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Journal articles on the topic 'Enhancer methylation'
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1
Pühringer, Katharina, Philipp Czarda, Sebastian Iluca, et al. "Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma." International Journal of Molecular Sciences 26, no. 7 (2025): 3390. https://doi.org/10.3390/ijms26073390.
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The methylation status of the MGMT gene promoter is recognized as a key predictive biomarker for glioblastoma patients, influencing treatment decisions and outcomes. Emerging evidence suggests that enhancer methylation may also play a role in gene regulation and is associated with various clinical parameters, genetic variants, and demographic factors. This study aimed to assess DNA methylation levels in intergenic and intragenic MGMT enhancers to investigate their relationship with MGMT promoter methylation, MGMT protein expression, and clinical and demographic characteristics in glioblastoma. We developed 18 pyrosequencing assays to analyze 54 CpGs, including 34 in intergenic and 20 in intragenic enhancers. The assays were applied to tumor cells derived from 38 glioma patients. Intragenic enhancer CpGs showed significantly higher methylation than intergenic enhancer CpGs. Intragenic enhancer methylation showed a strong negative correlation with MGMT promoter methylation. For several CpGs in intragenic enhancers, an inverse L-shaped relationship between methylation levels and MGMT expression was observed. We identified distinct associations between enhancer methylation and clinical and demographic parameters. Intergenic enhancer methylation was primarily linked to the TERT SNP rs2853669 genotype, Ki-67 expression, age, and sex, whereas intragenic enhancer methylation was associated with MGMT promoter methylation, MGMT expression, overall survival, and progression-free survival. Further studies with larger patient cohorts are needed to validate the clinical relevance of intergenic and intragenic MGMT enhancer methylation in glioblastoma.
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Alajem, Adi, Hava Roth, Sofia Ratgauzer, et al. "DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation." PLOS Genetics 17, no. 4 (2021): e1009498. http://dx.doi.org/10.1371/journal.pgen.1009498.
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In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.
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Fan, Zhiyu, Yingli Chen, Dongsheng Yan, and Qianzhong Li. "Effects of Differentially Methylated CpG Sites in Enhancer and Promoter Regions on the Chromatin Structures of Target LncRNAs in Breast Cancer." International Journal of Molecular Sciences 25, no. 20 (2024): 11048. http://dx.doi.org/10.3390/ijms252011048.
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Aberrant DNA methylation plays a crucial role in breast cancer progression by regulating gene expression. However, the regulatory pattern of DNA methylation in long noncoding RNAs (lncRNAs) for breast cancer remains unclear. In this study, we integrated gene expression, DNA methylation, and clinical data from breast cancer patients included in The Cancer Genome Atlas (TCGA) database. We examined DNA methylation distribution across various lncRNA categories, revealing distinct methylation characteristics. Through genome-wide correlation analysis, we identified the CpG sites located in lncRNAs and the distally associated CpG sites of lncRNAs. Functional genome enrichment analysis, conducted through the integration of ENCODE ChIP-seq data, revealed that differentially methylated CpG sites (DMCs) in lncRNAs were mostly located in promoter regions, while distally associated DMCs primarily acted on enhancer regions. By integrating Hi-C data, we found that DMCs in enhancer and promoter regions were closely associated with the changes in three-dimensional chromatin structures by affecting the formation of enhancer–promoter loops. Furthermore, through Cox regression analysis and three machine learning models, we identified 11 key methylation-driven lncRNAs (DIO3OS, ELOVL2-AS1, MIAT, LINC00536, C9orf163, AC105398.1, LINC02178, MILIP, HID1-AS1, KCNH1-IT1, and TMEM220-AS1) that were associated with the survival of breast cancer patients and constructed a prognostic risk scoring model, which demonstrated strong prognostic performance. These findings enhance our understanding of DNA methylation’s role in lncRNA regulation in breast cancer and provide potential biomarkers for diagnosis.
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Zappe, Katja, Katharina Pühringer, Simon Pflug, et al. "Association between MGMT Enhancer Methylation and MGMT Promoter Methylation, MGMT Protein Expression, and Overall Survival in Glioblastoma." Cells 12, no. 12 (2023): 1639. http://dx.doi.org/10.3390/cells12121639.
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The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01–03 and CpGs 09–13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.
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Bizet, Martin, Matthieu Defrance, Emilie Calonne, Gianluca Bontempi, François Fuks, and Jana Jeschke. "Abstract 6283: Reannotation and normalisation of Infinium 850k data for improved analysis of methylation at enhancers and non-coding RNAs." Cancer Research 82, no. 12_Supplement (2022): 6283. http://dx.doi.org/10.1158/1538-7445.am2022-6283.
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Abstract In this study, we evaluated the MethylationEPIC BeadChip (850k) technology for enhancer methylation analysis with respect to RRBS, both high-throughput technologies commonly used to screen large patient cohorts. We developed and applied a new approach to re-annotate the 850k data, which greatly improved the association of probes to enhancers and show that 850k targets more enhancers than RRBS. We further investigated the reproducibility of the two technologies and applied various existing normalization methods to 850k data that reduce variability between replicates and variability with other technologies. We thereby showed that normalization methods developed for 450k greatly reduced variability in 850k data to a level below that of highly variable RRBS data. We finally performed differential methylation analysis with 850k data from breast cancer samples applying our new re-annotation method and highlight that the majority of differentially methylated cytosines were detected with probes specific to the 850k mapping to enhancers, confirming the deregulation of enhancer methylation in breast cancer. In summary, our study provides a new annotation for the 850k array, which greatly increases the number of cytosines mapping to enhancers and therefore allows for the improved analysis of enhancer methylation. Overall, we conclude that the 850k array allows for detection of methylation changes in regions not covered by previous Infinium arrays and is the best choice, as compared to RRBS, for high-throughput analysis of enhancer methylation in large clinical cohorts. Citation Format: Martin Bizet, Matthieu Defrance, Emilie Calonne, Gianluca Bontempi, François Fuks, Jana Jeschke. Reannotation and normalisation of Infinium 850k data for improved analysis of methylation at enhancers and non-coding RNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6283.
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Zappe, Katja, Katharina Pühringer, Simon Pflug, et al. "Association of MGMT Promoter and Enhancer Methylation with Genetic Variants, Clinical Parameters, and Demographic Characteristics in Glioblastoma." Cancers 15, no. 24 (2023): 5777. http://dx.doi.org/10.3390/cancers15245777.
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The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.
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Margalit, Sapir, Yotam Abramson, Hila Sharim, et al. "Long reads capture simultaneous enhancer–promoter methylation status for cell-type deconvolution." Bioinformatics 37, Supplement_1 (2021): i327—i333. http://dx.doi.org/10.1093/bioinformatics/btab306.
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Abstract Motivation While promoter methylation is associated with reinforcing fundamental tissue identities, the methylation status of distant enhancers was shown by genome-wide association studies to be a powerful determinant of cell-state and cancer. With recent availability of long reads that report on the methylation status of enhancer–promoter pairs on the same molecule, we hypothesized that probing these pairs on the single-molecule level may serve the basis for detection of rare cancerous transformations in a given cell population. We explore various analysis approaches for deconvolving cell-type mixtures based on their genome-wide enhancer–promoter methylation profiles. Results To evaluate our hypothesis we examine long-read optical methylome data for the GM12878 cell line and myoblast cell lines from two donors. We identified over 100 000 enhancer–promoter pairs that co-exist on at least 30 individual DNA molecules. We developed a detailed methodology for mixture deconvolution and applied it to estimate the proportional cell compositions in synthetic mixtures. Analysis of promoter methylation, as well as enhancer–promoter pairwise methylation, resulted in very accurate estimates. In addition, we show that pairwise methylation analysis can be generalized from deconvolving different cell types to subtle scenarios where one wishes to resolve different cell populations of the same cell-type. Availability and implementation The code used in this work to analyze single-molecule Bionano Genomics optical maps is available via the GitHub repository https://github.com/ebensteinLab/Single_molecule_methylation_in_EP.
