CTCF is an architectural protein with a critical role in connecting higher-order chromatin folding in pluripotent stem cells. Recent reports have suggested that CTCF binding is more dynamic during development than previously appreciated. Here, we set out to understand the extent to which shifts in genome-wide CTCF occupancy contribute to the 3D reconfiguration of fine-scale chromatin folding during early neural lineage commitment. Unexpectedly, we observe a sharp decrease in CTCF occupancy during the transition from naï ve/primed pluripotency to multipotent primary neural progenitor cells (NPCs). Many pluripotency gene-enhancer interactions are anchored by CTCF, and its occupancy is lost in parallel with loop decommissioning during differentiation. Conversely, CTCF binding sites in NPCs are largely preexisting in pluripotent stem cells. Only a small number of CTCF sites arise de novo in NPCs. We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between NPC-specific genes and enhancers. Putative NPC-specific enhancers exhibit strong YY1 signal when engaged in 3D contacts and negligible YY1 signal when not in loops. Moreover, siRNA knockdown of Yy1 specifically disrupts interactions between key NPC enhancers and their target genes. YY1-mediated interactions between NPC regulatory elements are often nested within constitutive loops anchored by CTCF. Together, our results support a model in which YY1 acts as an architectural protein to connect developmentally regulated looping interactions; the location of YY1-mediated interactions may be demarcated in development by a preexisting topological framework created by constitutive CTCF-mediated interactions.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3–6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

3D genome organization regulates gene expression, and disruption of these long-range (>20kB) DNA-protein interactions results in pathogenic phenotypes. Chromosome conformation methods in conjunction with chromatin immunoprecipitation were used to decipher protein-directed chromatin interactions. However, these methods required abundant starting material (>500,000 cells), sizable number of sequencing reads (>100 million reads), and elaborate data processing methods to reduce background noise, which limited their use in primary cells. Hi-C Coupled chromatin cleavage and Tagmentation (HiCuT) is a new transposase-assisted tagmentation method that generates high-resolution protein directed long-range chromatin interactions as efficiently as existing methods, HiChIP and ChIA-PET, despite using 100,000 cells (5-fold less) and 12 million sequencing reads (8-fold fewer). Moreover, HiCuT generates high resolution fragment libraries with low background signal that are easily interpreted with minimal computational processing. We used HiCuT in human primary skin cells to link previously identified single nucleotide polymorphisms (SNPs) in skin disease to candidate genes and to identify functionally relevant transcription factors in an unbiased manner. HiCuT broadens the capacity for genomic profiling in systems previously unmeasurable, including primary cells, human tissue samples, and rare cell populations, and may be a useful tool for all investigators studying human genetics and personalized epigenomics.

More than 25 inherited neurological disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why specific STRs are susceptible to unstable expansion leading to severe pathology, whereas tens of thousands of normal-length repeat tracts across the human genome are relatively stable. Here, we unexpectedly discover that nearly all STRs associated with repeat expansion diseases are located at boundaries demarcating 3-D chromatin domains. We find that boundaries exhibit markedly higher CpG island density compared to loci internal to domains. Importantly, disease-associated STRs are specifically localized to ultra-dense CpG island-rich boundaries, suggesting that these loci might be hotspots for epigenetic instability and topological disruption upon unstable expansion. In Fragile X Syndrome, mutation-length expansion at the Fmr1 gene results in severe disruption of the boundary between TADs. Our data uncover higher-order chromatin architecture as a new dimension in understanding the mechanistic basis of repeat expansion disorders.