Table 2 Summary of the main results obtained for the Six3/Six2 locus

Δ6xCTCF (Figs. 5 and 6; Fig. S21)

36 Kb deletion that eliminates six out of the seven CTCF sites separating the Six3 and Six2 TADs.

Six3 induction is significantly impaired (~5-fold).

Six2 expression is significantly increased (~9-fold).

The contact frequency between the Six3 SE and Six3 is not affected.

Increased contact frequency between Six3 and Six2.

The contact frequency between the Six3 SE and Six2 is increased.

The positioning of Six3 close to the CTCF cluster might facilitate Six3 induction, although changes in Six3-SE contact frequency are not observed.

The CTCF cluster strengthens the physical insulation of the Six3 regulatory domain, preventing spurious enhancer-gene contacts.

156KbINV (Fig. 5)

Inversion that re-positions Six3 and its SE with respect to the CTCF cluster, placing the SE in between Six3 and Six2.

Six3 expression is not affected.

Six2 expression is not affected.

NA

The positioning of Six3 close to the CTCF cluster does not facilitate Six3 induction. The impaired induction of Six3 in Δ6xCTCF might involve alternative mechanisms.

Six3 does not contribute to the insulation of its regulatory domain through enhancer blocking.

Δ6xCTCF: 156KbINV (Fig. 5)

Cells with both the Δ6xCTCF deletion and the 156 Kb inversion.

Six3 induction is significantly impaired.

Six2 expression is increased to similar levels as in Δ6xCTCF.

NA

Six3 does not contribute to the insulation of its regulatory domain through enhancer blocking.

Δ6xCTCF:Six2^−/−(Fig. 5)

Cells with both the Δ6xCTCF deletion and a 11 Kb deletion spanning the Six2 gene.

Six3 induction is partially rescued (~2-fold reduction) in comparison to Δ6xCTCF cells (~5-fold reduction).

NA

The CTCF cluster might protect Six3 from promoter competition by Six2 and, potentially, from putative silencers found within the Six2 TAD (speculative).

Six3^−/− (Fig. 6; Fig. S21)

27 Kb deletion spanning the Six3 gene.

Six2 expression is not affected.

Minor impact on Six2-Six3 SE contacts.

Six3 does not contributes to the physical insulation of its regulatory domain.

Δ6XCTCF:Six3^−/− (Fig. 6; Fig. S21)

Cells with both the Δ6xCTCF deletion and the Six3^−/− deletion.

Strong and synergistic increase in Six2 expression (~70-fold).

The contact frequency between the Six3 SE and Six2 is mildly increased in comparison to Δ6xCTCF cells.

Six3 contributes to regulatory rather than physical insulation through promoter competition.

Six3 and the CTCF cluster cooperatively confer the nearby TAD boundary with strong insulator capacity.

Δ4xCTCF (Fig. 6)

36 Kb deletion that eliminates four out of the seven CTCF sites separating the Six3 and Six2 TADs.

Six3 induction is significantly impaired.

Six2 expression is not affected.

NA

The CTCF cluster might protect Six3 from promoter competition by Six2 and, potentially, from putative silencers found within the Six2 TAD (speculative).

Δ4XCTCF:Six3^−/− (Fig. 6)

Cells with both the Δ4xCTCF and Six3^−/− deletions.

Strong and synergistic increase in Six2 expression (~16-fold).

NA

Six3 and the CTCF cluster cooperatively confer the nearby TAD boundary with strong insulator capacity.