Mutation in Erythroid-Specific Transcription Factor EKLF/KLF1 Elevates Erythroid Metabolism and Induces Inflammation

EKLF/Klf1 a master transcriptional activator that regulates erythroid maturation and fate specification. The C-terminus of EKLF contains three Zn-fingers that bind to DNA. A semi-dominant mutation at a single position (E339D) in one allele of the mouse EKLF Zn-finger causes neonatal anemia (Nan). Interestingly, a mutation at the same position in human EKLF (E325K) causes type IV congenital dyserythropoietic anemia (CDA IV). Bulk RNA-seq analysis of Nan/+ fetal liver (FL) erythroid cells reveals that several enzymes involved in metabolic pathways are altered in their expression compared to wild-type (WT) littermates. Here we address metabolomic consequences in the erythroid cell of the Nan-EKLF genetic mutation to understand better the processes that regulate red cell metabolism.

Using liquid chromatography (LC) and gas chromatography (GC) mass spectrometry (MS) platforms, we analyzed the metabolome of erythroid cells isolated from embryonic day (E.13.5) Nan/+ FL compared with their WT littermates. After quality control and identification, PCA and PLS-DA plots of total metabolites demonstrated a clear separation between the 2 groups.

We find a significant elevation of the L-carnitine levels that normally decrease during erythroid differentiation besides other intermediate metabolites. L-carnitine is involved in lipid metabolism by transporting fatty acids into mitochondria, which oxidize and convert into energy through the tricarboxylic acid (TCA) cycle. Moreover, pyrimidine and purines, which are the basic components of nucleotides and provide the necessary energy and cofactors for cell survival and proliferation, are significantly reduced in Nan/+.

We find that intermediate metabolites related to the TCA cycle and glycolysis are significantly elevated in Nan/+. This, together with the increase of glucose and lactic acid, suggests rapid and high glucose consumption during glycolysis in Nan/+ compared to WT. TCA cycle converts acetyl-CoA from carbohydrates, fats, and amino acids into ATP, thus regulating oxidative metabolism in cells. Glycerolipid metabolism-related metabolites are significantly elevated in Nan/+. Dysregulation of glycerolipid may contribute to high glucose consumption.

We also find several essential and non-essential amino acids required for L-carnitine synthesis and in mitochondrial metabolism are elevated significantly in Nan/+. Furthermore, several metabolites essential for the transport of electrons in the mitochondrial respiratory chain are increased in Nan/+ significantly. Fatty acids are metabolites required for structural and functional membrane synthesis, and these are significantly elevated in Nan. Altogether these analyses of Nan/+ FL erythroid cells show that there is a general dysregulation of metabolites in the Nan/+ red cell resulting from the genetic changes that follow Nan-EKLF expression.

To complement these latter studies, we measured mitochondrial activities in FL erythroid cells isolated from Nan/+ or WT. We observe increased mitochondrial DNA (mtDNA), membrane potential, and reactive oxygen species in Nan/+ FL erythroid cells as compared to WT. Intracellular ATP levels are enhanced in Nan/+ FL erythroid cells, which also show a higher uptake of glucose and contain more 2NBDG+ cells as compared to WT. Electron and super resolution microscopy analysis of Nan/+ FL erythroid cells show extended, linked mitochondrial networks consistent with a hyperfused phenotype compared to WT. We discovered that cytosolic mtDNA release in Nan/+ FL erythroid cells results in higher expression of interferon-stimulated genes, activation of the cGAS-STING pathway, enhanced phosphorylation and nuclear accumulation of p-IRF-3 and -7, and increased transcription of IFNα, ß, and γ.

In sum, Nan-EKLF expression, even in the presence of WT EKLF, exerts dramatic, dominant effects on the metabolic properties of the erythroid cell. A range of amino acids, nucleotides, and intermediary metabolites of all types are altered from their normal levels. As a result: mutant EKLF-expressing cells require more energy for their survival and longevity; the increase in mitochondrial metabolism generates a greater uptake of glucose; mitochondrial morphology is altered; and an inflammatory response is generated. These observations model and are likely directly relevant to the dyserythropoiesis seen in CDA type IV patients.