TET3‐mediated DNA oxidation promotes ATR‐dependent DNA damage response

An efficient, accurate, and timely DNA damage response (DDR) is crucial for the maintenance of genome integrity. Here, we report that ten‐eleven translocation dioxygenase (TET) 3‐mediated conversion of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) in response to ATR‐dependent DDR regulates DNA repair. ATR‐dependent DDR leads to dynamic changes in 5hmC levels and TET3 enzymatic activity. We show that TET3 is an ATR kinase target that oxidizes DNA during ATR‐dependent DNA damage repair. Modulation of TET3 expression and activity affects DNA damage signaling and DNA repair and consequently cell death. Our results provide novel insight into ATR‐mediated DDR, in which TET3‐mediated DNA demethylation is crucial for efficient DNA repair and maintenance of genome stability.     

Playing TETris with DNA modifications

Methylation of the fifth carbon of cytosine was the first epigenetic modification to be discovered in DNA. Recently, three new DNA modifications have come to light: hydroxymethylcytosine, formylcytosine, and carboxylcytosine, all generated by oxidation of methylcytosine by Ten Eleven Translocation (TET) enzymes. These modifications can initiate full DNA demethylation, but they are also likely to participate, like methylcytosine, in epigenetic signalling per se. A scenario is emerging in which coordinated regulation at multiple levels governs the participation of TETs in a wide range of physiological functions, sometimes via a mechanism unrelated to their enzymatic activity. Although still under construction, a sophisticated picture is rapidly forming where, according to the function to be performed, TETs ensure epigenetic marking to create specific landscapes, and whose improper build‐up can lead to diseases such as cancer and neurodegenerative disorders.     

Low‐density lipoprotein receptor‐related protein 6 regulates alternative pre‐mRNA splicing

Low‐density lipoprotein receptor‐related protein 6 (LRP6) serves as a Wnt coreceptor. Although Wnt/LRP6 signalling is best known for the β‐catenin‐dependent regulation of target genes in tissue development and homeostasis, emerging evidence demonstrates the biological aspects of LRP6 beyond a Wnt coreceptor. Whether LRP6 modulates tissue development in a Wnt/β‐catenin signalling‐independent manner remains unknown. Using a model of striated muscle development, we observed that LRP6 was almost undetectable in proliferating myoblasts, whereas its expression gradually increased in the nucleus of myodifferentiating cells. During myodifferentiation, LRP6 modulated the muscle‐specific splicing of integrin‐β1D and consequent myotube maturation independently of the β‐catenin‐dependent Wnt signalling. Furthermore, we identified that the carboxy‐terminal serine‐rich region in LRP6 bond to the adenine‐rich sequence within alternative exon D (AED) of integrin‐β1 pre‐mRNA, and therefore, elicited AED inclusion when the spliceosome was recruited to the splice site. The interaction of LRP6 with the adenine‐rich sequence was sufficient to overcome AED exclusion by a splicing repressor, polypyrimidine tract binding protein‐1. Besides the integrin‐β1, deep RNA sequencing in different types of cells revealed that the LRP6‐mediated splicing regulation was widespread. Thus, our findings implicate LRP6 as a potential regulator for alternative pre‐mRNA splicing.     

The arginine and serine‐rich domains of Acinus modulate splicing

Apoptotic chromatin condensation inducer in the nucleus (Acinus) is an RNA‐binding protein that has a functional role in inducing apoptotic chromatin condensation and regulating messenger RNA (mRNA) processing. Acinus interacts with the spliceosomal machinery and is a member of the ASAP (apoptosis and splicing‐associated protein complex) as well as the EJC (exon junction complex), which gets deposited onto mRNA during splicing. In this study, we have used in vivo splicing assays to characterize the function of Acinus in pre‐mRNA splicing more closely. We show that full‐length Acinus‐S′, an isoform of Acinus, does not have a role in modulating splice site selection in human immunodeficiency virus 1 minigene reporter system. In contrast, we observed that the tethering of arginine/serine (RS) and RNPS1‐SAP18‐binding (RSB) domains of Acinus could regulate the selection of alternative splice sites, thereby revealing the potential of Acinus in stimulating alternative splicing. Altogether, our data suggest that the RS and RSB domains play a critical role in regulating splicing activity via selection of distinct splice sites during pre‐mRNA splicing.     

