and therefore ROS1 expression. We found that DDB2 represses enzymatic activity of ROS1. DNA demethylation intermediates generated by ROS1 are processed by the DNA 3'-phosphatase ZDP and the apurinic/apyrimidinic endonuclease APE1L, and we also show that DDB2 interacts with both enzymes and stimulates their activities. Taken together, our results indicate that DDB2 acts as a critical regulator of ROS1-mediated active DNA demethylation.

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain.

DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequence in vitro. We also found that transient in vivo expression of GBD-ROS1_CD in human cells specifically reactivates transcription of a methylation-silenced reporter gene, and that such reactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show that reactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpG sites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can be used for targeted active DNA demethylation in human cells.

An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis [corrected].

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.

Arabidopsis ZDP DNA 3'-phosphatase and ARP endonuclease function in 8-oxoG repair initiated by FPG and OGG1 DNA glycosylases.

Oxidation of guanine in DNA generates 7,8-dihydro-8-oxoguanine (8-oxoG), an ubiquitous lesion with mutagenic properties. 8-oxoG is primarily removed by DNA glycosylases distributed in two families, typified by bacterial Fpg proteins and eukaryotic Ogg1 proteins. Interestingly, plants possess both Fpg and Ogg1 homologs but their relative contributions to 8-oxoG repair remain uncertain. In this work we used Arabidopsis cell-free extracts to monitor 8-oxoG repair in wild-type and mutant plants. We found that both FPG and OGG1 catalyze excision of 8-oxoG in Arabidopsis cell extracts by a DNA glycosylase/lyase mechanism, and generate repair intermediates with blocked 3'-termini. An increase in oxidative damage is detected in both nuclear and mitochondrial DNA from double fpg ogg1 mutants, but not in single mutants, which suggests that a single deficiency in one of these DNA glycosylases may be compensated by the other. We also found that the DNA 3'-phosphatase ZDP (zinc finger DNA 3'-phosphoesterase) and the AP(apurinic/apyirmidinic) endonuclease ARP(apurinic endonuclease redox protein) are required in the 8-oxoG repair pathway to process the 3'-blocking ends generated by FPG and OGG1. Furthermore, deficiencies in ZDP and/or ARP decrease germination ability after seed deteriorating conditions. Altogether, our results suggest that Arabidopsis cells use both FPG and OGG1 to repair 8-oxoG in a pathway that requires ZDP and ARP in downstream steps.

Molecular characterization of a putative plant homolog of MBD4 DNA glycosylase.

Methyl-CpG-binding domain 4 (MBD4) DNA glycosylase is involved in excision of spontaneous deamination products of cytosine and 5-methylcytosine in animals, but it is unknown whether related proteins perform similar functions in plants. We report here the isolation and biochemical characterization of a putative MBD4 homolog from Arabidopsis thaliana, designated as MBD4L (MBD4-like). The plant enzyme lacks the MBD domain present in mammalian MBD4 proteins, but conserves a DNA glycosylase domain with critical residues for substrate recognition and catalysis, and it is more closely related to MBD4 homologs than to other members of the HhH-GPD superfamily. Arabidopsis MBD4L excises uracil and thymine opposite G, and the presence of halogen substituents at C5 of the target base greatly increases its excision efficiency. No significant activity is detected on cytosine derivatives such as 5-methylcytosine or 5-hydroxymethylcytosine. The enzyme binds to the abasic site product generated after excision, which decreases its catalytic turnover in vitro. Both the full-length protein and a N-terminal truncated version retaining the catalytic domain exhibit a preference for a CpG sequence context, where most plant DNA methylation is found. Our results suggest that an important function of Arabidopsis MBD4L is to protect the plant genome from the mutagenic consequences of cytosine and 5-methylcytosine deamination.

Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues.

Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.

The DNA repair protein XRCC1 functions in the plant DNA demethylation pathway by stimulating cytosine methylation (5-meC) excision, gap tailoring, and DNA ligation.

DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). ROS1 and its homologs remove 5-methylcytosine and incise the sugar backbone at the abasic site, thus initiating a base excision repair pathway that finally inserts an unmethylated cytosine. The DNA 3'-phosphatase ZDP processes some of the incision products generated by ROS1, allowing subsequent DNA polymerization and ligation steps. In this work, we examined the possible role of plant XRCC1 (x-ray cross-complementing group protein 1) in DNA demethylation. We found that XRCC1 interacts in vitro with ROS1 and ZDP and stimulates the enzymatic activity of both proteins. Furthermore, extracts from xrcc1 mutant plants exhibit a reduced capacity to complete DNA demethylation initiated by ROS1. An anti-XRCC1 antibody inhibits removal of the blocking 3'-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis.

Demethylation initiated by ROS1 glycosylase involves random sliding along DNA.

Active DNA demethylation processes play a critical role in shaping methylation patterns, yet our understanding of the mechanisms involved is still fragmented and incomplete. REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway. As ROS1 binds DNA non-specifically, we have critically tested the hypothesis that facilitated diffusion along DNA may contribute to target location by the enzyme. We have found that dissociation of ROS1 from DNA is severely restricted when access to both ends is obstructed by tetraloops obstacles. Unblocking any end facilitates protein dissociation, suggesting that random surface sliding is the main route to a specific target site. We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein. Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means. A model is proposed to explain how ROS1 finds its potential targets on DNA.

Using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro.

Base excision repair (BER) is a major pathway for the removal of endogenous and exogenous DNA damage. This repair mechanism is initiated by DNA glycosylases that excise the altered base, and continues through alternative routes that culminate in DNA resynthesis and ligation. In contrast to the information available for microbes and animals, our knowledge about this important DNA repair pathway in plants is very limited, partially due to a lack of biochemical approaches. Here we describe an in vitro assay to monitor BER in cell-free extracts from the model plant Arabidopsis thaliana. The assay uses labeled DNA substrates containing a single damaged base within a restriction site, and allows detection of fully repaired molecules as well as DNA repair intermediates. The method is easily applied to measure the repair activity of purified proteins and can be successfully used in combination with the extensive array of biological resources available for Arabidopsis.