Localization of R307C/H in an IDR with a role in nuclear localization and GATA1 dosage-dependent target gene expression. (A) Computational prediction of IDRs across the full GATA1 protein using the indicated algorithm. The dotted line represents the R307 residue. Predictions from 5 additional algorithms also all indicate that the residue is part of a disordered domain (supplemental Figure 4A). (B) Nuclear localization signal annotation and score for amino acid (aa) sequences in R307C/H mutants and GATA1 WT. (C) Western blot of cytoplasmic and nuclear fractions of G1E cells expressing indicated GATA1 constructs at day 2 postinfection (pi). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and LaminB were used as cytoplasmic and nuclear markers to validate purity of fractionation. (D) Schematic of experiment. G1E-ER4 cells were incubated with increasing β-estradiol concentrations to titrate GATA1 nuclear translocation, transcriptional activity, and erythroid differentiation as measured by Ter119⁺ surface marker expression and RNA-seq. (E) Depiction of 2 gene sets with impaired (left; n = 264) and equivalent (right; n = 502) induction of gene expression in R307C/H mutant compared with GATA1 WT transduced G1E cells (from Figure 3). Color bar: gene expression induction (percentage of GATA1 WT). (F) Log₂ fold change (FC) for each gene set as in panel E is shown for each β-estradiol–dependent RNA-seq sample from panel D. Error bars represent 1 standard error of the mean. Annotated points indicate the equivalent β-estradiol concentration representing the log₂ FC for each gene set as observed in R307C/H and GATA1 WT transduced G1E cells as in panel E and Figure 3. (G) Correlation of gene expression profiles for R307C/H and GATA1 WT transduced G1E cells compared with variable β-estradiol level–treated G1E-ER4 cells. The mean Pearson correlation per population is shown. NLS, nuclear localization signal.