Auswahl der wissenschaftlichen Literatur zum Thema „Site-specific DNA methylation“

Abstract Background: Epigenetic studies, such as the measurement of DNA methylation, are important in the investigation of syndromes influenced by imprinted genes. Quick and accurate quantification of methylation at such genes can be of appreciable diagnostic aid. Methods: We first digested genomic DNA with methylation-sensitive restriction enzymes and used DNA without digestion as a control and nonmethylated λ DNA as an internal control for digestion efficiency. We then performed quantitative real-time PCR analyses with 6 unique PCR assays to investigate 4 imprinting control regions on chromosomes 7 and 11 in individuals with uniparental disomy of chromosome 7 (UPD7) and in control individuals. Results: Our validation of the method demonstrated both quantitative recovery and low methodologic imprecision. The imprinted loci on chromosome 7 behaved as expected in maternal UPD7 (100% methylation) and paternal UPD7 (<10% methylation). In controls, the mean (SD) for percent methylation at 2 previously well-studied restriction sites were 46% (6%) for both H19 and KCNQ1OT1, a result consistent with the previously observed methylation rate of approximately 50%. The methylation percentages of all investigated imprinted loci were normally distributed, implying that the mean and SD can be used as a reference for screening methylation loss or gain. Conclusion: The investigated loci are of particular importance for investigating the congenital Silver–Russell and Beckwith–Wiedemann syndromes; however, the method can also be applied to other imprinted regions. This method is easy to set up, has no PCR bias, requires small amounts of DNA, and can easily be applied to large patient populations for screening the loss or gain of methylation.

Dynamic changes in DNA (hydroxy-)methylation are fundamental for stem cell differentiation. However, the signature of these epigenetic marks in specific cell types during corticogenesis is unknown. Moreover, site-specific manipulation of cytosine modifications is needed to reveal the significance and function of these changes. Here, we report the first assessment of (hydroxy-)methylation in neural stem cells, neurogenic progenitors, and newborn neurons during mammalian corticogenesis. We found that gain in hydroxymethylation and loss in methylation occur sequentially at specific cellular transitions during neurogenic commitment. We also found that these changes predominantly occur within enhancers of neurogenic genes up-regulated during neurogenesis and target of pioneer transcription factors. We further optimized the use of dCas9-Tet1 manipulation of (hydroxy-)methylation, locus-specifically, in vivo, showing the biological relevance of our observations for Dchs1, a regulator of corticogenesis involved in developmental malformations and cognitive impairment. Together, our data reveal the dynamics of cytosine modifications in lineage-related cell types, whereby methylation is reduced and hydroxymethylation gained during the neurogenic lineage concurrently with up-regulation of pioneer transcription factors and activation of enhancers for neurogenic genes.

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in ∼25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from −373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.

Abstract DNA methylation, an epigenetic modification, has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. To overcome this barrier, we utilized nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltranferase 3A (DNMT3A) (CRISPR-Cas SunTag-directed DNMT3A) to amplify local DNMT3A concentration and to methylate genomic sites of interest. Here, we demonstrated that CRISPR-Cas SunTag-directed DNMT3A not only dramatically increased CpG methylation but also, to our surprise, CpH (H =A or C or T) methylation at the HOXA5 lociin human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, CRISPR-Cas SunTag-directed DNMT3A was capable of methylating 4.5 kb genomic regions, surpassing previous targeted methylation tools whose activity is limited to 200bp. Using reduced representation bisulfite sequencing (RRBS) and RNA-seq, we concluded that CRISPR-Cas SunTag-directed DNMT3A methylated regions of interest without affecting global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship beteween DNA methylation and gene expression. Disclosures No relevant conflicts of interest to declare.