5‐Hydroxymethylcytosine: a new kid on the epigenetic block?

The discovery of the Ten‐Eleven‐Translocation (TET) oxygenases that catalyze the hydroxylation of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) has triggered an avalanche of studies aiming to resolve the role of 5hmC in gene regulation if any. Hitherto, TET1 is reported to bind to CpG‐island (CGI) and bivalent promoters in mouse embryonic stem cells, whereas binding at DNAseI hypersensitive sites (HS) had escaped previous analysis. Significant enrichment/accumulation of 5hmC but not 5mC can indeed be detected at bivalent promoters and at DNaseI‐HS. Surprisingly, however, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1. Our meta‐analysis of DNA methylation profiling points to potential issues with regard to the various methodologies that are part of the toolbox used to detect 5mC and 5hmC. Discrepancies between published studies and technical limitations prevent an unambiguous assignment of 5hmC as a ‘true’ epigenetic mark, that is, read and interpreted by other factors and/or as a transiently accumulating intermediary product of the conversion of 5mC to unmodified cytosines.     

Altering TET dioxygenase levels within physiological range affects DNA methylation dynamics of HEK293 cells

The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.     

TET2–BCLAF1 transcription repression complex epigenetically regulates the expression of colorectal cancer gene Ascl2 via methylation of its promoter

Ascl2 has been shown to be involved in tumorigenesis in colorectal cancer (CRC), although its epigenetic regulatory mechanism is largely unknown. Here, we found that methylation of the Ascl2 promoter (bp -1670 ∼ -1139) was significantly increased compared to the other regions of the Ascl2 locus in CRC cells and was associated with elevated Ascl2 mRNA expression. Furthermore, we found that promoter methylation was predictive of CRC patient survival after analyzing DNA methylation data, RNA-Seq data, and clinical data of 410 CRC patient samples from the MethHC database, the MEXPRESS database, and the Cbioportal website. Using the established TET methylcytosine dioxygenase 2 (TET2) knockdown and ectopic TET2 catalytic domain–expression cell models, we performed glucosylated hydroxymethyl–sensitive quatitative PCR (qPCR), real-time PCR, and Western blot assays to further confirm that hypermethylation of the Ascl2 promoter, and elevated Ascl2 expression in CRC cells was partly due to the decreased expression of TET2. Furthermore, BCLAF1 was identified as a TET2 interactor in CRC cells by LC-MS/MS, coimmunoprecipitation, immunofluorescence colocalization, and proximity ligation assays. Subsequently, we found the TET2–BCLAF1 complex bound to multiple elements around CCGG sites at the Ascl2 promoter and further restrained its hypermethylation by inducing its hydroxymethylation using chromatin immunoprecipitation-qPCR and glucosylated hydroxymethyl-qPCR assays. Finally, we demonstrate that TET2-modulated Ascl2-targeted stem gene expression in CRC cells was independent of Wnt signaling. Taken together, our data suggest an additional option for inhibiting Ascl2 expression in CRC cells through TET2–BCLAF1–mediated promoter methylation, Ascl2-dependent self-renewal of CRC progenitor cells, and TET2–BCLAF1–related CRC progression.     

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in leukemia. Using bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type CMML cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hypermethylated in TET2 mutant cases. However, total 5-methyl-cytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in CMML but most of the changes are likely to be outside gene promoters.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre‐leukaemic clone resulting in T2‐ALL and AML‐M0

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1^R174Q mutation who developed at the age of 42 years a T2‐ALL and, 2 years after remission, an AML‐M0. Both AML‐M0 and T2‐ALL blast populations demonstrated a loss of 1p36.32‐23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2‐ALL or in AML‐M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood‐derived CD34⁺ cells 5 years prior to T2‐ALL development revealed only the missense TET2^P1962T mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2‐ALL and AML‐M0 clones. This result suggests that TET2^P1962T mutation in association with germline RUNX1^R174Q mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.