Quantitative Proteomic and Transcriptomic Analysis Reveals Post-Transcriptional Regulation of Mitochondrial Biogenesis during Erythropoiesis
Authors: Liu Xin , Zhang Yuannyu , Zhou Feng , Shao Zhen , Signer Robert , Cao Hui , Hu Zeping , DeBerardinis Ralph , Xu Jian 
Published: October 2019

Abstract
The differentiation and maturation of erythroid cells require highly regulated patterns of gene expression and metabolism. Mitochondria are critical for heme biosynthesis and iron metabolism in erythroid cells, yet their regulation during normal erythroid maturation remains largely unexplored. Here we measured global protein and mRNA expression in primary human fetal liver and adult bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) and differentiated erythroid precursors (proerythroblasts or ProEs) by mass-spectrometry-based quantitative proteomics and RNA-seq analysis, respectively. In-depth proteomic profiling resulted in identification and high-quality quantification of proteins encoded by 14,502 genes, accounting for 72.4% of the total annotated protein-coding genes in humans. Through unbiased and comprehensive comparison of the proteomic and transcriptomic dynamics between primary HSPCs and committed erythroid precursors, we discovered a number of previously unrecognized regulatory pathways essential for normal erythropoiesis.
Importantly, we identified key pathways related to mitochondrial biogenesis, including ATP biosynthesis, electron transport chain, oxidative phosphorylation, and cellular respiration, whose protein expression was substantially induced during erythropoiesis. Strikingly, the increases in protein expression were not paralleled by changes in mRNA expression of these genes, suggesting that they are regulated through post-transcriptional mechanisms. Consistent with the enhanced mitochondrial protein expression, we observed substantial increases in mitochondrial membrane potential (MMP) and intracellular ATP level during early erythroid differentiation before clearance of mitochondria at terminal maturation stages. We next profiled metabolite levels in HSPCs and erythroid precursors at various differentiation stages, and observed progressive progressive alterations of metabolites from the tricarboxylic acid cycle and other mitochondrial pathways during erythroid maturation. Furthermore, we identified two principal mitochondria-associated transcription factors, mitochondrial transcription factor A (TFAM) and Prohibitin 2 (PHB2), whose protein but not mRNA expression was substantially increased during erythroid maturation. Depletion of TFAM or PHB2 expression markedly impaired erythroid differentiation from primary human CD34+ HSPCs. TFAM or PHB2-deficient cells displayed impaired erythroid differentiation, proliferation and hemoglobin expression, reduced mitochondrial mass, membrane potential and ATP synthesis, and increased apoptosis. Pharmacological inhibition of mitochondrial activities by targeting mitochondrial complex I (metformin, phenformin, and rotenone), complex III (antimycin A), or complex V (oligomycin) in CD34+ HSPCs impaired erythroid differentiation in a dose-dependent manner, consistent with an essential role of mitochondria for normal erythroid development.
To elucidate the regulatory mechanisms underlying the developmental control of mitochondrial biogenesis, we measured protein synthesis rate in CD34+ HSPCs and differentiated erythroid cells by analyzing the incorporation of the methionine analogue HPG (L-homopropargylglycine). The rate of HPG incorporation was the lowest in undifferentiated CD34+, and increased by 1.8 and 2.5-fold in CD71+CD235a- and CD71+CD235a+ erythroid progenitor cells, respectively. Consistent with the progressive increase in protein synthesis, we observed a gradual increase of mTORC1 signaling, as measured by the phosphorylated eIF4-E binding protein (4E-BP1) and S6 kinase (p70S6K), during erythroid differentiation of CD34+ HSPCs. Inhibition of mTORC1 signaling by active-site mTOR inhibitors markedly impaired expression of mitochondria-associated proteins, mitochondrial mass and membrane potential, and erythroid differentiation of CD34+ HSPCs. Thus, our results support a model that mitochondrial biogenesis is highly regulated through mTORC1-mediated activation of protein translation during erythroid lineage specification. Our studies also suggest a novel mechanism for proper regulation of mitochondrial biogenesis by post-transcriptional machinery, and may have direct relevance to the hematological defects associated with human mitochondrial diseases and aging.

Disclosures
DeBerardinis: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Peloton Therapeutics: Membership on an entity's Board of Directors or advisory committees.