Progressive digestion of limb-specific Shh regulatory region reveals critical sequences necessary for enhancer activity (541.5)

Regulation of anterior-posterior patterning of the limb bud during development is mediated by Sonic hedgehog (Shh), expressed in the zone of polarizing activity in the distal posterior region. Upstream of Shh lies a 1.8 kb limb-specific Shh regulatory region ( LSSRR ) that regulates the expression of Shh.  LSSRR  can be subdivided into four highly conserved regions, A, B1, B2, and B3. Our goal is to determine which sequences within  LSSRR  are necessary for enhancer activity. We progressively digested the  LSSRR  sequence from the 5’ end and the 3’ end. We generated expression constructs for each fragment containing the minimal HSV-tk promoter linked to a green fluorescent protein (GFP) reporter. These constructs were electroporated into the chick presumptive limb bud. After 48 hrs, we visualized and recorded the GFP fluorescence to determine enhancer activity. Removal of 217 3’ bases abolishes enhancer activity, while even after removal of 1161 bases from the 5’ end activity persists. Our progressive digestions indicate that enhancer activity is retained within 107 base pairs of the B3 conserved region. This region contains three highly conserved motif clusters and is rich with potential binding sites including overlapping sites for retinoic acid receptor/NFB/ETS and sites for E-Box and Blimp1. Although other sequences may be required for inhibiting activity and chromatin looping, we conclude that this 107 base pair region is essential for  LSSRR  enhancer activity.