GATA-1 regulates Fog1 directly. (A) Cis-regulatory modules in Fog1 as predicted by comparative genomics. The mouse Fog1 gene (Zfpm1) is plotted in the middle, using base positions on chromosome 8 from the February 2003 (mm3) assembly. Coding exons are taller boxes, untranslated regions are shorter boxes, and introns are shown as lines with arrowheads pointing in the direction of transcription. Positions of cytosine-phosphate-guanosine (CpG) islands are on the line underneath the gene map. The Conservation Score³⁸  estimates a log-likelihood that an alignment is in functional sequences. The RP score is a log-likelihood measurement that estimates the probability that a sequence is involved in regulating expression.⁴⁰  Matches to weight matrices for GATA-1 binding sites were identified in the mouse sequence (track labeled “all GATA-1_BS”), and these were filtered to find those that are conserved in mouse, rat, and human alignments (track labeled “conserved GATA-1_BS”). The DNA segments whose alignments exceed a calibrated threshold for RP score and also contain at least one conserved, predicted GATA-1 binding site are shown in the track labeled “hi RP & cGATA-1_BS”; these are the predicted cis-regulatory modules for the Fog1 locus. There were 2 tested for occupancy by GATA-1 in vivo (B); these are shown as ChIP amplicons R1 (preCRM1 in intron 1) and R2 (preCRM4 in intron 2) along with a negative control (“U”) located upstream of the Fog1 start site for transcription. The information in the first 3 tracks was obtained by queries to GALA⁴¹  and the data were visualized in the UCSC Genome Browser.³⁹  (B) Chromatin immunoprecipitation (ChIP) experiments showing GATA-1 occupancy at the Fog1 locus in G1E-ER4 cells at 24 hours after GATA-1 activation. The regions of the Fog1 gene examined are indicated as “ChIP amplicons” in line 4 of panel A.Antibodies used are indicated below the figure: G-1 indicates anti–GATA-1; ER, anti–estrogen receptor (used to detect the GATA-1/ER fusion protein). Control antibodies: rIgG indicates rat IgG; mIgG, mouse IgG. Each data point represents the average value obtained from 2 independent ChIP experiments. (C) Predicted cis-regulatory module 1 enhances expression from an erythroid promoter. The plasmid gammaLuc contains a firefly (FF) luciferase gene driven by the γ globin gene promoter, and preCRM1 (R1) was added upstream of the promoter to make the plasmid R1 gammaLuc. The normalized levels of expression are plotted for 2 experiments, with each sample tested in triplicate in both; the error bars indicate standard deviations. The activity from R1 gammaLuc is significantly higher than that from gammaLuc, with a P value no more than .005 for either by Student t test. (D) GATA1 and Fog1 act in a feed-forward loop to activate the β major globin gene.