A Potential Role For Genes Within The Mtorc1 Pathway In The Regulation Of Embryonic Globin Expression Revealed By Chip-Seq, Rna-Seq And Eqtl Mapping In The Nan Anemia Mouse Model

The Nan mouse model carries a mutation in the gene encoding the erythroid-specific transcription factor, Klf1 (Krüppel-like factor 1). KLF1 regulates the terminal development of erythroid cells via the transcriptional regulation of a diverse set of genes. We recently showed by ChIP-seq and RNA-seq that Nan-KLF1 fails to bind a subset of normal KLF1 gene targets and also ectopically binds a large set of genes not normally engaged by KLF1. The result is a corrupted transcriptome that produces anemia and a ~100-fold increase in βh1 expression in adult Nan spleen and bone marrow. Genetic modifiers regulating βh1 expression in mice can potentially be translated to the regulation of human fetal γ-globin expression and identify new drug targets for the treatment of Sickle Cell Disease and β-thalassemia, as increased fetal globin ameliorates disease symptoms. We identified genetic loci modifying βh1 expression by eQTL (expression quantitative trait locus/loci) mapping using two congenic Nan strains intercrossed with C57BL/6J. We identified several significant and suggestive eQTL in the two crosses, indicating that multiple genes regulate βh1 expression in adult Nan spleen. Notably, no previously described repressors of γ-globin expression mapped within the eQTL 95% confidence intervals. By integrating genes falling within eQTL confidence intervals on Chr 7, 4, 11, and 17 with ChIP-seq data obtained using an immortalized Klf1 -null fetal erythroid cell line (K1) expressing either WT or Nan Klf1, RNA-seq data from sorted WT and Nan adult spleen erythroid precursors (pro-, baso-, poly- and orthochromatic erythroblasts), and available functional annotations (DAVID and Ingenuity pathway analysis), we identified several strong candidate genes. Among them, several genes encoding components of the mTORC1 pathway are located in eQTL confidence intervals and/or have altered binding by Nan-KLF1 resulting in a dramatic decrease of their expression in Nan. The mTORC1 pathway regulates various cellular functions upon activation by nutrients and growth factors. In prior studies, decreased Mtor (mechanistic target of rapamycin) or Rptor (regulatory associated protein of MTOR complex) induced increased expression of fetal globin, providing compelling rationale that additional genes in this pathway may induce a significant increase in βh1 expression in Nan . In our eQTL dataset, Rptor, Tsc2 (tuberous sclerosis 2), and Mlst8 (MTOR associated protein, LST8 homolog), three genes that belong to the mTORC1 pathway, are located near eQTL peaks on chromosomes 11 and 17. Nan-KLF1 fails to bind Rragd (Ras-related GTP binding),a target of KLF1 required for mTOR activation, resulting in a dramatic and progressive decrease in its expression in Nan terminally differentiating erythroid precursors (6.4, 169, 1910, and 290 fold decrease during maturation from pro- to orthochromatic erythroblasts). Rptor expression is decreased 4.1 and 10.6 fold in polychromatic and orthochromatic erythroblasts, respectively. Interestingly, the top gene ontology terms annotated for the differentially expressed genes include translation, ribosome biogenesis, redox mechanisms, cell cycle control and DNA repair. These functions are regulated by the mTORC1 pathway and strongly suggest that the Nan phenotype is influenced by its downregulation. We conclude that (1) several genes from the mTORC1 pathway, including genes not previously described, are strong candidates that may contribute simultaneously to increased βh1 expression in Nan and (2) functions regulated by mTORC1 are strongly dysregulated by the Nan mutation and reinforce the hypothesis that this pathway plays a key role in the Nan phenotype. The identification of additional modifiers of βh1 in Nan that can be translated to human HbF may provide new therapeutic opportunities. Moreover, given the role of nutrition in the regulation of the mTORC1 pathway, new nutritional guidelines for patients with Sickle Cell Disease and β-thalassemia may emerge.