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Alteration of CTCF associated chromatin neighborhood inhibits TAL1-driven oncogenic transcription program and leukemogenesis

ABSTRACT: Aberrant activation of the TAL1 oncogene is associated with up to 60% of T-ALL patients and is involved in CTCF mediated genome organization within the TAL1 locus, suggesting the importance of the CTCF boundary in the molecular pathogenesis of T-ALL. Here, we show that deletion of CTCF binding site (CBS) or alternation of CTCF boundary orientation alters expression of the TAL1 oncogene in a cell context dependent manner. Deletion of the CTCF binding site located at -31 Kb upstream of TAL1 (-31CBS) reduces chromatin accessibility in the +51 enhancer and the TAL1 promoter I, and blocks long-range interaction between the +51 erythroid enhancer and TAL1 promoter 1b that inhibits expression of TAL1 in erythroid cells, but not in T-ALL cells. However, in the TAL1 expressed T-ALL primary patient samples or cell line, the T-ALL prone TAL1 promoter IV specifically interacts with the +19 stem cell enhancer that is located 19 kb downstream of the TAL1 promoter and required for TAL1 transcription in the hematopoietic stem cell (HSC) stage. Inversion of -31CBS orientation, but not deletion of -31CBS, alters chromatin accessibility, enhancer/promoter histone modifications, CTCF-mediated topological associated domain (TAD), and enhancer/promoter interaction in the TAL1 locus leading to inhibition of TAL1 oncogene expression and TAL1-driven T cell leukemogenesis. Thus, our data reveal that the TAL1 +19 stem cell enhancer acts not only as stem cell enhancer, but also as a leukemia specific enhancer to activate the TAL1 oncogene in T-ALL. Manipulation of CTCF defined chromatin boundary can alter TAL1 TAD and oncogenic transcription networks in leukemogenesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE135320 | GEO | 2020/04/06

REPOSITORIES: GEO

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Project description:The genome is partitioned into Topologically Associating Domains (TADs). About half of the boundaries of these TADs exhibit transcriptional activity and are correlated with better TAD insulation. However, the association between these transcripts and TAD insulation, enhancer:promoter interactions and transcription of genes remains unknown. Here we investigate the functional roles of these bRNAs (boundary-RNA) in boundary insulation and consequent effects on enhancer-promoter interactions and TAD transcription genome-wide and more specifically on the disease relevant INK4a/ARF TAD. Using a series of CTCF site deletions and bRNA knockdown experiments at this TAD boundary, we show a direct association of CTCF with the bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF at the TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. We used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD to better understand the regulation and origins of bRNA itself. We observe that the enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilize the CTCF even on weak motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation. This favors the intra-TAD enhancer:promoter interactions and leads to robust gene transcription. Functionally, bRNAs are more stable than eRNAs and their knockdown exactly mimics the CTCF site deletion. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumour datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries triggering CTCF clustering at the boundary which results in better insulation and favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.

Project description:CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops. In addition to insulation of euchromatin from heterochromatin, CTCF is an important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCF inactivating events have been found in human cancer, resulting in deregulation of global gene expression by altered methylated genomic states. In contrast to these studies, we here describe that inactivation of CTCF can drives subtle and local genomic effects that elevates oncogene expression levels from driving chromosomal rearrangements. For T cell acute lymphoblastic leukemia (T-ALL), heterozygous CTCF deletions or inactivating mutations are predominantly found in nearly 50 percent of t(5;14)(q35;q32.2) rearranged patients that couples the TLX3 oncogene in the vicinity of the BCL11B enhancer. This unique entity has been associated with γδ-lineage development. Functional CTCF loss results in diminished expression of the αβ-lineage commitment factor BCL11B from the non-rearranged allele, but unexpectedly drives higher levels of the TLX3 oncogene from the translocated allele. In line, Ctcf conditional knockout mice have reduced numbers of αβ T cells but increased numbers of γδ T cells, a phenotype identical to that of Bcl11b knockout mice and implying that CTCF is directly involved in the regulation of the BCL11B enhancer. We demonstrate that most TLX3-rearranged patients with heterozygous CTCF aberrations preserved single intervening CTCF bindings sites in the translocation breakpoint areas located in between the BCL11B enhancer and the TLX3 oncogene. These intervening CTCF sites insulate TLX3 from the enhancer by forming competitive loops with TLX3 regulatory sequences. Using reverse genetics, we provide evidence that heterozygous inactivation of CTCF diminishes competitive loop formation in favor of high-affinity TLX3 promoter loops to BCL11B enhancer sequences formed among multiple convergent CTCF binding sites. This boosts oncogene expression levels and leukemia burden in T-ALL patients.

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Alteration of CTCF associated chromatin neighborhood inhibits TAL1-driven oncogenic transcription program and leukemogenesis

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