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Yoon, Bongjune, Herry Herman, Benjamin Hu, et al. "Rasgrf1 Imprinting Is Regulated by a CTCF-Dependent Methylation-Sensitive Enhancer Blocker." Molecular and Cellular Biology 25, no. 24 (2005): 11184–90. http://dx.doi.org/10.1128/mcb.25.24.11184-11190.2005.
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ABSTRACT Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5′ of the promoter. A repeated sequence 3′ of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.
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Wang, Chaochen, Ji-Eun Lee, Binbin Lai, et al. "Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition." Proceedings of the National Academy of Sciences 113, no. 42 (2016): 11871–76. http://dx.doi.org/10.1073/pnas.1606857113.
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Transcriptional enhancers control cell-type–specific gene expression. Primed enhancers are marked by histone H3 lysine 4 (H3K4) mono/di-methylation (H3K4me1/2). Active enhancers are further marked by H3K27 acetylation (H3K27ac). Mixed-lineage leukemia 4 (MLL4/KMT2D) is a major enhancer H3K4me1/2 methyltransferase with functional redundancy with MLL3 (KMT2C). However, its role in cell fate maintenance and transition is poorly understood. Here, we show in mouse embryonic stem cells (ESCs) that MLL4 associates with, but is surprisingly dispensable for the maintenance of, active enhancers of cell-identity genes. As a result, MLL4 is dispensable for cell-identity gene expression and self-renewal in ESCs. In contrast, MLL4 is required for enhancer-binding of H3K27 acetyltransferase p300, enhancer activation, and induction of cell-identity genes during ESC differentiation. MLL4 protein, rather than MLL4-mediated H3K4 methylation, controls p300 recruitment to enhancers. We also show that, in somatic cells, MLL4 is dispensable for maintaining cell identity but essential for reprogramming into induced pluripotent stem cells. These results indicate that, although enhancer priming by MLL4 is dispensable for cell-identity maintenance, it controls cell fate transition by orchestrating p300-mediated enhancer activation.
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Cho, Jae-Won, Hyo Sup Shim, Chang Young Lee, et al. "The importance of enhancer methylation for epigenetic regulation of tumorigenesis in squamous lung cancer." Experimental & Molecular Medicine 54, no. 1 (2022): 12–22. http://dx.doi.org/10.1038/s12276-021-00718-4.
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AbstractLung squamous cell carcinoma (LUSC) is a subtype of non-small cell lung cancer (NSCLC). LUSC occurs at the bronchi, shows a squamous appearance, and often occurs in smokers. To determine the epigenetic regulatory mechanisms of tumorigenesis, we performed a genome-wide analysis of DNA methylation in tumor and adjacent normal tissues from LUSC patients. With the Infinium Methylation EPIC Array, > 850,000 CpG sites, including ~350,000 CpG sites for enhancer regions, were profiled, and the differentially methylated regions (DMRs) overlapping promoters (pDMRs) and enhancers (eDMRs) between tumor and normal tissues were identified. Dimension reduction based on DMR profiles revealed that eDMRs alone and not pDMRs alone can differentiate tumors from normal tissues with the equivalent performance of total DMRs. We observed a stronger negative correlation of LUSC-specific gene expression with methylation for enhancers than promoters. Target genes of eDMRs rather than pDMRs were found to be enriched for tumor-associated genes and pathways. Furthermore, DMR methylation associated with immune infiltration was more frequently observed among enhancers than promoters. Our results suggest that methylation of enhancer regions rather than promoters play more important roles in epigenetic regulation of tumorigenesis and immune infiltration in LUSC.
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Gimble, J. M., and E. E. Max. "Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution." Molecular and Cellular Biology 7, no. 1 (1987): 15–25. http://dx.doi.org/10.1128/mcb.7.1.15-25.1987.
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The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.
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Gimble, J. M., and E. E. Max. "Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution." Molecular and Cellular Biology 7, no. 1 (1987): 15–25. http://dx.doi.org/10.1128/mcb.7.1.15.
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The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.
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Hoivik, Erling A., Trine E. Bjanesoy, Oliver Mai, et al. "DNA Methylation of Intronic Enhancers Directs Tissue-Specific Expression of Steroidogenic Factor 1/Adrenal 4 Binding Protein (SF-1/Ad4BP)." Endocrinology 152, no. 5 (2011): 2100–2112. http://dx.doi.org/10.1210/en.2010-1305.
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The nuclear receptor steroidogenic factor 1/adrenal 4 binding protein (SF-1/Ad4BP) is an essential regulator of endocrine development and function, and the expression of the corresponding gene (sf-1/ad4bp) is precisely regulated in a time- and tissue-dependent manner. We previously demonstrated that the basal promoter of sf-1/ad4bp is controlled by DNA methylation and that its methylation status reflects the expression pattern of SF-1/Ad4BP. Recently, three intronic enhancers were identified in the sf-1/ad4bp gene that target SF-1/Ad4BP expression to the fetal adrenal (FAdE; fetal adrenal-specific enhancer), to pituitary gonadotropes (PGE; pituitary gonadotrope-specific enhancer), and to the ventromedial hypothalamic nucleus (VMHE; ventromedial hypothalamic nucleus-specific enhancer). Here, we demonstrate that the activity of these enhancers is correlated with their DNA methylation status. We show that they are hypomethylated in tissues where they are active and generally hypermethylated in tissues where they are not active. Furthermore, we demonstrate in transient transfection experiments that forced DNA methylation represses reporter gene activity driven by these enhancers. These data directly demonstrate a functional significance for the enhancers' methylation status. Intriguingly, further analyses of the basal promoter in gonadotropes revealed that it is methylated in these cells, in contrast to other SF-1/Ad4BP-expressing tissues. Consistent with this, sf-1/ad4bp is transcribed from an alternative promoter in gonadotropes. Taken together, our experiments show that the tissue-specific expression of SF-1/Ad4BP is epigenetically regulated and identify tissue-specific differentially methylated regions within the sf-1/ad4bp locus that are essential for its transcriptional control.
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Liu, Minmin, Guillermo A. Urrutia, Rachel Shereda, Gangning Liang, and Peter A. Jones. "Abstract A010: Dnmt3a2 ensures proper enhancer DNA methylation during embryonic development." Cancer Research 85, no. 3_Supplement (2025): A010. https://doi.org/10.1158/1538-7445.dnamethylation-a010.
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Abstract Proper enhancer function and switching is essential for the development of vertebrates and is known to be controlled by DNA cytosine methylation. We used isoform-specific knockouts of the two known DNA methyltransferase variants, Dnmt3a1 and Dnmt3a2 to probe their roles in enhancer methylation in embryogenesis and in postnatal development. Mouse embryos lacking Dnmt3a1 showed minimal loss of genomic methylation confirming that this isoform is not required for embryonic development. However, they were smaller than their littermates and died about 4 weeks after birth with considerable demethylation in the brain as has been previously reported. On the other hand, embryos lacking Dnmt3a2 showed widespread genomic hypomethylation which was particularly prevalent in enhancers and imprinted genes. Interestingly these methylation deficits were largely repaired after birth, presumably by the remaining Dnmt3a1 isoform. The mice were viable; however, they showed an increased prevalence of sporadic phenotypes including anophthalmia, hydronephrosis, hydrocephalus and male infertility which have previously been observed in laboratory mice. Hypomethylation of several imprinted genes was also observed in sperm which might potentially explain the infertility phenotype. There is therefore an interplay between the 2 isoforms which is developmentally regulated and a key role for Dnmt3a2 might be the stabilization of enhancer states which lessening the chance of sporadic phenotypes arising after birth. Citation Format: Minmin Liu, Guillermo A Urrutia, Rachel Shereda, Gangning Liang, Peter A Jones. Dnmt3a2 ensures proper enhancer DNA methylation during embryonic development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A010.
