Epigenetics and Oxidative Stress in Aging

Aging is a multifactorial process characterized by the progressive loss of physiological functions, leading to an increased vulnerability to age-associated diseases and finally to death. Several theories have been proposed to explain the nature of aging. One of the most known identifies the free radicals produced by the mitochondrial metabolism as the cause of cellular and DNA damage. However, there are also several evidences supporting that epigenetic modifications, such as DNA methylation, noncoding RNAs, and histone modifications, play a critical role in the molecular mechanism of aging. In this review, we explore the significance of these findings and argue how the interlinked effects of oxidative stress and epigenetics can explain the cause of age-related declines

Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop

Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an similar to 1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions similar to 5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3' untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locu

Hypermethylation of the alternative AWT1 promoter in hematological malignancies is a highly specific marker for acute myeloid leukemias despite high expression levels

Background: Wilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A) WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/ CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown. Methods: To determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies. Results: Despite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling. Conclusions: We describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer

Human imprinted retrogenes exhibit non-canonical imprint chromatin signatures and reside in non-imprinted host genes

Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic ‘host’ gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters

Hypermethylation of the alternative AWT1 promoter in hematological malignancies is a highly specific marker for acute myeloid leukemias despite high expression levels

Abstract Background: Wilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A)WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/ CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown. Methods: To determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies. Results: Despite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling. Conclusions: We describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer

Characterization of Novel Paternal ncRNAs at the Plagl1 Locus, Including Hymai, Predicted to Interact with Regulators of Active Chromatin

Genomic imprinting is a complex epigenetic mechanism of transcriptional control that utilizes DNA methylation and histone modifications to bring about parent-of-origin specific monoallelic expression in mammals. Genes subject to imprinting are often organised in clusters associated with large non-coding RNAs (ncRNAs), some of which have cis-regulatory functions. Here we have undertaken a detailed allelic expression analysis of an imprinted domain on mouse proximal chromosome 10 comprising the paternally expressed Plagl1 gene. We identified three novel Plagl1 transcripts, only one of which contains protein-coding exons. In addition, we characterised two unspliced ncRNAs, Hymai, the mouse orthologue of HYMAI, and Plagl1it (Plagl1 intronic transcript), a transcript located in intron 5 of Plagl1. Imprinted expression of these novel ncRNAs requires DNMT3L-mediated maternal DNA methylation, which is also indispensable for establishing the correct chromatin profile at the Plagl1 DMR. Significantly, the two ncRNAs are retained in the nucleus, consistent with a potential regulatory function at the imprinted domain. Analysis with catRAPID, a protein-ncRNA association prediction algorithm, suggests that Hymai and Plagl1it RNAs both have potentially high affinity for Trithorax chromatin regulators. The two ncRNAs could therefore help to protect the paternal allele from DNA methylation by attracting Trithorax proteins that mediate H3 lysine-4 methylation

