Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability

Abstract

The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development

“…(Gillinder et al, 2016) support the initial genotypic analysis of the Nan mouse (Siatecka et al, 2010b), showing a dysregulated erythroid transcript pattern in spite of the heterozygosity of the KLF1 mutation. For example (Fig.…”

“…We found additional evidence for disarray in iron utilization components within the Nan/+ erythroid cell (Gillinder et al, 2016). For example, when comparing Nan/+ cells with wild type we see a slight decrease in transferrin receptor (Tfrc) levels, and a slight increase in ferroportin (Slc40a1) in spite of the increase in hepcidin (Fig.…”

“…5′CCMCGCCCN) compared with wild type (i.e. 5′CCMCRCCCN) (Siatecka et al, 2010b), our recent studies show that there is a second consequence of the E339D mutation in Nan-KLF1, namely that a different recognition sequence (5′CCMNGCCCN) is newly recognized (Gillinder et al, 2016). We considered whether this might play an additional role in the anemia and the phenotypic properties observed throughout development in Nan/+ embryos and adults, specifically whether the new targets would give rise to aberrant expression of unusual/unexpected proteins in Nan erythroid cells that yield deleterious effects.…”

“…We began by focusing on the relatively small number (∼80) of genes that are newly and most highly expressed in Nan/+ cells compared with wild type (Gillinder et al, 2016), and further parsed this to genes that are secreted and thus could systemically affect normal erythropoiesis via the circulation. Two genes in particular caught our attention.…”

“…The molecular explanation behind this remains unclear. However, the change also confers recognition of an altered sequence that is not normally associated with KLF1 recognition (Gillinder et al, 2016), such that genes that are never seen in the erythroid cell are ectopically expressed in the Nan/+ mutant.…”