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.

Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence.

Cell senescence is a state of limited cell proliferation during a stress response or as part of a programmed process. When a senescent cell stops dividing, maintaining metabolic activity contributes to cellular homeostasis maintenance. In this process, the cell cycle is arrested at the G0/G1 phase. p16INK4A protein is a key regulator of this process via its cyclin­dependent kinase inhibitor (CDKI) function. CDKI 2A (CDKN2A)/p16 gene expression is regulated by DNA methylation and histone acetylation. Sirtuins (SIRTs) are nicotinamide dinucleotide (NAD+)­dependent deacetylases that have properties which prevent diseases and reverse certain aspects of aging (such as immune, metabolic and cardiovascular diseases). By performing quantitative PCR, Western blot, ChIP, and siRNAs assays, in this study it was demonstrated that CDKN2A/p16 gene transcriptional activation and repression were accompanied by selective deposition and elimination of histone acetylation during the senescence of MRC5 cells. Specifically, significant H3K9Ac and H3K18Ac enrichment in cells with a senescent phenotype concomitant with CDKN2A/p16 gene overexpression was demonstrated compared with the non­senescent phenotype. Furthermore, the presence of H3K18Ac in deacetyl­transferase SIRT7 knockdown MRC5 cells allowed CDKN2A/p16 promoter activation. These results suggested that SIRT7 served as a critical component of an epigenetic mechanism involved in senescence mediated by the CDKN2A/p16 gene.

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.

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.

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.

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.

A DNA 3' phosphatase functions in active DNA demethylation in Arabidopsis.

DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3'- and 5'-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3' phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and colocalize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway.

Methylation-independent DNA binding modulates specificity of Repressor of Silencing 1 (ROS1) and facilitates demethylation in long substrates.

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC.G, but not for T.G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.

Single-nucleotide and long-patch base excision repair of DNA damage in plants.

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell-free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5'- or 3'-blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short-patch BER) or several nucleotides (long-patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks.

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.

DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases.

Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.

Arabidopsis thaliana Ogg1 protein excises 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from oxidatively damaged DNA containing multiple lesions.

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.

ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase.

Mutations in the Arabidopsis ROS1 locus cause transcriptional silencing of a transgene and a homologous endogenous gene. In the ros1 mutants, the promoter of the silenced loci is hypermethylated, which may be triggered by small RNAs produced from the transgene repeats. The transcriptional silencing in ros1 mutants can be released by the ddm1 mutation or the application of the DNA methylation inhibitor 5-aza-2'-deoxycytidine. ROS1 encodes an endonuclease III domain nuclear protein with bifunctional DNA glycosylase/lyase activity against methylated but not unmethylated DNA. The ros1 mutant shows enhanced sensitivity to genotoxic agents methyl methanesulfonate and hydrogen peroxide. We suggest that ROS1 is a DNA repair protein that represses homology-dependent transcriptional gene silencing by demethylating the target promoter DNA.