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Pfeifer, Gerd P. "Switching enhancer methylation in metastatic melanoma." Pigment Cell & Melanoma Research 29, no. 5 (2016): 491–93. http://dx.doi.org/10.1111/pcmr.12505.
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Ordoñez, Martínez-Calle, Agirre, and Prosper. "DNA Methylation of Enhancer Elements in Myeloid Neoplasms: Think Outside the Promoters?" Cancers 11, no. 10 (2019): 1424. http://dx.doi.org/10.3390/cancers11101424.
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Gene regulation through DNA methylation is a well described phenomenon that has a prominent role in physiological and pathological cell-states. This epigenetic modification is usually grouped in regions denominated CpG islands, which frequently co-localize with gene promoters, silencing the transcription of those genes. Recent genome-wide DNA methylation studies have challenged this paradigm, demonstrating that DNA methylation of regulatory regions outside promoters is able to influence cell-type specific gene expression programs under physiologic or pathologic conditions. Coupling genome-wide DNA methylation assays with histone mark annotation has allowed for the identification of specific epigenomic changes that affect enhancer regulatory regions, revealing an additional layer of complexity to the epigenetic regulation of gene expression. In this review, we summarize the novel evidence for the molecular and biological regulation of DNA methylation in enhancer regions and the dynamism of these changes contributing to the fine-tuning of gene expression. We also analyze the contribution of enhancer DNA methylation on the expression of relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms. The characterization of the aberrant enhancer DNA methylation provides not only a novel pathogenic mechanism for different tumors but also highlights novel potential therapeutic targets for myeloid derived neoplasms.
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Izzi, Benedetta, Mariaelena Pistoni, Katrien Cludts, et al. "Allele-specific DNA methylation reinforces PEAR1 enhancer activity." Blood 128, no. 7 (2016): 1003–12. http://dx.doi.org/10.1182/blood-2015-11-682153.
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Key Points Rs12041331 is the first functional CpG-SNP related to platelet function whose regulatory mechanism depends on DNA methylation. Rs12041331 marks allele-specific methylation at the CpG island encompassing the first untranslated exon during megakaryopoiesis.
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Okhovat, M., S. M. Maguire, and S. M. Phelps. "Methylation of avpr1a in the cortex of wild prairie voles: effects of CpG position and polymorphism." Royal Society Open Science 4, no. 1 (2017): 160646. http://dx.doi.org/10.1098/rsos.160646.
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DNA methylation can cause stable changes in neuronal gene expression, but we know little about its role in individual differences in the wild. In this study, we focus on the vasopressin 1a receptor ( avpr1a ), a gene extensively implicated in vertebrate social behaviour, and explore natural variation in DNA methylation, genetic polymorphism and neuronal gene expression among 30 wild prairie voles ( Microtus ochrogaster ). Examination of CpG density across 8 kb of the locus revealed two distinct CpG islands overlapping promoter and first exon, characterized by few CpG polymorphisms. We used a targeted bisulfite sequencing approach to measure DNA methylation across approximately 3 kb of avpr1a in the retrosplenial cortex, a brain region implicated in male space use and sexual fidelity. We find dramatic variation in methylation across the avrp1a locus, with pronounced diversity near the exon–intron boundary and in a genetically variable putative enhancer within the intron. Among our wild voles, differences in cortical avpr1a expression correlate with DNA methylation in this putative enhancer, but not with the methylation status of the promoter. We also find an unusually high number of polymorphic CpG sites (polyCpGs) in this focal enhancer. One polyCpG within this enhancer (polyCpG 2170) may drive variation in expression either by disrupting transcription factor binding motifs or by changing local DNA methylation and chromatin silencing. Our results contradict some assumptions made within behavioural epigenetics, but are remarkably concordant with genome-wide studies of gene regulation.
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Silva, Tiago C., Simon G. Coetzee, Nicole Gull, et al. "ELMER v.2: an R/Bioconductor package to reconstruct gene regulatory networks from DNA methylation and transcriptome profiles." Bioinformatics 35, no. 11 (2018): 1974–77. http://dx.doi.org/10.1093/bioinformatics/bty902.
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Abstract Motivation DNA methylation has been used to identify functional changes at transcriptional enhancers and other cis-regulatory modules (CRMs) in tumors and other disease tissues. Our R/Bioconductor package ELMER (Enhancer Linking by Methylation/Expression Relationships) provides a systematic approach that reconstructs altered gene regulatory networks (GRNs) by combining enhancer methylation and gene expression data derived from the same sample set. Results We present a completely revised version 2 of ELMER that provides numerous new features including an optional web-based interface and a new Supervised Analysis mode to use pre-defined sample groupings. We show that Supervised mode significantly increases statistical power and identifies additional GRNs and associated Master Regulators, such as SOX11 and KLF5 in Basal-like breast cancer. Availability and implementation ELMER v.2 is available as an R/Bioconductor package at http://bioconductor.org/packages/ELMER/. Supplementary information Supplementary data are available at Bioinformatics online.
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Angeloni, Allegra, and Ozren Bogdanovic. "Enhancer DNA methylation: implications for gene regulation." Essays in Biochemistry 63, no. 6 (2019): 707–15. http://dx.doi.org/10.1042/ebc20190030.
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Abstract DNA methylation involves the addition of a methyl group to the fifth carbon of the pyrimidine cytosine ring (5-methylcytosine, 5mC). 5mC is widespread in vertebrate genomes where it is predominantly found within CpG dinucleotides. In mammals, 5mC participates in long-term silencing processes such as X-chromosome inactivation, genomic imprinting, somatic silencing of germline genes, and silencing of repetitive DNA elements. The evidence for 5mC as a dynamic gene-regulatory mechanism is mostly limited to specific examples, and is far from being completely understood. Recent work from diverse model systems suggests that 5mC might not always act as a dominant repressive mechanism and that hypermethylated promoters and enhancers can be permissive to transcription in vivo and in vitro. In this review, we discuss the links between 5mC and enhancer activity, and evaluate the role of this biochemical mechanism in various biological contexts.
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Fluhr, Silvia, Christopher Felix Krombholz, Angelina Meier, Christoph Plass, Charlotte Niemeyer, and Christian Flotho. "Epigenetic Changes Accompany Disordered Hemoglobin Regulation in Juvenile Myelomonocytic Leukemia." Blood 126, no. 23 (2015): 438. http://dx.doi.org/10.1182/blood.v126.23.438.438.
