Table 1 Summary of the main results obtained for the Gbx2/Asb18 locus

Δ3xCTCF (Fig. 3; Fig. S13)

10 Kb deletion that eliminates the three CTCF sites separating the Gbx2 and Asb18 TADs.

Gbx2 expression is not affected.

Asb18 expression is significantly increased (~5-fold).

The contact frequency between the Gbx2 SE and Gbx2 is reduced.

The contact frequency between the Gbx2 SE and Asb18 is increased.

The positioning of Gbx2 close to the 3XCTCF cluster does not play a major role in the maintenance of Gbx2 expression in ESC.

The CTCF cluster strengthens the physical insulation of the Gbx2 regulatory domain, preventing spurious enhancer-gene contacts.

71Kb INV (Fig. 3; Fig. S13)

Inversion that re-positions Gbx2 and its SE with respect to the CTCF cluster, placing the SE in between Gbx2 and Asb18.

Gbx2 expression is not affected.

Asb18 expression is significantly increased (~16-fold).

The contact frequency between the Gbx2 SE and Gbx2 is reduced.

Minor impact on Asb18-Gbx2 SE contacts.

Strong increase in the contact frequency between the Gbx2 SE and the 3XCTCF cluster.

The positioning of Gbx2 close to the 3xCTCF cluster does not play a major role in the maintenance of Gbx2 expression in ESC.

Gbx2 does not contribute to physical insulation through enhancer blocking. The increased expression of Asb18 might be explained by the reduced linear distance between Asb18 and the Gbx2 SE in 71Kb INV cells.

Δ3xCTCF:71Kb INV (Fig. 3; Fig. S13)

Cells with both the Δ3xCTCF deletion and the 71 Kb inversion.

Gbx2 expression is not affected.

Asb18 expression is increased to similar levels as in 71Kb INV cells.

The contact frequency between the SE and Gbx2 is reduced.

The contact frequency between the Gbx2 SE and Asb18 is strongly increased.

Non-linear relationship between gene activity and E-P contact frequency: the increase in Gbx2 SE-Asb18 contacts does not lead to a further increase in Asb18 expression.

ΔProm Gbx2 (Fig. 4; Fig. S15)

1 Kb deletion spanning the Gbx2 promoter.

Asb18 expression is significantly increased (~7-fold).

Minor impact on Asb18-Gbx2 SE contacts.

Gbx2 contributes to regulatory rather than physical insulation through promoter competition.

Δ3XCTCF:ΔProm Gbx2 (Fig. 4; Fig.  S15)

Cells with both the Δ3xCTCF and ΔPromGbx2 deletions.

Strong and synergistic increase in Asb18 expression (~60-fold).

The contact frequency between the Gbx2 SE and Asb18 is increased to a similar extent as in Δ3xCTCF cells.

Gbx2 contributes to regulatory rather than physical insulation through promoter competition. Gbx2 and the CTCF cluster cooperatively confer the nearby TAD boundary with strong insulator capacity.