TET-dependent DNA methylation patterns in mammalian development and disease

TET enzymes are relatively novel players in the epigenetic regulation of mammalian DNA methylation. They participate in DNA demethylation, but their precise roles in different developmental and disease scenarios are not fully understood. The aim of this work was to investigate the biological roles of TET enzymes in lineage-committed normal and cancer cells. To this end, murine primary cells with genetic deletion of TET enzymes and human cancer cells with recurrent mutations in the cofactor providing isocitrate dehydrogenases (IDH), provoking competitive inhibition of TET enzymes, were analyzed. By characterizing mouse embryonic fibroblasts adipogenic differentiation defects, inefficient activation of genes relevant to adipogenesis and widespread gene deregulation upon TET1/2-deficiency were discovered. Examination of the genome-wide DNA methylation landscape demonstrated the hypermethylation of DNA methylation canyons as a main characteristic of the TET1/2-deficient methylome. Canyons were associated with developmentally important genes and canyon collapse due to hypermethylation coincided with developmental gene deregulation, defective induction of adipogenic markers and the hypermethylation of their promoters. Together, these findings uncovered a novel epigenetic regulatory role in the maintenance of DNA methylation canyons for TET1 and TET2 that is essential for epigenetic programming during differentiation. In the second part of this thesis, published array-based DNA methylation profiles of a large acute myeloid leukemia (AML) patient cohort were used to examine mutant IDH- (mIDH) and TET-dependent DNA methylation changes. This confirmed the known association between mIDH and genome-wide hypermethylation. However, similar global methylation changes were not present in TET2 mutant patients and mIDH carrying patients lacked specific canyon hypermethylation. Intriguingly, neither overexpression of mIDH, nor treatment of a leukemia cell line with D-2-hydroxyglutarate, which is a putative TET inhibitor produced by mIDH, recapitulated the mIDH-associated hypermethylation. Instead, comparison with hematopoietic reference methylomes revealed high similarity between mIDH-associated and myeloid progenitor methylation profiles, suggesting the involvement of differentiation state rather than TET inhibition in the hypermethylation phenotype. These findings implicate a previously unnoted factor in the epigenomic changes of AML cells with mIDH, which may be critical to understand and therapeutically target mIDH-dependent pathogenesis

DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries

Coordinated changes of DNA (de)methylation, nucleosome positioning and chromatin binding of the architectural protein CTCF play an important role for establishing cell type specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the TET1 and TET2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC) or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation and gene expression in DKO ESCs, and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, while unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild type cells but not from 5fC enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, e.g. at promoters, boundaries of chromatin loops and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to downregulation of neighbouring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions and DNA sequence that determines differential CTCF binding and regulates gene expression

T‐cell prolymphocytic leukemia is associated with deregulation of oncogenic microRNAs on transcriptional and epigenetic level

Deregulation of micro(mi)-RNAs is a common mechanism in tumorigenesis. We investigated the expression of 2083 miRNAs in T-cell prolymphocytic leukemia (T-PLL). Compared to physiologic CD4+ and CD8+ T-cell subsets, 111 miRNAs were differentially expressed in T-PLL. Of these, 33 belonged to miRNA gene clusters linked to cancer. Genomic variants affecting miRNAs were infrequent with the notable exception of copy number aberrations. Remarkably, we found strong upregulation of the miR-200c/-141 cluster in T-PLL to be associated with DNA hypomethylation and active promoter marks. Our findings suggest that copy number aberrations and epigenetic changes could contribute to miRNA deregulation in T-PLL

Reconstruction of rearranged T-cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is an aggressive tumor with leukemic presentation of mature T-lymphocytes. Here, we aimed at characterizing the initial events in the molecular pathogenesis of T-PLL and particularly, at determining the point in T-cell differentiation when the hallmark oncogenic events, that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) occur. To this end, we mined whole genome and transcriptome sequencing data of 17 and 11 T-PLL cases, respectively. Mapping of the 14q32.1 locus breakpoints identified only TCL1A, which was moreover significantly overexpressed in T-PLL as compared to benign CD4+ and CD8+ T-cells, as the only common oncogenic target of aberrations. In cases with t(14;14), the breakpoints mapped telomeric and in cases with inv(14) centromeric or in the 3 '-untranslated region of TCL1A. Regarding the T-cell receptor alpha (TRA) locus-TCL1A breakpoint junctions, all 17 breakpoints involved recombination signal sequences and 15 junctions contained nontemplated (N-) nucleotides. All T-PLL cases studied carried in-frame TRA rearrangements on the intact allele, which skewed significantly toward usage of distal/central TRAV/TRAJ gene segments as compared to the illegitimate TRA rearrangements. Our findings suggest that the oncogenic TRA-TCL1A/MTCP1 rearrangements in T-PLL occur during opening of the TRA locus, that is, during the progression from CD4+ immature single positive to early double positive thymocyte stage, just before physiologic TCL1A expression is silenced. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Thereafter, it switches off RAG and DNTT expression in line with the mature T-cell phenotype at presentation of T-PLL