Part:BBa_K080001
A20/alpha cardiac actin miniPro-BMP4
Induction of BMP4 during early stages of cardiomyocyte differentiation results in inhibitory effects. From previous studies it is not clear whether BMP4 is necessary for cardiomyocyte differentiation. In our circuit, nkx2.5 and gata4 will be induced and initiate the pathways of cardiac differentiation. During normal cardiac muscle differentiation BMP4 is a downstream target of nkx-2.5 and gata4, the expression of which turns on at later stages of cardiac differentiation. To ensure that BMP4 has a delayed expression, we designed BMP4 under the control of minimal promoters of late markers of cardiac muscle differentiation, TCF and alpha cardiac actin.
Contractile proteins are expressed very late in the cardiomyocyte differentiation pathway making it an ideal choice as a timer for the induction of BMP4. One of these cardiac specific contractile proteins is alpha cardiac actin. We have designed delayed BMP4 expression under the control of an alpha cardiac actin minimal promoter.
A20+ linker Triple repeat of A20, an nkx2.5 consensus binding sequence that nkx2.5 binds with high affinity to, plus linker sequence. A20 sequence was initially identified through random oligonucleotide selection as described in Chen, C.Y. and Schwartz, R.J. (1995) JBC 270(30) 15628-15633
alpha cardiac actin minpro Previous work by Schwartz and collegues revealed that the interactions of nkx2.5, serum response factor (SRF), gata4, and other transcription factors play an important role during cardiomyocyte differentiation. One of the downstream targets of these transcription factors in cardiogenic pathways was alpha cardiac actin, whose promoter they identified as being under direct control of nkx2.5 as reviwed in Chen, C. and R.J. Schwartz. (1997) Competition between negative acting YY1 versus positive acting serum response factor and tinman homologue nkx-2.5 regulates cardiac alpha-actin promoter activity 11(6) 812-822.
To create a late strong minimal promoter of cardiomyogenesis we designed 3 repeats of A20 fused to the promoter sequence of cardiac alpha-actin with known cardiac specific regulation (Sepulveda, J.L., et al. GATA-4 and Nkx-2.5 Coactivate Nkx-2 DNA Binding Targets: Role for Regulating Early Cardiac Gene Expression (1998) 18(6) 3405-3415).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 13
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Illegal PstI site found at 813 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1385
Illegal SpeI site found at 766
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Illegal NotI site found at 70 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 13
Illegal SpeI site found at 766
Illegal PstI site found at 813 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 13
Illegal SpeI site found at 766
Illegal PstI site found at 813
Illegal NgoMIV site found at 109 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 239
Illegal SapI.rc site found at 443
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