Fig. 6. BMP responsiveness of SR3/CAR3 and cardiac expression of a CAR3-driven transgene requires intact binding sites for transcription factor YY1. (A) Sequence of SR3 region encompassing site D (blue box) and SBE3 (green box). Sense and antisense YY1 consensus binding sites are shown above putative YY1-binding sites within SR3. Point mutations in putative YY1 binding sites A and B are shown in red below their cognate nucleotides. (B) YY1 binds to SR3. EMSA with oligos encoding either WT SR3 (lanes 1 and 5) or SR3 with mutations in putative YY1-binding site A (lane 2), site B (lane 3) or sites A+B (lane 4) with either P19 cell nuclear extract (top panel), whole cell extracts from day3 (HH Stage 24) chick hearts (middle panel) or purified HA-tagged YY1 protein (lower panel). Point mutation of the YY1 consensus binding site B in the SR3 oligomer significantly decreased or abolished DNA-binding activities in all samples (lane 3), as did mutations of both sites A and B (lane 4). Anti-YY1 polyclonal antibody added to the EMSA disrupted the interaction of factors in both P19 cells and day 3 chick hearts with the SR3 oligomer (lane 5). (C) Point mutation of the YY1 consensus site B in the context of an Nkx2.5-lux-CAR3 reporter (schematized at top) results in loss of BMP response in P19 cells. Results are shown in duplicate for wild-type Nkx2.5-lux-CAR3 (left) and Nkx2.5-lux-CAR3 mutB (right). (D-G) YY1-binding sites are required for CAR3-driven transgene expression in the heart. Representative X-gal staining patterns are shown for transgenic mice embryos containing Nkx2.5-lacZ-CAR3 mut B (shown in D). lacZ expression is abrogated at all stages assayed, indicating a requirement for YY1-binding sites in CAR3 to drive transgene expression in the heart (compare with the robust cardiac expression of the parental Nkx2.5-lacZ-CAR3 construct in Fig. 2D-F). Embryonic stages are shown in the lower left-hand corner. Results are representative of 3/3 E7.5 and 5/5 E10.5 transgenic embryos. Abbreviations are as in previous figures.