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Abstract Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by massive proliferation of not only the monocytic and granulocytic lineage but also of erythropoietic precursors. Elevated levels of fetal hemoglobin (HbF) are found at time of diagnosis in more than half of JMML cases and are associated with poor outcome. We and others previously found that a key molecular feature of many JMML cases is DNA hypermethylation of distinct target genes, which also has adverse prognostic relevance. It is long known that epigenetic processes are involved in hemoglobin regulation. We therefore hypothesized that epigenetic dysregulation would also be involved in aberrant hemoglobin expression in JMML erythroblasts. ɣ-globin silencing, which is completed one year after birth under physiological conditions, is associated with dense CpG methylation of the ɣ-globin promoter. Consistent with re-expression of ɣ-globin in JMML cases with elevated HbF, CpG methylation at the ɣ-globin promoter was decreased in purified erythroblasts from JMML patients with elevated HbF compared to JMML with normal HbF (average methylation 40.0% ±4.2%, N=6 vs. 72.0% ±3.5%, N=4) (P<0.001). CpG methylation at the ɣ-globin promoter inversely correlated with the relative amount of ɣ-globin expression (ɣ/(β+ɣ)) as determined by RT-qPCR in JMML erythroblasts. Correspondingly, the β-globin promoter is normally unmethylated in erythroblasts from healthy adults, and was also found to be unmethylated in erythroblasts from a JMML patient with normal HbF, whereas it was hypermethylated in JMML with elevated HbF (average methylation 67.0% ±26.0%, N=2). We also assessed CpG methylation at the KLF1 gene which encodes an erythroid-specific transcription factor and strong β-globin activator. We found that the KLF1 enhancer (-593 to -312 relative to the transcription start site of KLF1) and promoter (-182 to +203) regions were unmethylated in healthy erythroblasts irrespective of neonatal or adult origin (N=11; enhancer, 4.5% ±0.9%; promoter, 2.2% ±0.6%), whereas both regions were densely methylated (>80%) in healthy non-erythroid cell populations. By contrast, the KLF1 enhancer and promoter were aberrantly methylated in JMML erythroblasts (N=11; enhancer, 21.2% ±4.7%, P=0.002; promoter, 11.5% ±2.9%, P=0.004). This suggested a regulatory role of KLF1 methylation in aberrant globin expression in JMML. To explore this functionally, we used a dual luciferase reporter assay, ligating the KLF1 enhancer into a CpG-free reporter vector and comparing the reporter activity of unmethylated vector with that of in vitro methylated vector after transfection into K562 cells. CpG methylation at the KLF1 enhancer abolished its activity, supporting methylation-associated silencing of KLF1 in JMML erythroblasts. Compatible with this, KLF1 transcript levels were reduced 2.1-fold in JMML erythroblasts (N=12) compared to healthy erythroblasts (N=9) as assessed by RT-qPCR (P=0.018). We additionally assessed the protein expression of the direct KLF1 target CD44 on the surface of JMML erythroblasts by FACS analysis. CD44 expression was 2.4-fold lower in JMML erythroblasts with high KLF1 enhancer methylation compared to JMML erythroblasts with normal KLF1 enhancer methylation. In summary, we show that multiple layers of hemoglobin regulation are affected by epigenetic changes in JMML erythroblasts. The globin genes themselves are targets of aberrant DNA methylation in JMML erythroblasts with elevated HbF. Moreover, the β-globin activator KLF1 is aberrantly methylated and repressed in JMML erythroblasts. These findings provide for the first time a mechanistic explanation for the strong correlation and uniform prognostic relevance of elevated HbF and gene hypermethylation in JMML. Disclosures No relevant conflicts of interest to declare.
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He, Yupeng, David U. Gorkin, Diane E. Dickel, et al. "Improved regulatory element prediction based on tissue-specific local epigenomic signatures." Proceedings of the National Academy of Sciences 114, no. 9 (2017): E1633—E1640. http://dx.doi.org/10.1073/pnas.1618353114.
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Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared with existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types. REPTILE is available at https://github.com/yupenghe/REPTILE/.
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Lorincz, Matthew C., Dirk Schübeler, Shauna R. Hutchinson, David R. Dickerson, and Mark Groudine. "DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation." Molecular and Cellular Biology 22, no. 21 (2002): 7572–80. http://dx.doi.org/10.1128/mcb.22.21.7572-7580.2002.
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ABSTRACT DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.
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Schübeler, Dirk, Matthew C. Lorincz, Daniel M. Cimbora, et al. "Genomic Targeting of Methylated DNA: Influence of Methylation on Transcription, Replication, Chromatin Structure, and Histone Acetylation." Molecular and Cellular Biology 20, no. 24 (2000): 9103–12. http://dx.doi.org/10.1128/mcb.20.24.9103-9112.2000.
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ABSTRACT We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.
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Jain, Siddhant U., Sima Khazaei, Dylan M. Marchione, et al. "Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression." Proceedings of the National Academy of Sciences 117, no. 44 (2020): 27354–64. http://dx.doi.org/10.1073/pnas.2006076117.
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A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.
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Mosella, M. Q. S., T. S. Sabedot, T. M. Malta, et al. "P13.15 DNA methylation abnormalities in non-promoter regulatory regions are associated with invasive behavior in pituitary tumors." Neuro-Oncology 21, Supplement_3 (2019): iii65—iii66. http://dx.doi.org/10.1093/neuonc/noz126.236.
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Abstract BACKGROUND Despite histologically benign, pituitary tumors (PT) may invade important adjacent neurovascular structures which can incur in significant comorbidities preventing a complete surgical resection and contributing to resistance to medical treatment. DNA methylation clearly stratified PT based on their functional status i.e. nonfunctioning PTs (NFPTs) from functioning PT (FPTs). However associations of methylation aberrations with invasive behavior is less clear. MATERIAL AND METHODS In order to evaluate whether DNA methylation alterations in regulatory regions other than promoter and coding regions are associated with invasive behavior we performed a meta-analysis of the genome-wide methylome of three public available PT cohorts plus our own (Illumina HumanMethylation platforms- 450K/EPIC). Pituitary specimens comprised of 43 invasive pituitary tumors (InvPT) and 37 noninvasive (NInvPT); 12 FPT and 68 NFPTs, in addition to 20 non-tumor pituitaries. RNA-seq data were available for one cohort (n=23, 12 InvPT,11NInvPT) and integrated with DNA methylation. Invasiveness criteria was based on Knosp grade >= 2 and/or sphenoid or dural invasion. RESULTS Wilcoxon Rank-sum test; Δβ=0.15; p-value <0.001 identified 58 differentially methylated CpG sites in InvPT that were mainly hypomethylated (95%) in relation to NInvPT. NInvPT methylation profile was similar to non-tumor specimens, despite its heterogeneity. Thirty-four percent (n=20) of the differentially methylated CpG sites were located within predicted enhancer regions distributed in intronic (40%), intergenic (40%) and promoter (20%) regions. Predicted enhancer-target genes were enriched for actin filament cell movement, response to starvation, growth factor stimulus and protein autophosporilation pathways. Among them, ZNF625 and INO80E were found mostly negative correlated among methylation and expression data (-0.50 and -0.48, respectively), besides DOC2A found to be one potentially differentially expressed gene under enhancer control (log2FC > 0.2, pvalue <0.05). CONCLUSION Our results suggest that methylation alterations in predicted regulatory regions, such as enhancers, annotated in non-promoter regions (introns and intergenic) may contribute to the invasive behavior of PT.
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Michelle, Rönnerblad, Olofsson Tor, Iyadh Douagi, et al. "Analysis of DNA Methylation and Transcription During Granulopoiesis Reveals Timed Methylation Changes in Low CpG Areas That Correlate with Changed Transcriptional Activity." Blood 120, no. 21 (2012): 2334. http://dx.doi.org/10.1182/blood.v120.21.2334.2334.
