In this enhancer blocking assay, putative boundary elements are placed in between two
fushi tarazu (
ftz) enhancers (UPS and NE) and an
hsp70:LacZ reporter. The UPS enhancer is active in early embryos and drives LacZ expression a seven stripe pair-rule pattern. The NE enhancer is active later in development and drives LacZ expression in the central nervous system. (
A) When a ‘non-specific’ DNA sequence is placed between the
ftz enhancers and the
hsp70:LacZ reporter, no blocking is observed. In early embryos (stage 10–11 in this assay), the
ftz UPS enhancers drive LacZ expression in seven stripes. In older embryos (stage 13–14) the NE enhancer drives LacZ expression in the CNS. (
B) When
Fab-7 is place between the
ftz enhancers and the reporter, it blocks both enhancers from activating the
hsp70 promoter and there is little if any LacZ expression in either early or late embryos. (
C) When pHS1×4 (four copies of the 236-bp pHS1: see
Figure 1) is placed between the enhancers and the reporter, it blocks the UPS enhancer from activating stripe expression. However, boundary activity is stage specific and pHS1×4 does not block the NE from activating LacZ expression in the CNS in older embryos (
Aoki et al., 2008). (
D) When mutations (M3 in
Figure 1) that disrupt Elba binding in nuclear extracts are introduced into the Elba sequence of pHS1, the enhancer blocking activity of the mutated pHS1×4 is compromised. In this case, both UPS stripe and NE CNS LacZ expression is observed (
Aoki et al., 2008). (
E) When a 27-bp oligo containing the Elba binding sequence is multimerized (Elba×8) and placed in between the
ftz enhancers and the reporter, it blocks UPS driven LacZ stripe expression. It also causes a reduction in the extent of NE activation. This effect is position dependent. The blocking activity seen at this stage with the 8× multimer is likely due to the presence of some residual Elba activity in older embryos as seen in EMSA experiments with 6–12 hr nuclear extracts (
Aoki et al., 2008).