Figure 4.

GATA2 mutations impair their DNA binding properties on rat preproTRH and human TSHβ promoters. (A,B) Schematic representation of rat preproTRH and human TSHβ promoter. The positions of primers for the oligo DNAs for gel shift assay are indicated. GATA-RE, GATA responsive element; AP-1, AP-1-binding site; GRE, glucocorticoid responsive element; GRE1/2, glucocorticoid responsive element half-site; STAT, signal transducer and activator of transcription; CRE, cyclic AMP response element; GCA, GCA site; KEM1, KEM1 site; TSS, transcription start site; CAT, chloramphenicol acetyltransferase. Pit1-US; Pit1-binding site upstream of GATA-REs; SR, suppressor region; nTRE, negative T₃ responsive element. (C,D) Gel shift assays demonstrated single bands (arrows) when ³²P-labeled probe-G or probe-PG were incubated with nuclear extract of CV1 cells transfected with mouse GATA2 expression plasmid. The specific band signal (lane 3) was abolished by a 50-fold amount of cold probe-G or probe-PG (lane 4) but not nonspecific double strand oligo DNA (NS; lane 5). These signals were supershifted when GATA2 protein was mixed with the anti-GATA2 antibody (ab) before incubation with ³²P-labeled probes (lane 6). (E,F) Gel shift analysis with probe-G or probe-PG was conducted using nuclear extract of CV1 cells transfected with expression plasmids for various GATA2 mutants.