Beyond transcription : a post-transcriptional role of 3D chromatin crosstalk in oncogene regulation

Abstract

This thesis explores how stochastic chromatin fibre interactions, chromatin organization in the 3D nuclear architecture, and environmental signals collaborate to regulate MYC oncogene expression in human colon cancer cells. In Paper I, we employ the ultra-sensitive Nodewalk technique to uncover the dynamic and stochastic nature of chromatin networks impinging on MYC. The analyses revealed that the MYC interactome mainly consists of stochastic pairwise interactions between MYC and its flanking enhancers in two neighbouring topologically associated domains (TADs), which are insulated self-interacting genomic domains. The limits of Nodewalk were also pushed to enable the detection of interactions in very small cell populations, corresponding to the genomic content of ~7 cells. Comparing the frequency of interactions detected in such small input samples with ensemble interactomes of large cell populations uncovered that the enhancer hubs of the ensemble interactomes that appear to simultaneously interact with MYC likely represent virtual events, which are not present in reality at the single cell level. These data support a model where MYC interacts with its enhancers in a mutually exclusive way, with MYC screening for enhancer contacts, rather than the other way around.

Paper II provides a detailed understanding of a novel post-transcriptional mechanism of enhancer action on MYC expression. We have thus uncovered that the cancer-specific recruitment of the MYC gene to nuclear pores and ensuing rapid nuclear export of MYC transcripts - a process that increases MYC expression by enabling the escape of MYC mRNAs from rapid decay in the nucleus - require a CTCF binding site positioned within the colorectal oncogenic super-enhancer. Genetic editing by CRISPR-Cas9 was thus commissioned to establish two clones of human colon cancer cells with a mutated sequence in the OSE-specific CTCFBS. Comparing the mutant cells to the parental cell line, we uncovered that the WNT-dependent increase in the nuclear export rate of MYC transcripts was abrogated in the CTCFBS mutant clones, providing the first genetic evidence of super- enhancer-mediated gene gating in human cells. In line with this finding, the OSE-specific CTCFBS thus conferred a significant growth advantage to the parental colon cancer cells, compared to the mutant clones. Moreover, we found that WNT-dependent CCAT1 eRNA transcription is mediated by the OSE-specific CTCFBS that is required for recruitment of AHCTF1 to the OSE to mediate the positioning of the OSE to the nuclear periphery, enabling the subsequent facilitation of MYC mRNA export. A multistep molecular process including WNT signalling and the OSE-specific CTCFBS thus underlies the gene gating of MYC in human colon cancer cells, and could potentially be targeted for diagnostic or therapeutic uses.

In summary, this thesis explores the dynamics of the stochastic interactomes impinging on the MYC oncogene, and provides new insights on the role of 3D chromatin orchestration in the transcriptional regulation of MYC. Our analyses uncovered the molecular factors involved in the gene gating of MYC, and thus increase our understanding of tumour development. These findings could potentially be beneficial for future diagnostic approaches, or for targeted therapeutic strategies in the treatment of cancer.