Epigenetic deregulation of WT1 and AWT1 in hematological maligancies

En l’última dècada, s’ha posat en evidència que el càncer és tant una malaltia genètica com epigenètica. La desregulació epigenètica pot afectar molts processos cel·lulars, com són el silenciament de gens supressors de tumors, l’activació d’oncògens, l’inestabilitat genòmica i/o la desregulació d’expressió de gens impremtats. Aquesta última, és l’expressió monoal·lèlica parental dictada a través de regions de metilació diferencial, heretades de la línia germinal. Aquests gens, en càncer es troben pertorbats, contribuint a la iniciació i progressió del càncer. En neoplàsies hematològiques es desconeix la desregulació d’aquests transcrits. El gen WT1, segons els seu context cel·lular, pot actuar com un suppressor de tumors o com un oncogen, a més a més, presenta unes isoformes d’expressió paterna, el transcrit alternatiu AWT1 i l’antisense WT1-AS. Aquesta expressió al·lèlica es creu que és regulada per una regió diferencialment metilada (DMR), coneguda com la regió reguladora de WT1 antisense. Curiosament, l'expressió de WT1 està desregulada en neoplàsies hematològiques, i s'utilitza rutinàriament com un marcador residual de la malaltia en leucèmies mieloides agudes. S’ha caracteritzat l’epigenètica de les regions promotores de WT1 i AWT1, per identificar el mecanisme que condueix a l'expressió aberrant, i si la pèrdua d'empremta n’és la causa. Utilitzant diverses tècniques moleculars no es va ser capaç d'identificar la metilació al·lèlica dins de l'interval del promotor de WT1, però tot i això s’observa un augment temporal en la metilació, coherent amb resultats publicats prèviament. Malgrat no trobar aquesta regió impremtada, s’observa que aquesta regió s’hipermetila amb freqüència en leucèmies i limfomes, i que la hipermetilació del promotor AWT1 es produeix en el 100% de línies cel·lulars d’AML, malgrat els alts nivells d’expressió. A més a més, la hipermetilació s'associa amb un canvi concomitant en les modificacions d'histones, d’un estat permissiu a un estat d’heterocromatina. En l’anàlisi de metilació de DNA en mes de 169 mostres de leucèmia primària, es va observar una hipermetilació en un 89% de les mostres de AML, independentment de mutacions i/o translocacions/fusions, a més a més de ser un marcador molecular prometedor per a la AML, amb un valor predictiu positiu de 100% i valor predictiu negatiu de 87,6%. Malgrat aquest perfil epigenètic extens, tant a nivell de promotor com a nivell de miRNA, l'única correlació que s’observa amb l’expressió de WT1/AWT1 en AML és la co-expressió del factor de transcripció, GATA-2, que s'uneix a un enhancer situat al 3'UTR de WT1. Aquesta observació, porta a la hipòtesi que GATA-2 indueix l’expressió de WT1/AWT1, i aquest inicia un mecanisme autoregulador en què interactua WT1 i recluta les metiltransferases d'DNA als llocs d’unió de WT1/EGR-1 al promotor d’AWT1 hipermetilant-lo.Intense research over the past decade has revealed that cancer is much an epigenetic as a genetic disease. The epigenetic deregulation can disrupt many cellular processes resulting in silencing of tumor suppressor genes, activation of oncogenes, genome instability and inappropriate imprinted gene expression. The latter process is the parental of origin monoallelic expression that is dictated by regions of differential methylation inherited from the germline, that when disrupted contributes to the initiation and progression of cancer. In hematological malignancies very little is known about the deregulation of imprinted transcripts. The WT1 gene has various transcripts that act as a tumor suppressor and an oncogene depending on the cellular context, and for which paternal expression of the alternative AWT1 and antisense isoforms has been reported. This allelic expression is postulated to be regulated by a tissue-specific differential methylation region (DMR), known as the WT1-antisense regulatory region. Interestingly, WT1 expression is aberrantly up regulated in hematological malignancies and is routinely used as a molecular marker for minimal residue of disease in acute myeloid leukemia. Herein, we have fully characterized the epigenetic landscape encompassing the WT1 and AWT1 promoter regions in an attempt to identify the mechanism leading to aberrant expression, and whether loss-of-imprinting is routinely observed. Using various molecular techniques we fail to identify allelic methylation within the WT1 promoter interval, but we observe a temporal increase in methylation that is consistent with previous reports. Despite conclusive evidences for lack of imprinting, we found that the region frequently becomes hypermethylated in leukemias and lymphomas, and that hypermethylation of the AWT1 promoter occurs in 100 % of AML cell lines despite the high expression levels. Further characterization of myeloid derived leukemia cell lines revealed that this cancer-associated hypermethylation is associated with a concomitant switch in histone modifications, from a permissive to a heterochromatic state. DNA methylation analysis in more than 169 primary leukemia samples revealed that this hypermethylated signature occurs in 89% of AML samples, independent of underlying mutations or translocation/fusion proteins, and is a promising molecular marker for AML, having a positive predictive value of 100% and a negative predictive value of 87.6%. Despite this extensive epigenetic profiling, both at the promoter and at the miRNA level, the only correlation we observe with WT1/AWT1 expression in AML is the co-expression of the transcription factor, GATA-2, which binds to the enhancer located in the 3’UTR of WT1. This observation, lead us to hypothesize that GATA-2 induced expression of WT1 initiating an auto-regulative mechanism in which WT1 interacts and recruits the DNA methyltransferases to degenerative WT1/EGR-1 binding sites within the AWT1 promoter, leading to its hypermethylation

Epigenetic deregulation of WT1 and AWT1 in hematological maligancies [i.e. malignancies]

