Fig 1. The enhancer of Furin M10 is repressed by NKX2-5.

(A) ChIP-seq trace at the Furin locus. The region identified as enriched for NKX2-5 binding (M10) and subsequently tested in transgenic studies is indicated (red bar). Note the 3’ terminus part of the Blm gene. 20kb scale is shown. (B) ChIP analysis with an anti-NKX2-5 antibody, using chromatin purified from E9.5 AHF and heart. Three independent preparations of chromatin extracts were used for the real time PCR analysis with primers against the M10 region. For relative enrichment, a region of the gamma-crystallin gene (gcrys) was used as a negative control. (C) Electrophoretic mobility shift assay (EMSA) showing that NKX2-5 binds its motif (see methods) in the M10 probe (black arrowhead). Inclusion of a specific antibody against NKX2-5 results in a supershift of the band (white arrowhead, lane 3). Presence of unlabelled probe oligonucleotide results in specific competition (M10-c, lane 4 to lane 7). No competition is observed with a mutated unlabelled probe (lane 8). (D) Expression of Furin mRNA as detected using in situ hybridisation in wildtype and Nkx2-5 knock out mouse embryo at E9.5. Note the increase staining for Furin in the heart of the mutants (white arrowhead). The transgene M10 recapitulates Furin expression in the heart (compare white arrows). Note the increase staining for the transgene in the heart of the mutants (black arrowhead) while the expression in the limbs remains comparable to wild type (compare black arrows). Scale bar 1mm. (E) Quantification of Furin and LacZ mRNA in heart and limb using qPCR in wildtype (n = 4) and Nkx2-5 knock out embryos (n = 4). Furin and LacZ mRNA expression is increased by 1.84 and 1.64-fold respectively in mutants while LacZ expression remains similar in the limbs. (F) Relative luciferase activity in control and NKX2-5 deficient HL-1 cells using the M10-pGL3 fragment. Graph represents 3 different experiments. (G) M10-pGL3 Luciferase reporter activity decreases with cotransfection of increasing amounts of an Nkx2-5 expressing vector. In contrast, luciferase activity from the M10-mut-pGL3 reporter vector is unaffected by Nkx2-5 overexpression in HL-1 cells. (Only significant changes are indicated. Graph represents 3 independent transfections). (H) Whole mount in situ hybridization showing a reduction in Furin mRNA expression in the heart of a E9.5 mutant mouse embryo carrying a homozygous deletion of the enhancer M10 (compare black arrows). (I) Furin mRNA. expression is significantly reduced in the hearts of homozygous mutants’ embryos (n = 4) for M10 deletion. *: p<0.05, **: p<0.001 according to a two-tailed Student’s t-test.