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

Les LAL-T sont des proliférations malignes de précurseurs lymphoïdes T bloqués à un stade précis de leur maturation. Si les anomalies génétiques impliquées dans la leucémogenèse T sont de mieux en mieux connues, les modifications de la régulation épigénétique sont beaucoup moins étudiées. Mon travail de thèse a consisté à étudier la dérégulation épigénétique intervenant dans les LAL-T au travers de 3 projets principaux. Dans le premier travail, nous avons identifié un mécanisme original de dérégulation de l’oncogène TAL1 consistant en la création d’un « neo-enhancer » oncogénique. TAL1 est l’un des oncogènes les plus fréquemment dérégulés dans les LAL-T. Cette dérégulation résulte principalement de translocations avec le locus du TCR ou des micro-délétions interstitielles SIL-TAL1, anomalies chromosomiques altérant des éléments de cis-régulation engendrant une expression ectopique monoallélique de TAL1. Mais dans une proportion importante de cas (environ 50%) de LAL-T TAL1+, une expression aberrante de TAL1 est observée sans que le mécanisme en cause ne soit identifié suggérant l’existence de mécanismes génétiques ou épigénétiques non connus. Nous avons découvert une nouvelle anomalie somatique consistant en une micro-insertion focale et récurrente 7 kpb en amont de TAL1, dans une région intergénique non-codante, responsable de la création d’un « neo-enhancer » oncogénique accompagné d’une modification des marques épigénétiques d’histones i.e. une substitution des marques répressives H3H27me3 par des marques activatrices H3K27ac. Ces micro-insertions sont un événement récurrent dans les LAL-T et ont été retrouvées dans 20% des LAL-T TAL1+ inexpliquées. Au travers du deuxième projet, j’ai tenté de mieux comprendre les bases biologiques à l’origine de différences de réponse au traitement. En effet, considérant deux groupes oncogéniques proches, le pronostic des patients TLX1+, déjà plutôt favorable dans le protocole LALA-94, n’a pas été significativement amélioré dans le protocole thérapeutique intensifié d’inspiration pédiatrique GRAALL 2003-2005 alors que les patients TLX3+ semblent avoir particulièrement bénéficié de ce dernier; les deux protocoles différant majoritairement par les doses de L-asparaginase. Nous avons montré que les patients TLX1+ exprimaient moins d’ASNS (Asparagine synthétase) que les patients TLX3+ et TLX- et que cette moindre expression résultait d’une inhibition épigénétique d’ASNS, à la fois par méthylation du promoteur et diminution des marques histones activatrices. Un niveau de méthylation d’ASNS bas est par ailleurs associé à une moindre sensibilité in vitro à la L-asparaginase. Enfin, la méthylation d’ASNS est un facteur pronostic indépendant pour les patients inclus dans le protocole GRAALL 2003-2005 suggérant que le statut de la méthylation d’ASNS au diagnostic pourrait intervenir dans l’adaptation des doses de L-asparaginase. Dans le troisième projet, je me suis intéressée à la méthylation globale de l’ADN. L’étude de la méthylation par MeDIP-array d’une série de 24 LAL-T nous a permis d’identifier des signatures de méthylation différentielle et de définir un panel minimum de 9 promoteurs de gènes dont l’étude de la méthylation par MS-MLPA a permis de déterminer un ratio de méthylation pour une large série de LAL-T adultes du GRAALL 2003-2005. Le statut de méthylation semble dicté par l’oncogène dérégulé avec en particulier les LAL-T TLX1+ ou TLX3+ associées à une signature d’hyperméthylation et les LAL-T SIL-TAL1+ à une signature d’hypométhylation. Ce statut de méthylation est par ailleurs un facteur pronostique indépendant. Ainsi, les patients hypométhylés ont un pronostic significativement plus défavorable que les patients hyperméthylés. Ensemble, ces résultats illustrent comment des perturbations de la régulation épigénétique peuvent intervenir à la fois dans l’oncogénèse des LAL-T mais aussi dans la réponse au traitement
T-ALLs are rare lymphoid neoplasms characterized by the proliferation of immature T precursors arrested at specific stages of maturation. While the genetic abnormalities involved in T-ALL leukemogenesis are becoming better known, alterations in epigenetic regulation, a very important component of the cellular homeostasis, are much less studied. My work was to tsudy the epigenetic deregulation in T-ALL through 3 main projects. In the first project, we identified an original mechanism of TAL1 oncogene deregulation. TAL1 is one of the most frequently deregulated oncogenes in T-ALL. This deregulation results mainly from translocations with the TCRδ locus or micro-deletions SIL-TAL1, two chromosomal abnormalities altering cis-regulatory elements leading to monoallelic TAL1 expression. But in a significant proportion of cases (about 50%) of TAL1+ T-ALL, an aberrant expression of TAL1 is observed without recognized mechanism suggesting unknown genetic or epigenetic mechanisms. We discovered a new somatic alteration consisting of a focal and recurrent microinsertion 7 kbp upstream of TAL1, in a non-coding intergenic region, responsible for the creation of an oncogenic "neo-enhancer" accompanied by a modification of epigenetic histone marks i.e. a “switch” from H3H27me3 repressive marks to H3K27ac activating marks. These microinsertions are a recurrent event in T-ALL and have been found in 20% of “unresolved” TAL1+ T-ALL. Through the second project, I tried to better understand the biological bases for discrepancies in patients related response to treatment. Indeed, considering two close oncogenic groups, the prognosis of TLX1+ patients, already rather favourable in the LALA-94 protocol, has not been significantly improved in the paediatric-inspired GRAALL2003-2005 trial , whereas TLX3+ patients seem to have benefited particularly from the latter; the two protocols differing mainly by L-asparaginase doses. We showed that TLX1+ patients expressed less ASNS (Asparagine synthetase) than TLX3+ and TLX- patients and that this lower expression resulted from ASNS epigenetic silencing, both by methylation of the promoter and reduction of active histone marks. A low level of ASNS methylation is also associated with lower in vitro sensitivity to L-asparaginase. Finally, ASNS methylation is an independent prognostic factor for patients included in the 2003-2005 GRAALL trial suggesting that the ASNS methylation status may be relevant for the adaptation of L-asparaginase doses. In the third project, I was interested in the global DNA methylation. MeDIP-array methylation data of a series of 24 T-ALLs allowed us to identify differential methylation signatures. We then studied the methylation status in a large series of adult T-ALL by MS-MLPA using a predictor containing 9 gene promoters. We observed that main driver oncogenes dictated methylation status. TLX1+ and TLX3+ T-ALLs displayed a hypermethylated profile and conversely, SIL-TAL1+ cases were associated with a hypomethylated profile. This methylation status is also an independent prognostic factor and hypomethylated patients have a significantly unfavorable prognosis compared to hypomethylated patients. Together, these results illustrate how disruptions in epigenetic regulation can be involved both in the T-ALL oncogenesis and in the response to treatment

