Nprl3, a gene that topologically neighbors the α-globins and was conserved for more than 500 million years among animals and fungi, was called into question. Why is it important enough to have been maintained for all these years? Alexandra Preston, a graduate student, from Professor Hal Drakesmith’s laboratory at MRC Weatherall Institute of Molecular Medicine, tackled this question with astonishing results, discovering a new mechanism that controls erythropoiesis.
The current work on Nprl3-deficient erythropoiesis in mouse models and human peripheral blood led to the discovery that Nprl3 is essential for erythropoiesis and adapting to fluctuating nutrient and growth factor conditions. Their work revealed the phenotypes of Nprl3-deficient mouse fetal liver and bone marrow as well as human peripheral blood. Notably there was severe impairment of erythroid development - failing to develop beyond the proerythroblast stage.
Nprl3 functions as a negative regulator of mTORC1, controlling cellular metabolism based on environmental input from available amino acids, iron, and erythropoietin. “This is important because red blood cell synthesis must continue while the supply of nutrients like amino acids and iron fluctuates,” stated Professor Drakesmith. In their model, loss of Nprl3 was accompanied by elevated mTORC1 signaling and, in turn, suppressed autophagy in erythroblasts. They also explored whether this was related to hematopoietic-intrinsic Nprl3 requirements by evaluating the ability of Nprl3-/- mouse fetal liver cells to restore radiated bone marrow without success, leading to the conclusion that Nprl3 regulates baseline erythropoiesis.
The CRISPR-Cas9 RNP-editing system was used to knock out Nprl3 in human CD34+ progenitors to strengthen their case. These progenitors were impaired in producing enucleated erythroid cells and were shown to have defective mTORC1 signaling responses to iron deficiency, amino acid withdrawal, and erythropoietin stimulation.
After demonstrating that Nprl3 is essential for erythropoiesis, its linkage to the α-globin genes was assessed. Regulation of the α-globin genes by the α-globin enhancers is well known. Interestingly, during erythroid lineage commitment, Nprl3 expression is elevated approximately 30-fold due to α-globin enhancer activity. The group created a model to evaluate whether increased Nprl3 expression is needed for functional erythropoiesis. Genetic engineering was used to delete the Nprl3 promoter on one allele and all α-globin enhancers on the other, eliminating interplay between Nprl3 and α-globin regulatory elements. "Embryos heterozygous for both alleles (Nprl3+/-α-globin-enhancers+/-, 'Nalph') retain non-erythroid 'un-enhanced' Nprl3 expression levels and enhancer-regulated α-globin expression,” Professor Drakesmith revealed. These embryos showed impaired erythropoiesis similar to that observed in Nprl3-/- embryos. In layman’s terms – the area of the genome within α-globin, the enhancer elements, and Nprl3, allows the metabolism of developing red blood cells to be synced with the production of hemoglobin.
Drakesmith’s group also conclude that Nprl3 function depends on transcriptional support from the α-globin enhancers. He describes this work as “a collaboration between two laboratories with different expertise: Professor Doug Higgs and his group with knowledge on transcriptional regulation of α=globin, and our laboratory with a background in metabolism.” He commends Ms. Preston, as she “managed to connect the two to make original insights into basic, and ancient, mechanisms of red blood cell synthesis.” Ms. Preston expressed that she is excited to find out whether the concept of enhancer sharing applies elsewhere in the genome. “There is a certain magic to this project that has made it a joy to pursue,” she said.
Go Alexandra!
Dr. Blackmon and Dr. Amanam indicated no relevant conflicts of interest.
