Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency.

BACKGROUND: Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. RESULTS: We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. CONCLUSION: Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

A subset of topologically associating domains fold into mesoscale core-periphery networks.

Mammalian genomes are folded into a hierarchy of compartments, topologically associating domains (TADs), subTADs, and long-range looping interactions. The higher-order folding patterns of chromatin contacts within TADs and how they localize to disease-associated single nucleotide variants (daSNVs) remains an open area of investigation. Here, we analyze high-resolution Hi-C data with graph theory to understand possible mesoscale network architecture within chromatin domains. We identify a subset of TADs exhibiting strong core-periphery mesoscale structure in embryonic stem cells, neural progenitor cells, and cortical neurons. Hyper-connected core nodes co-localize with genomic segments engaged in multiple looping interactions and enriched for occupancy of the architectural protein CCCTC binding protein (CTCF). CTCF knockdown and in silico deletion of CTCF-bound core nodes disrupts core-periphery structure, whereas in silico mutation of cell type-specific enhancer or gene nodes has a negligible effect. Importantly, neuropsychiatric daSNVs are significantly more likely to localize with TADs folded into core-periphery networks compared to domains devoid of such structure. Together, our results reveal that a subset of TADs encompasses looping interactions connected into a core-periphery mesoscale network. We hypothesize that daSNVs in the periphery of genome folding networks might preserve global nuclear architecture but cause local topological and functional disruptions contributing to human disease. By contrast, daSNVs co-localized with hyper-connected core nodes might cause severe topological and functional disruptions. Overall, these findings shed new light into the mesoscale network structure of fine scale genome folding within chromatin domains and its link to common genetic variants in human disease.

CTCF regulates the FoxO signaling pathway to affect the progression of prostate cancer.

The present research focuses on the influence of CCCTC-binding factor (CTCF) on prostate cancer (PC) via the regulation of the FoxO signalling pathway. A bioinformatics analysis was conducted to screen out target genes for CTCF in LNCaP cells and to enrich the relevant pathways in LNCaP cells. It was found that the FoxO pathway was enriched according to the ChIP-seq results of CTCF. The expression of CTCF, pFoxO1a, FoxO1a, pFoxO3a and FoxO3a was tested by RT-qPCR and Western blot. Inhibition of CTCF could lead to the up-regulation of the FoxO signalling pathway. The rates of cell proliferation, cell invasion and apoptosis were examined by MTT assay, cell invasion assay and flow cytometry under different interference conditions. Down-regulation of CTCF could suppress cell proliferation, cell invasion and facilitate cell apoptosis. Lastly, the effect of CTCF on tumour growth was determined in nude mice. Inhibition of CTCF regulated the FoxO signalling pathway, which retarded tumour growth in vivo. In conclusion, CTCF regulates the FoxO signalling pathway to affect the progress of PC.

Selective inhibition of CTCF binding by iAs directs TET-mediated reprogramming of 5-hydroxymethylation patterns in iAs-transformed cells.

Methylation at cytosine (5mC) is a fundamental epigenetic DNA modification recently associated with iAs-mediated carcinogenesis. In contrast, the role of 5-hydroxymethylcytosine (5hmC), the oxidation product of 5mC in iAs-mediated carcinogenesis is unknown. Here we assess the hydroxymethylome in iAs-transformed cells, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks. Moreover, this pathologic iAs-mediated carcinogenesis is characterized by a shift toward a higher hydroxymethylation pattern genome-wide. At specific promoters, hydroxymethylation correlated with increased gene expression. Furthermore, this increase in hydroxymethylation occurs concurrently with an upregulation of ten-eleven translocation (TET) enzymes that oxidize 5-methylcytosine (5mC) in DNA. To gain an understanding into how iAs might impact TET expression, we found that iAs inhibits the binding of CTCF at the proximal, weak CTCF binding sites of the TET1 and TET2 gene promoters and enhances CTCF binding at the stronger distal binding site. Further analyses suggest that this distal site acts as an enhancer, thus high CTCF occupancy at the enhancer region of TET1 and TET2 possibly drives their high expression in iAs-transformed cells. These results have major implications in understanding the impact of differential CTCF binding, genome architecture and its consequences in iAs-mediated pathogenesis.

CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair.

CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs). CTCF depletion increased chromosomal instability, marked by chromosome breakage and end fusions, elevated genotoxic stress-induced genomic DNA fragmentation, and activated the ataxia telangiectasia mutated (ATM) kinase. We show that CTCF could be recruited to drug-induced 53BP1 foci and known fragile sites, as well as to I-SceI endonuclease-induced DSBs. Laser irradiation analysis revealed that this recruitment depends on ATM, Nijmegen breakage syndrome (NBS), and the zinc finger DNA-binding domain of CTCF. We demonstrate that CTCF knockdown impaired homologous recombination (HR) repair of DSBs. Consistent with this, CTCF knockdown reduced the formation of γ-radiation-induced Rad51 foci, as well as the recruitment of Rad51 to laser-irradiated sites of DNA lesions and to I-SceI-induced DSBs. We further show that CTCF is associated with DNA HR repair factors MDC1 and AGO2, and directly interacts with Rad51 via its C terminus. These analyses establish a direct, functional role of CTCF in DNA repair and provide a potential link between genome organization and genome stability.