Figure 6

Lhx9 gene is a direct target of the GATA4/FOG2 transcription complex. (A). A map of the mouse Lhx9 genomic locus. Exons are shown as blue and yellow boxes; the blue boxes correspond to the protein coding regions while yellow bars depict the UTRs. Both Lhx9 and 2310009B15Rik (an uncharacterized ORF flanking the Lhx9 locus) are oriented 5'-3' left-to-right. The Lhx9 introns are shown as thin blue lines. The DNA sequences corresponding to the intergenic space for all three isoforms are shown in green. The positions of the evolutionary conserved GATA sites are shown as vertical red bars and their distances with respect to the translation start sites +1(ATG) are indicated; the non-functional GATA sites are situated 3' from the start sites and ("+2x") are shown as black bars. The arrowheads are indicating the sequences amplified in the ChIP assay (see Materials and Methods for primer sequences). The horizontal red lines correspond to the DNA fragments used to generate the luciferase reporter constructs. (B). Alignement of the conserved reverse GATA sites (emboldened) in five genomes; the distance is shown from the first nucleotide (T) of the GATA site relative to the ATG codone of the respective isoform: β (top) and α (bottom). (C). Immunoprecipitations of the cross-linked chromatin from E11.5 hearts were performed with antibodies against FOG2, GATA4, RNA polymerase II or with normal IgG. Following DNA purification, samples were subjected to PCR with primers designed for the regions of the Lhx9 gene that contain GATA sites or the Gapdh promoter as a control; the PCR products were visualized on ethidium bromide gels. The PCR products were consistently observed with the total input chromatin aliquot before the immunoprecipitation step (not shown). The figure is a representative of four experiments performed.(D) The ChIP reactions corresponding to the α GATA4 and α IgG (same as in (C)) were subjected to PCR with primers designed to amplify the internal region of Lhx9 with two perfect consensus GATA sites (2xAGATAG;Additional File 2B) and were analyzed directly by the ethidium bromide gel (top panel). α FOG2 and α IgG ChIPs (same as in (C) were subjected to real-time PCR either with primers corresponding to the regulatory GATA-containing sequences or with internal primers. Primers corresponding to the two regulatory sequences amplify the α FOG2 ChIP reaction 70 and 18 times more efficiently than the α IgG reaction (left panel); they also amplify the α FOG2 reaction ~18 and 8 more efficiently than internal primers (middle panel); the amplification efficiency for the three sets of primers with respect to the control IgG reaction is close to 1 (right panel). The Y axis is showing the fold change. (E-F) GATA4 and FOG2 cooperate to inhibit Lhx9 promoters. (E) Luciferase reporter assays using Lhx9-luciferase reporters pGL3_Lhx9_1121 (black bars) or pGL3_Lhx9_1121G^m (white bar) or (F) pGL3_SV40_Lhx9_938 (black bars) in HEK 293-T cells with GATA4, FOG2 and GATA4^ki expression vectors. pGL3_Lhx9_1121G^m contains a GAAA sequence instead of the wild-type GATA sequence in position -521; the differences between the control and the mutant plasmid are not significant except for the transfection shown (the white bar). **p < 0.01, paired t-test vs control.