L'épissage alternatif de l'ARN précurseur est un processus co-transcriptionnel qui affecte la grande majorité des gènes humains et contribue à la diversité des protéines. Le dérèglement d'un tel processus est impliqué dans diverses maladies, y compris la tumorigènes. Cependant, les mécanismes qui régulent ces processus restaient à caractériser. Dans cette étude, nous avons montré que les perturbations de l'épissage alternatif étaient corrélées aux dérèglements de la transcription convergente et de la méthylation de l'ADN. Une transcription convergente peut être générée entre des paires de gènes voisins en en orientation opposée, ou entre des amplificateurs intragéniques et leur gène hôte. CENPO et ADCY3 ont été identifiés comme une paire de gènes de transcription convergentes. Nous avons constaté, dans un modèle de progression tumorale du cancer du sein, que le changement d'épissage de l'exon22 variant d'ADCY3 était corrélé à une augmentation de sa transcription qui allait contre celle de CENPO. En utilisant le système de répression ciblée de la transcription CRISPRi, nous avons démontré que l’inhibition de la transcription de CENPO ne pouvait pas inverser l'altération d'épissage alternatif d'ADCY3 dans les cellules cancéreuses (DCIS). Un activateur intragénique actif a été identifié dans l'intron16 du gène CD44, en aval de ses exons alternatifs. En utilisant le système d'activation ciblée de transcription CRISPRa, nous avons montré que l’augmentation de la transcription de CD44 ne pouvait pas modifier l'épissage alternatif de CD44 dans les cellules DCIS. Ces résultats suggèrent que la modification de transcription convergente par des changements d’activité des promoteurs ne permet pas d’altérer l'épissage alternatif de ADCY3 et CD44 dans les cellules DCIS. Cependant, en remplaçant l'amplificateur intragénique par un promoteur inductible, nous avons constaté que l'activation de la transcription intragénique augmentait le niveau d'inclusion de plusieurs exons alternatifs de CD44 dans les cellules HCT116. Ce résultat suggère que la transcription convergente local pourrait avoir un impact direct sur l'épissage alternatif de CD44. De plus, en utilisant le système de méthylation de l'ADN ciblée CRISPR/dCas9-DNMT3b, nous avons démontré que la méthylation de l'ADN au niveau des exons variants pouvait modifier l'épissage alternatif de CD44. Ce travail de thèse a également exploré les limites et la faisabilité de l'étude de l'épissage alternatif avec des outils moléculaires basés sur le système CRISPR
Alternative splicing of precursor RNA is a co-transcriptional process that affects the vast majority of human genes and contributes to protein diversity. Dysregulation of such process is implicated in various diseases, including tumorigenesis. However, the mechanisms regulating these processes were still to be characterized. In this study, we showed that perturbations of alternative splicing correlated with dysregulations of convergent transcription and DNA methylation. Convergent transcription could be generated between pairs of neighboring genes in opposite orientation, or between intragenic enhancers and their host gene. CENPO and ADCY3 was identified as a convergent transcription gene pair. We found, in a tumor progression model of breast cancer, that the splicing change of the ADCY3 variant exon22 correlated with an increase of its transcription that went against that of CENPO. By using targeted transcription repression system CRISPRi, we demonstrated that downregulating the transcription of CENPO could not reverse the alternative splicing alteration of ADCY3 in cancer cells (DCIS). An active intragenic enhancer was identified in the intron16 of CD44, at the downstream of its alternative exons. By using targeted transcription activation system CRISPRa, we showed that upregulating the transcription of CD44 could not alter the alternative splicing of CD44 in DCIS cells. These results suggest that convergent transcription modulation through changes of promoter activity does not alter the alternative splicing of ADCY3 and CD44 in DCIS cells. However, through replacing the intragenic enhancer by an inducible promoter, we found that intragenic transcription activation increased the inclusion level of several alternative exons of CD44 in HCT116 cells. This result suggested that local convergent transcription could have a direct impact on the alternative splicing of CD44. Furthermore, by using targeted DNA methylation system CRISPR/dCas9-DNMT3b, we showed that DNA methylation at variant exons could directly modify CD44 alternative splicing. This thesis work also explored the limitation and feasibility of studying alternative splicing with repurposed CRISPR systems