Mitochondria Coordinate Intracellular Metabolism and Epigenetic Gene Regulation during Erythropoiesis

The developing hematopoietic system requires an exquisite coordination of gene expression and metabolism. Mitochondria are critical for heme and iron metabolism in erythroid cells, yet their regulation during normal erythropoiesis remains largely unknown. Previously we determined the genome-scale protein and mRNA expression in human primary CD34+ hematopoietic stem/progenitor cells (HSPCs) and erythroid precursors (proerythroblasts or ProEs) by mass-spectrometry-based quantitative proteomics and RNA-seq analysis, respectively. In-depth proteomic profiling resulted in identification and quantification of proteins encoded by 14,502 genes, 13,341 of which are assigned to their corresponding transcripts detected by RNA-seq.

Through unbiased comparison of the proteomic and transcriptomic changes between HSPCs and ProEs, we unexpectedly uncovered major pathways related to mitochondrial biogenesis enhanced through post-transcriptional mechanisms. Mitochondrial activities, as measured by mitochondrial mass, membrane potential and intracellular ATP, and associated metabolites are dynamically regulated during erythroid development. Expression of mitochondrial factors including mitochondrial transcription factor A (TFAM), a key regulator of mitochondrial DNA replication and transcription, is markedly increased without changes in the cognate mRNAs during erythropoiesis. Consistently, through analysis of protein synthesis by polysome profiling in HSPCs and ProEs, we demonstrated that mitochondria-associated proteins are specifically increased through enhanced protein translation.

To establish the in vivo role of mitochondria in normal erythroid development, we generated Tfam conditional knockout (KO) mice by an erythroid-specific EpoR-Cre allele. Tfam KO embryos are anemic and display defective erythroid differentiation of CD71⁺Ter119^- progenitor cells to lineage-committed CD71⁺Ter119⁺ erythroid cells. By RNA-seq analysis of Tfam WT and KO E13.5 fetal liver Ter119⁺CD71⁺ cells, we found that Tfam KO erythroid cells are severely impaired in mitochondrial biogenesis and metabolism. Specifically, mitochondrial signature genes and genes associated with reactive oxygen species pathway, TCA cycle and heme metabolism are significantly downregulated, whereas HSPC-specific genes are markedly upregulated in Tfam KO erythroid cells. Changes in intracellular metabolism often results in altered epigenetic gene regulation. We then measured the levels of histone modifications in Tfam KO erythroid cells. Strikingly, the levels of acetylated histones including H3K9ac and H3K27ac are markedly increased, whereas the levels of methylated or unmodified histones remain largely unchanged, indicating that Tfam loss leads to a hyperacetylated chromatin. By ChIP-seq analysis of H3K27ac in Tfam WT vs KO erythroid cells, we found that histone acetylation is significantly increased at HSPC-specific genes, consistent with the derepression of HSPC genes in Tfam-deficient erythroid cells. To establish the causality, we found by metabolomic profiling that Tfam-deficient erythroid cells display defective intracellular metabolism affecting several major metabolic pathways. Most importantly, the metabolic intermediate β-hydroxybutyrate (βOHB), a ketone body synthesized from acetyl-CoA, is significantly upregulated and functions as a potent HDAC inhibitor, resulting in histone hyperacetylation in Tfam-deficient erythroid cells. Treatment of human HSPCs with βOHB significantly impairs erythroid differentiation and gene expression, similar to the phenotypes observed in Tfam KO erythroid cells.

Collectively, our studies support a mechanism for proper regulation of mitochondrial biogenesis by post-transcriptional machinery, and establish a new molecular link between mitochondrial biogenesis, metabolism and epigenetic gene regulation indispensable for normal erythropoiesis. Mitochondrial dysfunction leads to a variety of human disorders with impaired erythropoiesis being a severe manifestation in a subset of these diseases. Therefore, our findings provide a mechanistic explanation for the broad notion that red blood cells are uniquely sensitive to perturbations that affect protein translation and/or mitochondrial activities, and may have direct relevance to the hematological defects associated with mitochondrial diseases and aging.