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Abstract Abstract 2334 Accumulating evidence demonstrates that epigenetic changes, including DNA methylation play a central role in differentiation, providing cellular memory and stabilizing lineage choice in hematopoiesis1–3. DNA methylation is an important epigenetic mechanism involved in transcriptional regulation, heterochromatin formation and the normal development of many organisms. In this study we investigated the DNA methylome and transcriptome of human cells in four separate differentiation stages in granulopoiesis, ranging from the multipotent Common Myeloid progenitor (CMP) to terminally differentiated bone marrow neutrophils (PMN). To this end we employed HumanMethylation 450 BeadChip (450K array) from Illumina with extensive genomic coverage and mRNA expression arrays (Illumina). Temporally distinct methylation changes during granulopoiesis Differential methylation between two cell stages was defined as an average difference in β value of at least 0.17 (p ≤ 0.05). We detected 12132 DMSs during granulopoiesis. Of these the majority showed decreased methylation during granulopoeisis (10771 CpGs) and a smaller set gained methylation (1658 CpGs). Strikingly, increases in methylation predominantly occur between CMP and GMP, the two least mature cell types. Some CpGs also show increased methylation in the GMP-PMC transition, while very few CpG sites increase at the final stage of differentiation from PMC to PMN. Although reduction of methylation occurs at all stages of granulopoiesis, the greatest change is between GMP and PMC. It is striking that the DNA methylation patterns preferentially change at points of lineage restriction, and that the greatest change occurs upon loss of oligopotency between GMP and PMC. DMSs within CGIs were greatly underrepresented (p<0.001 with chi-square test), while DMSs were overrepresented in shelves (p<0.001) and open sea (p<0.001). Thus, methylation appears to be more dynamic outside of CGIs during granulocytic development. For all regions the variation within enhancers was greater than outside of enhancers indicating greater methylation changes in enhancers compared to non-enhancers. In addition, CpGs in enhancer regions are significantly enriched in the list of DMSs (p<0.001, chi-square test) further supporting the observation that enhancer regions display dynamic DNA methylation changes during granulopoiesis. Changes in gene expression correlate with DNA methylation changes There was a significant overlap between genes showing decreased methylation and genes with increased expression as well as for the reverse comparison between genes with increased methylation and decreased expression. Thus, support a general anticorrelation between DNA methylation and gene expression. Azurophilic granule proteins showed increased expression peaking in PMC and a rapid decrease toward PMN. CpG methylation levels for those genes decreased concomitantly with the peak in expression. We report cell population specific changes of DNA methylation levels. The main reduction of CpG methylation coincides with the loss of oligopotency at the transition from GMP-PMC. This suggests a role of DNA methylation in regulating cell plasticity and lineage choice. Disclosures: No relevant conflicts of interest to declare.
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Chu, Michelle, and Mark H. Kaplan. "TL1A promotes a multi-cytokine Th9 cell phenotype." Journal of Immunology 208, no. 1_Supplement (2022): 56.06. http://dx.doi.org/10.4049/jimmunol.208.supp.56.06.
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Abstract The TNF superfamily member TL1A is a costimulatory molecule that signals through its receptor DR3 on T lymphocytes. The Th9 subset of T lymphocytes secretes the pleiotropic cytokine IL-9 which has functions in allergic airway disease, helminth infections, and tumor immunity. TL1A signaling has been shown to increase IL-9 production by Th9 cells. However, its role in regulating other functions of Th9 cells is unknown. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 as well as the frequency of IL-9 and IL-13 co-expressing cells in Th9 cell cultures through flow cytometric analyses. We also show that the Il9 and Il13 promoter and enhancer regions are differentially accessible in response to TL1A over a five-day culture period through chromatin accessibility assays. At the Il9 locus, TL1A enhances binding of IRF4, BATF, and PPARg. Mechanistically, this is linked to decreased H3K9 tri-methylation and increased H3K4 tri-methylation at Il9 enhancer region CNS2. At the Il13 locus, TL1A enhances binding of BATF at the Il13 promoter alongside decreased H3K9 tri-methylation and increased H3K4 tri-methylation. Ongoing experiments define the function of TL1A-induced multi-cytokine producing cells. Together, these data indicate that TL1A contributes to heterogeneity of IL-9-secreting T cell populations. Supported by NIH Combined Adult and Pediatrics Pulmonary Research Training Program T32
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Satyamoorthy, K., K. Park, M. L. Atchison, and C. C. Howe. "The intracisternal A-particle upstream element interacts with transcription factor YY1 to activate transcription: pleiotropic effects of YY1 on distinct DNA promoter elements." Molecular and Cellular Biology 13, no. 11 (1993): 6621–28. http://dx.doi.org/10.1128/mcb.13.11.6621-6628.1993.
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Murine intracisternal A-particle long terminal repeats contain an intracisternal A-particle upstream enhancer (IUE) element that binds to a 65-kDa IUE binding protein (IUEB) present in both undifferentiated F9 embryonal carcinoma cells and differentiated parietal endoderm-like PYS-2 cells. This IUE element confers a CpG methylation-sensitive IUEB binding and enhancer activity. Using gel retardation, methylation interference, CpG methylation sensitivity binding, and cotransfection assays, we have now identified the 65-kDa IUEB as YY1 (also called NF-E1, delta, or UCRBP), a zinc finger protein related to the Krüppel family. YY1 binds to a number of similar but distinct DNA motifs, and cotransfection assays indicate that these motifs have different enhancer potentials in PYS-2 cells. The relative strengths of these elements are as follows: IUE > kappa E3' from the human immunoglobulin kappa light-chain 3' enhancer > upstream conserved region from the Moloney murine leukemia virus promoter. Results of DNA binding assays suggest that the differences in enhancer potentials are due to the different binding affinities of YY1 to the various motifs and the binding of two other transcription factors to the IUE sequence.
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Satyamoorthy, K., K. Park, M. L. Atchison, and C. C. Howe. "The intracisternal A-particle upstream element interacts with transcription factor YY1 to activate transcription: pleiotropic effects of YY1 on distinct DNA promoter elements." Molecular and Cellular Biology 13, no. 11 (1993): 6621–28. http://dx.doi.org/10.1128/mcb.13.11.6621.
Full textAbstract:
Murine intracisternal A-particle long terminal repeats contain an intracisternal A-particle upstream enhancer (IUE) element that binds to a 65-kDa IUE binding protein (IUEB) present in both undifferentiated F9 embryonal carcinoma cells and differentiated parietal endoderm-like PYS-2 cells. This IUE element confers a CpG methylation-sensitive IUEB binding and enhancer activity. Using gel retardation, methylation interference, CpG methylation sensitivity binding, and cotransfection assays, we have now identified the 65-kDa IUEB as YY1 (also called NF-E1, delta, or UCRBP), a zinc finger protein related to the Krüppel family. YY1 binds to a number of similar but distinct DNA motifs, and cotransfection assays indicate that these motifs have different enhancer potentials in PYS-2 cells. The relative strengths of these elements are as follows: IUE > kappa E3' from the human immunoglobulin kappa light-chain 3' enhancer > upstream conserved region from the Moloney murine leukemia virus promoter. Results of DNA binding assays suggest that the differences in enhancer potentials are due to the different binding affinities of YY1 to the various motifs and the binding of two other transcription factors to the IUE sequence.
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Cheng, Donghang, Vidyasiri Vemulapalli, Yue Lu, et al. "CARM1 methylates MED12 to regulate its RNA-binding ability." Life Science Alliance 1, no. 5 (2018): e201800117. http://dx.doi.org/10.26508/lsa.201800117.
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The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain–containing effector molecule, TDRD3. Chromatin immunoprecipitation–seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.
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Bueno-Costa, Alberto, David Piñeyro, Marta Soler, Biola Maria Javierre, José Angel Martínez-Climent, and Manel Esteller. "Induced Transdifferentiation of Leukemia B-Cells to Macrophages Involves Reconfiguration of the DNA Methylome." Blood 132, Supplement 1 (2018): 5115. http://dx.doi.org/10.1182/blood-2018-99-111580.
