The Nodewalk assay to quantitate chromatin fiber interactomes in very small cell populations.

The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to <300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.

Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site.

Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer-(OSE)-mediated gating of the active MYC to the nuclear pore in a poorly understood process. We show here that the principal tenet of the WNT-regulated MYC gating, facilitating nuclear export of the MYC mRNA, is regulated by a CTCF binding site (CTCFBS) within the OSE to confer growth advantage in HCT-116 cells. To achieve this, the CTCFBS directs the WNT-dependent trafficking of the OSE to the nuclear pore from intra-nucleoplasmic positions in a stepwise manner. Once the OSE reaches a peripheral position, which is triggered by a CTCFBS-mediated CCAT1 eRNA activation, its final stretch (≤0.7 µm) to the nuclear pore requires the recruitment of AHCTF1, a key nucleoporin, to the CTCFBS. Thus, a WNT/ß-catenin-AHCTF1-CTCF-eRNA circuit enables the OSE to promote pathological cell growth by coordinating the trafficking of the active MYC gene within the 3D nuclear architecture.

MYC as a driver of stochastic chromatin networks: implications for the fitness of cancer cells.

The relationship between stochastic transcriptional bursts and dynamic 3D chromatin states is not well understood. Using an innovated, ultra-sensitive technique, we address here enigmatic features underlying the communications between MYC and its enhancers in relation to the transcriptional process. MYC thus interacts with its flanking enhancers in a mutually exclusive manner documenting that enhancer hubs impinging on MYC detected in large cell populations likely do not exist in single cells. Dynamic encounters with pathologically activated enhancers responsive to a range of environmental cues, involved <10% of active MYC alleles at any given time in colon cancer cells. Being the most central node of the chromatin network, MYC itself likely drives its communications with flanking enhancers, rather than vice versa. We submit that these features underlie an acquired ability of MYC to become dynamically activated in response to a diverse range of environmental cues encountered by the cell during the neoplastic process.