The functional analysis of insulator interactions in the Drosophila embryo.

Chromatin boundaries or insulators modulate enhancer-promoter interactions in complex genetic loci. However, the mechanism underlying insulator activity is not known. Previous studies showed that the activity of the Drosophila suHw insulator is abolished by the tandem arrangement (pairing) of the insulator elements, suggesting that interactions between insulators or like elements may be involved in their enhancer-blocking mechanism. To test whether such phenomenon reflects a general property of chromatin insulators, we tested the effect of pairing on enhancer-blocking activity of 11 homologous and heterologous insulator combinations using suHw, scs, or SF1 insulators. We found that, unlike the homologous pairing of suHw, the heterologous combinations of suHw with other insulators do not reduce their enhancer-blocking activity. Rather, paired insulators exhibit a higher level of enhancer-blocking activity than either single insulator alone, suggesting that they can function independently or additively. Furthermore, the analyses of two additional chromatin boundaries, scs and SF1, in homologous or heterologous pairing with other boundary elements, also showed no reduction but rather enhancement of insulator activity. We propose that diverse mechanisms may underlie insulator activity, and selective interactions among insulators could influence their function as well as the formations of independent chromatin domains.