Tet2-dependent processing of 5-methylcytosine and the maintenance of active higher order structures during differentiation.
Final Report Abstract
Lineage specific differentiation in the developing embryo is accompanied by extensive reprogramming of the epigenome, resulting in the repression of sternness specific factors, regulators of other lineages and the transcriptional maintenance of activated lineage-specific genes. DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification. We analysed the role of DNA methylation and the enzymes necessary for regulating this mark during mammalian development. The major goal was to elucidate how this modification affects the expression of stem cell specific and lineage specific genes via higher order chromatin structures. In previous studies we have used the mammalian Hoxa cluster as a model system to follow epigenetic changes during differentiation. We found the inactive cluster to be marked by defined patterns of 5-methylcytosine (5mC), that where progressively converted to 5hmC during induced differentiation by proteins of the Ten-Eleven-Translocation (Tet) family. We found the maintenance of activated Hoxa transcription significantly disturbed in Tet-deficient cells, suggesting that gene specific 5mC-5hmC conversion by Tet proteins is crucial for the maintenance of active chromatin states, most likely by triggering active demethylation pathways. Using Tet1/2 double knockout (DKO) mice and Tet1/2/3 triple knockout (TKO) embryonic stem cells (ESCs) we found that combined deficiencies of Tet proteins greatly reduced 5hmC levels, increased global 5mC and impaired differentiation. DKO embryos exhibited various developmental abnormalities, whereas TKO ESCs showed severe differentiation defects caused by promoter hypermethylation and deregulation of developmental genes. The loss of Tet enzymes promotes global hypermethylation and leads to general epigenetic instability. Most CpG dinucleotides in the vertebrate genome are methylated, rendering the subset of the genome inaccessible that is not functionally relevant. Therefore, lineage specific regulative regions and promoters often reside in lowly methylated and accessible domains, termed DNA methylation canyons. Using whole-genome bisulfite and transcriptome sequencing we identified hypermethylation of such regions as the key feature of Tet-deficient MEFs. Hypermethylation significantly disturbed the regulation of associated genes, providing a mechanistic explanation for the observed differentiation defects and suggesting that the prevention of invasive DNA methylation is a major function of Tet-dependent demethylation. The combined action of DNA methyltransferases and demethylating Tet dioxygenases therefore is essential to maintain regulative genomic domains that contain major effector genes. One factor that specifically interacts with unmethylated recognition sites on the DNA is CTCF. In a genome wide approach, combining high-throughput sequencing in wt and Tet-deficient ESCs to map changes in nucleosome positions, CTCF binding, DNA methylation and gene expression, we found in a final set of experiments that deficiency in DNA demethylating proteins reduces CTCF binding, altering higher order structures and nucleosome locations, affecting the expression of nearby genes, in particular at topologically associated domains (TADs). The maintenance of hypomethylated genomic domains thus represents a major activity of Tet proteins in differentiating cells and provides a key for understanding DNA methylation and Tet-dependent epigenetic gene regulation.
Publications
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Combined Deficiency of Tet1 and Tet2 Causes Epigenetic Abnormalities but Is Compatible with Postnatal Development. . (2013). Dev Cell 24: 310-323
Dawlaty MM, Breiling A, Le T, Raddatz G, Barrasa MI, Cheng AW, Gao Q, Powell BE, Li Z, Xu M, Faull KF, Lyko F, Jaenisch R
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Loss of Tet enzymes compromises proper differentiation of embryonic stem cells. (2014) Dev. Cell 29: 102-111
Dawlaty MM, Breiling A, Le T, Barrasa MI, Raddatz G, Gao Q, Powell BE, Cheng AW, Faull KF, Lyko F, Jaenisch R
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Epigenetic regulatory functions of DNA modifications: 5-methylcytosine and beyond. (2015) Epigenetics Chromatin 21; 8:24
Breiling A, Lyko F.
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Chromatin Immunoprecipitation. (2016) Methods Mol. Biol. 1480:721
Wiehle L, Breiling A.
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Tet1 and Tet2 Protect DNA Methylation Canyons against Hypermethylation. (2016) Mol Cell Biol. 36:452-461
Wiehle L, Raddatz G, Musch T, Dawlaty MM, Jaenisch R, Lyko F, Breiling A
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TET-dependent DNA methylation patterns in mammalian development and disease, Dissertation, Heidelberg, Universität Heidelberg, 2017. VIII, 123 S.
Laura Wiehle