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Abstract The epigenomic changes that occur during the process of cellular differentiation, such as in the blood cell lineage, are currently not well understood, especially at distant regulatory regions such as enhancer sequences. To study the effects of DNA methylation on cellular (de)differentiation, we have used a human B Cell Acute Lymphoblastic Leukemia model of transdifferentiation (BLaER1), which has an estradiol-inducible CEBPA construct that allows the conversion of leukemic B cells to non-tumorigenic macrophage-like cells. By analyzing the DNA methylation landscape of these cells at different time points of transdifferentiation with an Illumina EPIC methylation array, we have found enhancer-associated CpGs that shifts their methylation levels at the end of the transdifferentiation. By merging these results with the data obtained by Genome-wide Chromosome Conformation Capture Capture (Hi-C) in naive B cells and macrophages, we studied the putative interaction between several gene-promoters and our differentially methylated CpGs. We then proceed further to characterize the impact of the observed interactions on gene expression. We have identified the DNA methylation dependent enhancer interactomes of B-cells and macrophages. These target genes are related with vesicle trafficking, endocytosis and immune response. Our data highlight the role of DNA methylation to determine cell identity in the blood cell lineage. Disclosures No relevant conflicts of interest to declare.
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Xuan, C., L. Jiankang, J. Tao, and L. Hailong. "P16.09.A TGFB1 INDUCED TET3 DEPENDENT REGULATION OF OTX2 SUPER ENHANCER HYPOMETHYLATION PROMOTES GROUP3 MEDULLOBLASTOMA PROGRESSION." Neuro-Oncology 26, Supplement_5 (2024): v85—v86. http://dx.doi.org/10.1093/neuonc/noae144.284.
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Abstract BACKGROUND DNA methylation patterns have been extensively utilized for medulloblastoma (MB) classification. However, the functional implications of enhancer methylation in MB remain poorly understood. METHODS We conducted bisulfite-based genome-wide sequencing on 189 human MB cases. Upon molecular classification, 80 cases was compared against 8 normal cerebellums, revealing differentially methylated regions (DMRs). Integration with gene expression data facilitated the identification of DMR target genes, subsequently validated in the 109 cases. Further, leveraging sn-ATACseq, we evaluated the association between DMRs in chromatin accessibility regions and tumorigenesis. Finally, we investigatedd the impact of upstream epigenetic events involving aberrant methylation on tumor development. RESULTS We observed a negative correlation between the methylation levels of DMR overlapping super enhancers (seDMRs) and the expression of targeted genes, particularly in Group_3 MB (G3-MB), the most aggressive subgroup. Genes targeted by hypomethylated seDMRs were significantly enriched in early-stage cerebellum development hallmarks, specifically the rhombic lip subventricular zone (RLSVZ), but not in the late stage. Hypomethylated seDMRs of OTX2 were associated with elevated chromatin accessibility and oncogenic activation of progenitor-like MB tumor cells, leading to worse prognosis. Integrative epigenetic analysis revealed highly active OTX2 binding sites within its own hypomethylated seDMRs, indicating feedback auto-regulation. Deletion of motif at this enhancer region reduced OTX2 expression and attenuated tumor progression. Further, we found that ten-eleven translocation 3 (TET3), a DNA methylation eraser, was recruited by OTX2 to catalyze active DNA demethylation at binding motifs, thereby organizing chromatin and stimulating gene expression. TET3 was upregulated by TGF-β1/Smad2 signaling. Based on these findings, nanoparticle-coated small interfering RNAs (siRNA) targeting TET3 were designed, effectively suppressing xenograft progression. CONCLUSION Our study highlights the increased transcriptional activity of OTX2 through cell-type-specific super enhancer demethylation, which is dependent on TGF-β1/Smad-induced TET3. This identifies a potential therapeutic strategy for G3-MB.
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Yoon, Young Soo, Sangkyun Jeong, Qi Rong, Kye-Yoon Park, Jae Hoon Chung, and Karl Pfeifer. "Analysis of the H19ICR Insulator." Molecular and Cellular Biology 27, no. 9 (2007): 3499–510. http://dx.doi.org/10.1128/mcb.02170-06.
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ABSTRACT Transcriptional insulators are specialized cis-acting elements that protect promoters from inappropriate activation by distal enhancers. The H19 imprinting control region (ICR) functions as a CTCF-dependent, methylation-sensitive transcriptional insulator. We analyzed several insertional mutations and demonstrate that the ICR can function as a methylation-regulated maternal chromosome-specific insulator in novel chromosomal contexts. We used chromosome conformation capture and chromatin immunoprecipitation assays to investigate the configuration of cis-acting elements at these several insertion sites. By comparing maternal and paternal organizations on wild-type and mutant chromosomes, we hoped to identify mechanisms for ICR insulator function. We found that promoter and enhancer elements invariably associate to form DNA loop domains at transcriptionally active loci. Conversely, active insulators always prevent these promoter-enhancer interactions. Instead, the ICR insulator forms novel loop domains by associating with the blocked promoters and enhancers. We propose that these associations are fundamental to insulator function.
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Pacis, Alain, Florence Mailhot-Léonard, Ludovic Tailleux, et al. "Gene activation precedes DNA demethylation in response to infection in human dendritic cells." Proceedings of the National Academy of Sciences 116, no. 14 (2019): 6938–43. http://dx.doi.org/10.1073/pnas.1814700116.
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DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.
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Oka, Rurika, Johan Zicola, Blaise Weber, et al. "Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize." Genome Biology 18, no. 1 (2017): 137. https://doi.org/10.1186/s13059-017-1273-4.
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<strong>Background: </strong>While most cells in multicellular organisms carry the same genetic information, in each cell type only a subset of genes is being transcribed. Such differentiation in gene expression depends, for a large part, on the activation and repression of regulatory sequences, including transcriptional enhancers. Transcriptional enhancers can be located tens of kilobases from their target genes, but display characteristic chromatin and DNA features, allowing their identification by genome-wide profiling. Here we show that integration of chromatin characteristics can be applied to predict distal enhancer candidates in <i>Zea mays</i>, thereby providing a basis for a better understanding of gene regulation in this important crop plant.<strong>Result: </strong>To predict transcriptional enhancers in the crop plant maize (<i>Zea mays</i> L. ssp. mays), we integrated available genome-wide DNA methylation data with newly generated maps for chromatin accessibility and histone 3 lysine 9 acetylation (H3K9ac) enrichment in young seedling and husk tissue. Approximately 1500 intergenic regions, displaying low DNA methylation, high chromatin accessibility and H3K9ac enrichment, were classified as enhancer candidates. Based on their chromatin profiles, candidate sequences can be classified into four subcategories. Tissue-specificity of enhancer candidates is defined based on the tissues in which they are identified and putative target genes are assigned based on tissue-specific expression patterns of flanking genes.<strong>Conclusions: </strong>Our method identifies three previously identified distal enhancers in maize, validating the new set of enhancer candidates and enlarging the toolbox for the functional characterization of gene regulation in the highly repetitive maize genome.
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Shull, Austin Y., Junfeng Luo, Lirong Pei, et al. "DNA Hypomethylation within B-Cell Enhancers and Super Enhancers Reveal a Dependency on Immune and Metabolic Mechanisms in Chronic Lymphocytic Leukemia." Blood 128, no. 22 (2016): 1049. http://dx.doi.org/10.1182/blood.v128.22.1049.1049.
