R216Q and D218G mutations disrupt LMO2 binding and produce unique transcriptional signatures. (A) Anti-GATA1 and anti-LMO2 ChIP in G1E cells expressing wild-type or mutant GATA1 after 24 hours of E2 treatment using primers as in Figure 3D and E. LMO2 ChIP signals were normalized to GATA1 ChIP signals at each site. Error bars denote SEM (n = 3). (B) A portion of the ¹⁵N-HSQC spectra of ¹⁵N-LMO2_LIM2-Ldb1_LID (red peaks) following addition of 1 equivalent of either GATA1 NF (green), R216Q (cyan), R216W (gold), or D218G (purple). R216W caused peak shifts similar to those induced by wild type, while D218G induced qualitatively similar shifts that were smaller in magnitude, and R216Q did not result in significant shifts to any peaks. (C) Relative weighted average change in chemical shift position of resonances from LMO2_LIM2-Ldb1_LID (B) following addition of wild-type or mutant GATA1 NF. Shown are the average shifts (±SD) of 3 separate resonances for each series of titrations. *P < .05. (D) Unsupervised hierarchical clustering of G1E cells expressing wild-type or mutant GATA1 based on expression profiling with microarrays. One D218G replicate is indistinguishable from the R216Q replicates. (E) Venn diagrams of direct GATA1-activated target genes significantly downregulated when compared with wild-type GATA1. (F) Expression of GATA1-regulated genes highly sensitive to TAL1 complex disruption was validated by RT-qPCR, normalized to β-actin, and plotted as fold change from uninfected samples. Error bars denote SEM (n = 3). (G) Anti-GATA1 and anti-LMO2 ChIP in G1E cells expressing wild-type or mutant GATA1 after 24 hours of E2 treatment using primers against genes significantly impaired in response to R216Q and D218G mutations. LMO2 ChIP signals were normalized to GATA1 ChIP signals at each site. Error bars denote SEM (n = 3).