TADs as Organizational Framework

TADs are typically bounded by head-to-head pairs of CTCF binding sites that act as insulating barriers, preventing inappropriate interactions between regulatory elements in adjacent domains.

CTCF's Central Role in Domain Organization

CTCF functions as the primary architectural protein that establishes and maintains TAD boundaries. CTCF binding sites are strategically interwoven with enhancers within TADs, creating a network that stabilizes enhancer-promoter interactions.Approximately 70% of CTCF binding sites are located within 20 kb of p300 binding sites (an enhancer-associated protein), demonstrating the close spatial relationship between CTCF and active regulatory elements.

The strength of CTCF binding at TAD boundaries correlates directly with transcriptional activity. Transcribed TAD boundaries show significantly higher CTCF enrichment than non-transcribed boundaries, and this enrichment is associated with stronger insulation between domains

Enhancer-Promoter Interaction Mechanisms

Within TADs, enhancers and promoters establish functional contacts that drive gene transcription. CTCF binding sites facilitate these interactions by stabilizing enhancer-promoter contacts. When CTCF sites are deleted, the interactions between promoters and their associated enhancers are substantially decreased, leading to reduced gene expression and increased cell-to-cell variation in transcription

The relationship between enhancer-promoter contact and transcription follows a nonlinear pattern, where transcriptional output depends on contact probabilities rather than simple physical distance. This suggests that transient enhancer-promoter interactions are translated into sustained transcriptional bursting in individual cells.

Sub-TAD Formation and Function

Within larger TADs, sub-TADs form smaller regulatory neighborhoods that provide more precise control over enhancer-promoter interactions. These sub-domains are essential for proper gene regulation, as disruption of sub-TAD structure impairs enhancer-promoter interactions even when the larger TAD remains intact

Functional Consequences of Disruption

When this coordinated system is disrupted, several consequences emerge:

• Loss of enhancer-promoter specificity: Deletion of CTCF sites or depletion of CTCF protein leads to weakened interactions between enhancers and their target promoters

• Increased transcriptional noise: CTCF disruption results in greater cell-to-cell variation in gene expression

• Domain merging: Weakened boundaries allow adjacent TADs to merge into larger, less functional domains

• Genome-wide effects: More than half of enhancer-promoter loops disappear when CTCF is depleted, accompanied by reduced gene transcription

  The correlation between these elements creates a hierarchical regulatory system where TADs provide the structural framework, CTCF establishes boundaries and facilitates contacts, enhancers drive transcription and strengthen boundaries, and promoters respond to enhancer signals within their local chromatin environment. This system ensures that genes are activated by their appropriate regulatory elements while being insulated from inappropriate influences from neighboring domains.

  CTCF at TAD Boundaries: The General Pattern

  The vast majority of TAD boundaries do contain CTCF binding sites. Research shows that over 95% of all TAD boundaries have at least one CTCF binding site, and 41% of conserved CTCF binding sites are associated with TAD boundaries

  Clustered CTCF Sites, Not single sites

  However, TAD boundaries typically don't consist of single CTCF binding sites. Instead, TAD boundaries frequently harbor clusters containing multiple CTCF binding sites. These clusters contain both evolutionarily old and young CTCF sites as a result of repeated acquisition of new species-specific sites close to conserved ones

  The clustering is functionally important - CTCF sites belonging to clusters are significantly more enriched at TAD borders than singleton CTCF sites. Strong TAD boundaries show enrichment of strongly bound and closely spaced CTCF binding peaks, with further enrichment of DNA-binding motifs within these peaks.

Exceptions to the CTCF-TAD Rule

Importantly, not all TAD boundaries require CTCF:

Nearly 5% of TAD boundaries apparently do not overlap with any CTCF occupancy

12% of boundaries had CTCF sites conserved only in one, two, or three out of five studied species

This suggests that although the connection between CTCF binding and TAD boundaries is consistently observed, it may not be a strictly necessary feature for demarcation of all TAD boundaries.

How to determine Enhancer and promoter region

There are several approached to determin enhancer and promoters, and you don't necessarily need to rely soley on database or chromatin contact matrix annoration

• we can use Chip-Seq data to determin
• we can also use histone marks