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Abstract Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response mediated by epigenetic mechanisms. In this study, we sought to better characterize the complexities of the CLL transcriptional profile by conducting an integrative analysis between the B cell enhancer and super enhancer signatures defined from 3 B cell H3K27Ac ChIPseq samples (CD19+ B cell, GM12878, and MEC1), the DNA methylation signatures defined from reduced-representation bisulfite sequencing (RRBS) of 42 CLL patient and 8 healthy donor samples, and the mRNA expression signatures defined from RNA sequencing of 47 CLL patient and 5 healthy donor samples. From our analysis, we identified super enhancers (SEs) in each of the ChIPseq profiles (approximately 4% of called enhancers) and discovered 741 SEs in GM12878, 374 SEs in MEC1, and 523 SEs in the CD19+ B cell profiles, respectively. Based on MSigDB gene ontology analysis, many of the genes corresponding with SEs were involved in pathways regulating immune signaling activation (e.g. TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY RESPONSE) or metabolic homeostasis (e.g. MTORC1_SIGNALING, FATTY_ACID_METABOLISM). By further analyzing the corresponding expression level of SE-associated genes in CLL patients, we identified 190 transcripts associated with SEs that were significantly overexpressed in CLL patient B cells (Student's t-test p<0.05), and this overexpressed subset of SE-associated transcripts was enriched in genes involved in either immune signaling (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). Based on the differential expression of genes associated with enhancers occurring between CLL patient and healthy donor B cells, we then wanted to determine whether differential DNA methylation within enhancers corresponded with upregulation of CLL transcripts. Based on differential DNA methylation (DM) analysis (methylation difference +/- .25; Student's t-test p-value<0.05) from our RRBS samples, we discovered 744 DM CpG sites that overlapped within our identified B cell enhancers, and most of the DM CpG sites in CLL were significantly hypomethylated (avg. DM GpG difference: enhancer = -0.40; non-enhancer = -0.08). Examples of hypomethylated enhancers included super enhancers corresponding with overexpressed transcripts ENO2, SEPT9, RXRA, and CCR7 as well as a typical enhancer that corresponded with the overexpressed transcript PDCD1. Based on the derived information from our integrative analysis of B cell enhancers, we then compared the effects of preferentially targeting enhancer-mediated expression with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. Based on in vitro assays and RNAseq expression analysis comparing THZ1 and JQ1-treated CLL cell lines MEC1 and MEC2, we saw that JQ1 could inhibit CLL cell line proliferation, suppress IgM-mediated primary CLL proliferation, and differentially disrupt transcription of genes involved in immune signaling cascades. Contrastingly, we saw that THZ1 elicited a different response in CLL cell lines and primary cells by disrupting cell viability, inducing apoptosis, and differentially downregulating genes involved in metabolic homeostasis. The specific enhancer-associated genes disrupted by the respective treatments further highlight the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 selectively suppressed the B cell activation marker gene FCER2 and the PD-1 receptor gene PDCD1, whereas THZ1 selectively suppressed the glycolytic enolase gene ENO2 and the proto-oncogene FGR. Collectively, these results reveal how CLL DNA hypomethylation within B cell enhancers can mediate immune signaling and metabolic expression signatures in CLL and can differentially be disrupted by BET bromodomain or CDK7 inhibition. Disclosures Awan: Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy.
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Saha, Debapriya, Allison B. Norvil, Nadia A. Lanman, and Humaira Gowher. "Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells." Epigenomes 4, no. 4 (2020): 24. http://dx.doi.org/10.3390/epigenomes4040024.
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Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.
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Koroleva, Yu A., A. V. Markov, I. A. Goncharova, et al. "Deoxyribonucleic acid methylation in the enhancer region of the CDKN2A/2B and CDKN2B-AS1 genes in blood vessels and cells in patients with carotid atherosclerosis." Russian Journal of Cardiology 25, no. 10 (2020): 4060. http://dx.doi.org/10.15829/1560-4071-2020-4060.
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Aim. Comparative analysis of the deoxyribonucleic acid (DNA) methylation level in the enhancer region of the CDKN2A/2B and CDKN2B-AS1 genes (9p21.3 locus) in vessels with/without atherosclerotic lesions, as well as in leukocytes of patients with clinically relevant carotid artery (CA) atherosclerosis and healthy individuals.Material and methods. The group of patients with clinically relevant atherosclerosis included 22 individuals with severe stenosis (>80%) of CA. Samples of atherosclerotic plaques, presenting CA regions, and great saphenous veins, as well as peripheral blood samples (leukocytes) were obtained from patients. The control group consisted of 14 individuals with the mild CA stenosis (£24%) and without hemodynamically relevant changes; peripheral blood samples were obtained from each of them. DNA methylation level was assessed by targeted bisulfite sequencing of amplicons.Results. The tissue-specific methylation of 31 CpG-site in the CDKN2A/2B and CDKN2B-AS1 gene enhancer was established: the vascular tissues significantly differed from the peripheral blood leukocytes. At the same time, there was an increase in the methylation level of both certain CpG sites and whole analyzed CA region affected by atherosclerosis (48,6 [34,8; 62,0]%), compared with intact vessels, both arteries (25,2 [23,1; 41,60]%, p=0,0001) and veins (35,0 [31,6; 40,0]%, p=0,0039). Patients had lower methylation levels in all CpG sites in blood leukocytes compared to blood vessel samples (8,7 [6,1; 9,7]%; p<0,05). At the same time, the level of DNA methylation in the blood leukocytes of atherosclerotic patients does not differ from that in healthy individuals (9,3 [8,3; 13,6]%; p>0,8).Conclusion. In the present study, the relationship between an increase in the DNA methylation in the enhancer of the CDKN2A/2B and CDKN2B-AS1 genes in CA and their atherosclerotic lesions was revealed, as well as the tissue-specific DNA methylation between vessels and peripheral blood leukocytes.
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Rönnerblad, Michelle, Robin Andersson, Tor Olofsson, et al. "Analysis of the DNA methylome and transcriptome in granulopoiesis reveals timed changes and dynamic enhancer methylation." Blood 123, no. 17 (2014): e79-e89. http://dx.doi.org/10.1182/blood-2013-02-482893.
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Key Points In granulopoiesis, changes in DNA methylation preferably occur at points of lineage restriction in low CpG areas. DNA methylation is dynamic in enhancer elements and appears to regulate the expression of key transcription factors and neutrophil genes.
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Chang, Chu-Yuan, Jui-Hung Hung, Liang-Wei Huang, et al. "Epigenetic Regulation of WNT3A Enhancer during Regeneration of Injured Cortical Neurons." International Journal of Molecular Sciences 21, no. 5 (2020): 1891. http://dx.doi.org/10.3390/ijms21051891.
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Traumatic brain injury is known to reprogram the epigenome. Chromatin immunoprecipitation-sequencing of histone H3 lysine 27 acetylation (H3K27ac) and tri-methylation of histone H3 at lysine 4 (H3K4me3) marks was performed to address the transcriptional regulation of candidate regeneration-associated genes. In this study, we identify a novel enhancer region for induced WNT3A transcription during regeneration of injured cortical neurons. We further demonstrated an increased mono-methylation of histone H3 at lysine 4 (H3K4me1) modification at this enhancer concomitant with a topological interaction between sub-regions of this enhancer and with promoter of WNT3A gene. Together, this study reports a novel mechanism for WNT3A gene transcription and reveals a potential therapeutic intervention for neuronal regeneration.
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Kelley, D. E., B. A. Pollok, M. L. Atchison, and R. P. Perry. "The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated." Molecular and Cellular Biology 8, no. 2 (1988): 930–37. http://dx.doi.org/10.1128/mcb.8.2.930-937.1988.
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Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.
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Kelley, D. E., B. A. Pollok, M. L. Atchison, and R. P. Perry. "The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated." Molecular and Cellular Biology 8, no. 2 (1988): 930–37. http://dx.doi.org/10.1128/mcb.8.2.930.
Full textAbstract:
Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.
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Canon, E., L. Jouneau, T. Blachère, et al. "Progressive methylation of POU5F1 regulatory regions during blastocyst development." Reproduction 156, no. 2 (2018): 145–61. http://dx.doi.org/10.1530/rep-17-0689.