En l'última dècada, s'ha posat en evidència que el càncer és tant una malaltia genètica com epigenètica. La desregulació epigenètica pot afectar molts processos cel·lulars, com són el silenciament de gens supressors de tumors, l'activació d'oncògens, l'inestabilitat genòmica i/o la desregulació d'expressió de gens impremtats. Aquesta última, és l'expressió monoal·lèlica parental dictada a través de regions de metilació diferencial, heretades de la línia germinal. Aquests gens, en càncer es troben pertorbats, contribuint a la iniciació i progressió del càncer. En neoplàsies hematològiques es desconeix la desregulació d'aquests transcrits. El gen WT1, segons els seu context cel·lular, pot actuar com un suppressor de tumors o com un oncogen, a més a més, presenta unes isoformes d'expressió paterna, el transcrit alternatiu AWT1 i l'antisense WT1-AS. Aquesta expressió al·lèlica es creu que és regulada per una regió diferencialment metilada (DMR), coneguda com la regió reguladora de WT1 antisense. Curiosament, l'expressió de WT1 està desregulada en neoplàsies hematològiques, i s'utilitza rutinàriament com un marcador residual de la malaltia en leucèmies mieloides agudes. S'ha caracteritzat l'epigenètica de les regions promotores de WT1 i AWT1, per identificar el mecanisme que condueix a l'expressió aberrant, i si la pèrdua d'empremta n'és la causa. Utilitzant diverses tècniques moleculars no es va ser capaç d'identificar la metilació al·lèlica dins de l'interval del promotor de WT1, però tot i això s'observa un augment temporal en la metilació, coherent amb resultats publicats prèviament. Malgrat no trobar aquesta regió impremtada, s'observa que aquesta regió s'hipermetila amb freqüència en leucèmies i limfomes, i que la hipermetilació del promotor AWT1 es produeix en el 100% de línies cel·lulars d'AML, malgrat els alts nivells d'expressió. A més a més, la hipermetilació s'associa amb un canvi concomitant en les modificacions d'histones, d'un estat permissiu a un estat d'heterocromatina. En l'anàlisi de metilació de DNA en mes de 169 mostres de leucèmia primària, es va observar una hipermetilació en un 89% de les mostres de AML, independentment de mutacions i/o translocacions/fusions, a més a més de ser un marcador molecular prometedor per a la AML, amb un valor predictiu positiu de 100% i valor predictiu negatiu de 87,6%. Malgrat aquest perfil epigenètic extens, tant a nivell de promotor com a nivell de miRNA, l'única correlació que s'observa amb l'expressió de WT1/AWT1 en AML és la co-expressió del factor de transcripció, GATA-2, que s'uneix a un enhancer situat al 3'UTR de WT1. Aquesta observació, porta a la hipòtesi que GATA-2 indueix l'expressió de WT1/AWT1, i aquest inicia un mecanisme autoregulador en què interactua WT1 i recluta les metiltransferases d'DNA als llocs d'unió de WT1/EGR-1 al promotor d'AWT1 hipermetilant-lo.Intense research over the past decade has revealed that cancer is much an epigenetic as a genetic disease. The epigenetic deregulation can disrupt many cellular processes resulting in silencing of tumor suppressor genes, activation of oncogenes, genome instability and inappropriate imprinted gene expression. The latter process is the parental of origin monoallelic expression that is dictated by regions of differential methylation inherited from the germline, that when disrupted contributes to the initiation and progression of cancer. In hematological malignancies very little is known about the deregulation of imprinted transcripts. The WT1 gene has various transcripts that act as a tumor suppressor and an oncogene depending on the cellular context, and for which paternal expression of the alternative AWT1 and antisense isoforms has been reported. This allelic expression is postulated to be regulated by a tissue-specific differential methylation region (DMR), known as the WT1-antisense regulatory region. Interestingly, WT1 expression is aberrantly up regulated in hematological malignancies and is routinely used as a molecular marker for minimal residue of disease in acute myeloid leukemia. Herein, we have fully characterized the epigenetic landscape encompassing the WT1 and AWT1 promoter regions in an attempt to identify the mechanism leading to aberrant expression, and whether loss-of-imprinting is routinely observed. Using various molecular techniques we fail to identify allelic methylation within the WT1 promoter interval, but we observe a temporal increase in methylation that is consistent with previous reports. Despite conclusive evidences for lack of imprinting, we found that the region frequently becomes hypermethylated in leukemias and lymphomas, and that hypermethylation of the AWT1 promoter occurs in 100 % of AML cell lines despite the high expression levels. Further characterization of myeloid derived leukemia cell lines revealed that this cancer-associated hypermethylation is associated with a concomitant switch in histone modifications, from a permissive to a heterochromatic state. DNA methylation analysis in more than 169 primary leukemia samples revealed that this hypermethylated signature occurs in 89% of AML samples, independent of underlying mutations or translocation/fusion proteins, and is a promising molecular marker for AML, having a positive predictive value of 100% and a negative predictive value of 87.6%. Despite this extensive epigenetic profiling, both at the promoter and at the miRNA level, the only correlation we observe with WT1/AWT1 expression in AML is the co-expression of the transcription factor, GATA-2, which binds to the enhancer located in the 3'UTR of WT1. This observation, lead us to hypothesize that GATA-2 induced expression of WT1 initiating an auto-regulative mechanism in which WT1 interacts and recruits the DNA methyltransferases to degenerative WT1/EGR-1 binding sites within the AWT1 promoter, leading to its hypermethylation

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

[Background] Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9.[Results] Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression.[Conclusions] We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locus.This work was supported by Spanish Ministerio de Educacion y Ciencia (grant number BFU2011-27658 to DM); Fundació La Marató de TV3 (101130 to DM); Telethon-Italia grant number GGP11122 (to AR). DM and AR are members of the COST action BM1208. DM is a Ramon y Cajal research fellow and AGA was funded by a FPU studentship.Peer Reviewe