Abstract
The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here we test the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-specific or allele-specific manner. In contrast, Firre’s deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.
Highlights
The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs)
By analyzing publicly available chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C datasets, we determined that the genomic region around the Firre locus harbors one of the highest densities of CTCF binding on chromosome X across multiple cell types (Fig. 1a,b)
We find that a TAD boundary is directly located at the Firre locus (Supplementary Figure 1)
Summary
The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. The deletion of a single CTCF site or of a minimal genomic region is sufficient to perturb a TAD boundary in some studies[9,17,18]; others reported that disruptions of a TAD boundary occur only upon deleting very large genomic regions (e.g., 200–400 kb)[1,18] To address this conundrum, we focused on a locus on chromosome X that exhibits dense CTCF binding, and that harbors the Firre long non-coding RNA (lncRNA)[19,20]. RPE1 K562 HMEC NHEK FIRRE TAD boundary mESC CH12 Patski mNSC Firre TAD boundary qRT-PCR Relative Firre expression RNA-seq Firre expression (TPM)
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