Dissecting Molecular Pathways That Underlie Disease-Causing Gata1 Mutations

Each mammalian cell type has a unique gene expression pattern that supports its specialized function. Mutations in factors that regulate gene expression can disrupt normal function and cause human disease, though the mechanistic consequences of these defects are often unknown. Here, we address how alterations in the transcription factor GATA1 lead to distinct hematologic disorders by combining structural, biochemical, and genomic approaches with gene complementation systems that examine GATA1 function in biologically relevant cellular contexts. We first investigated missense mutations in the GATA1 N-terminal zinc finger (NF) and found that NF mutations impair association with essential GATA1 cofactors. Several NF mutations diminish FOG1 binding, resulting in greatly reduced transcriptional activation and repression. This severely impairs erythroid and megakaryocyte maturation and correlates with pronounced clinical phenotypes. Notably, clinical severity parallels the degree of FOG1 disruption. Unexpectedly, NF mutations shown to disrupt DNA binding of GATA1 in vitro did not measurably affect target gene occupancy in vivo. Rather, one of these falls into a subset of mutations that diminish TAL1 complex binding. Reduced association with the TAL1 complex moderately impairs transcriptional activation, resulting in subtle defects in erythroid and megakaryocyte development that correlate with relatively mild disease presentations. Remarkably, different substitutions at the same amino acid position can selectively inhibit TAL1 complex or FOG1 binding, producing distinct cellular and clinical phenotypes. We next examined splice site mutations in the second exon of GATA1 that lead to the expression of an amino-truncated protein called GATA1 short (GATA1s). We found that GATA1s was significantly impaired in binding to erythroid-specific target genes, while occupancy at megakaryocyte-specific genes was normal. This results in a strongly diminished erythroid gene expression program and inhibits erythroid maturation, similar to the phenotype observed in patients. In concert, our findings uncover novel molecular mechanisms that link genetic defects in GATA1 to cellular and human phenotypes. Applying this knowledge to the clinic should improve patient diagnosis, classification, and treatment. More broadly, this work highlights the power of gene complementation assays for elucidating the underlying basis of disease, and serves as a model for the study of other disease-causing mutations.