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ThePOU5F1gene encodes one of the ‘core’ transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5′ regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere withPOU5F1expression and function inin vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends onde novomethylation of its promoter. However, little is known about the epigenetic regulation ofPOU5F1expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of thePOU5F15′ upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5′ regulatory region and the first exon accompanying differentiation and the gradual repression ofPOU5F1. Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.
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Qu, Ying, Lee Siggens, Lina Cordeddu, et al. "Cancer-specific changes in DNA methylation reveal aberrant silencing and activation of enhancers in leukemia." Blood 129, no. 7 (2017): e13-e25. http://dx.doi.org/10.1182/blood-2016-07-726877.
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Key Points DNA demethylation activates new and poised enhancers in AML that cause a leukemic transcriptome. Only a subset of DNA demethylated enhancers becomes activated. A specific additional activation step is required for enhancer activation.
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Sugimoto, Jun, Danny J. Schust, Makiko Sugimoto, Yoshihiro Jinno, and Yoshiki Kudo. "Controlling Trophoblast Cell Fusion in the Human Placenta—Transcriptional Regulation of Suppressyn, an Endogenous Inhibitor of Syncytin-1." Biomolecules 13, no. 11 (2023): 1627. http://dx.doi.org/10.3390/biom13111627.
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Cell fusion in the placenta is tightly regulated. Suppressyn is a human placental endogenous retroviral protein that inhibits the profusogenic activities of another well-described endogenous retroviral protein, syncytin-1. In this study, we aimed to elucidate the mechanisms underlying suppressyn’s placenta-specific expression. We identified the promoter region and a novel enhancer region for the gene encoding suppressyn, ERVH48-1, and examined their regulation via DNA methylation and their responses to changes in the oxygen concentration. Like other endogenous retroviral genes, the ERVH48-1 promoter sequence is found within a characteristic retroviral 5′ LTR sequence. The novel enhancer sequence we describe here is downstream of this LTR sequence (designated EIEs: ERV internal enhancer sequence) and governs placental expression. The placenta-specific expression of ERVH48-1 is tightly controlled by DNA methylation and further regulated by oxygen concentration-dependent, hypoxia-induced transcription factors (HIF1α and HIF2α). Our findings highlight the involvement of (1) tissue specificity through DNA methylation, (2) expression specificity through placenta-specific enhancer regions, and (3) the regulation of suppressyn expression in differing oxygen conditions by HIF1α and HIF2α. We suggest that these regulatory mechanisms are central to normal and abnormal placental development, including the development of disorders of pregnancy involving altered oxygenation, such as preeclampsia, pregnancy-induced hypertension, and fetal growth restriction.
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Schmidl, C., M. Klug, T. J. Boeld, et al. "Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity." Genome Research 19, no. 7 (2009): 1165–74. http://dx.doi.org/10.1101/gr.091470.109.
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Britsemmer, Jan H., Christin Krause, Natalie Taege, et al. "Fatty Acid Induced Hypermethylation in the Slc2a4 Gene in Visceral Adipose Tissue Is Associated to Insulin-Resistance and Obesity." International Journal of Molecular Sciences 24, no. 7 (2023): 6417. http://dx.doi.org/10.3390/ijms24076417.
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De novo lipogenesis (DNL) in visceral adipose tissue (VAT) is associated with systemic insulin sensitivity. DNL in VAT is regulated through ChREBP activity and glucose uptake through Glut4 (encoded by Slc2a4). Slc2a4 expression, ChREBP activity, and DNL are decreased in obesity, the underlying cause however remains unidentified. We hypothesize that increased DNA methylation in an enhancer region of Slc2a4 decreases Slc2a4 expression in obesity and insulin resistance. We found that SLC2A4 expression in VAT of morbidly obese subjects with high HbA1c (>6.5%, n = 35) is decreased, whereas DNA methylation is concomitantly increased compared to morbidly obese subjects with low HbA1c (≤6.5%, n = 65). In diet-induced obese (DIO) mice, DNA methylation of Slc2a4 persistently increases with the onset of obesity and insulin resistance, while gene expression progressively decreases. The regulatory impact of DNA methylation in the investigated enhancer region on SLC2A4 gene expression was validated with a reporter gene assay. Additionally, treatment of 3T3 pre-adipocytes with palmitate/oleate during differentiation decreased DNA methylation and increased Slc2a4 expression. These findings highlight a potential regulation of Slc2a4 by DNA methylation in VAT, which is induced by fatty acids and may play a role in the progression of obesity and insulin resistance in humans.
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Ankill, Jørgen, Zhi Zhao, Xavier Tekpli, et al. "Integrative pan-cancer analysis reveals a common architecture of dysregulated transcriptional networks characterized by loss of enhancer methylation." PLOS Computational Biology 20, no. 11 (2024): e1012565. http://dx.doi.org/10.1371/journal.pcbi.1012565.
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Aberrant DNA methylation contributes to gene expression deregulation in cancer. However, these alterations’ precise regulatory role and clinical implications are still not fully understood. In this study, we performed expression-methylation Quantitative Trait Loci (emQTL) analysis to identify deregulated cancer-driving transcriptional networks linked to CpG demethylation pan-cancer. By analyzing 33 cancer types from The Cancer Genome Atlas, we identified and confirmed significant correlations between CpG methylation and gene expression (emQTL) in cis and trans, both across and within cancer types. Bipartite network analysis of the emQTL revealed groups of CpGs and genes related to important biological processes involved in carcinogenesis including proliferation, metabolism and hormone-signaling. These bipartite communities were characterized by loss of enhancer methylation in specific transcription factor binding regions (TFBRs) and the CpGs were topologically linked to upregulated genes through chromatin loops. Penalized Cox regression analysis showed a significant prognostic impact of the pan-cancer emQTL in many cancer types. Taken together, our integrative pan-cancer analysis reveals a common architecture where hallmark cancer-driving functions are affected by the loss of enhancer methylation and may be epigenetically regulated.
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Johnson, Kevin C., Kevin Anderson, Elise Courtois, et al. "GENE-40. CHARACTERIZING EPIGENETIC INTRATUMORAL HETEROGENEITY IN GLIOMA USING SINGLE-CELL BISULFITE SEQUENCING." Neuro-Oncology 21, Supplement_6 (2019): vi106. http://dx.doi.org/10.1093/neuonc/noz175.442.
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Abstract Genetic and epigenetic alterations contribute to the observed intratumoral heterogeneity in adult glioma. Current glioma classification, based on genotype (e.g., IDH1 mutations) and DNA methylation profiles (e.g., glioma CpG Island Methylator Phenotype), can provide clinically relevant tumor subgroups. However, traditional bulk sampling fails to adequately capture the full complement of epigenomic heterogeneity, and may mask deadly features present in less abundant glioma cells. To more precisely characterize the glioma epigenome, we separately profiled single-cell DNA methylation (Reduced Representation Bisulfite Sequencing, RRBS), single-cell RNA expression (10X genomics), and bulk whole genome sequencing in nine gliomas. The genomic regions profiled by scRRBS were primarily gene promoters, but adequate coverage was also reached for glioma-specific enhancer elements and binding sites of chromatin remodelers. Unsupervised clustering of single-cell DNA methylation data revealed intratumoral variability in epigenetic classification and these cell types were distinguished by regulatory element DNA methylation. We further integrated single-cell epigenetic, single-cell transcriptomic, and genomic features to better understand gene regulation and reconstruct each tumor’s lineage history. Together, our study aims to generate a glioma cellular hierarchy shaped by the epigenetic programs that drive tumor growth.