Science Spotlight

Methylation marks make the difference in erythropoiesis

From the Paddison lab, Human Biology Division

Two hundred-billion red blood cells are renewed in our body every single day. These erythrocytes (also known as red blood cells) are generated from bi-potent megakaryocyte-erythroid progenitor cells during a maturation process called erythropoiesis. Erythropoiesis is controlled very tightly and defects in the key elements of this step-wise maturation can lead to life-threatening conditions such as severe anemia or myeloproliferative disease. Transcriptional RNA interference-dependent and translational regulations are part of the core mechanisms required for proper erythropoiesis. Recently, an mRNA modification called N6-methyladenosine (m6A) has been found to control the expansion and self-renewal of hematopoietic stem cells. The addition of the methyl group on mRNA adenosine is mediated by a methyl-transferase (MTase) complex composed of METTL3, METTL14 and Wilms’ tumor 1-associated protein (WTAP). m6A is involved in pre-mRNA processing, mRNA translation efficiency and mRNA stability. Dr. Patrick Paddison’s group (Human Biology Division) investigated the role of m6A in erythropoiesis and communicated their findings in a recent publication in Nature Communications1.

The authors first performed a genetic screen based on the CRIPSR-Cas9 system to identify regulators of erythropoiesis, using human erythroid leukemia (HEL) cells as a surrogate model for erythropoiesis. After lentiviral transduction with a library of sgRNAs, HEL cells were outgrown and CD235-negative cells were sorted (CD235 is a marker of the mature erythrocytes). Deep sequencing of the CD235--enriched population allowed them to identify genes required for proper erythroid differentiation. METTL3, METTL14 and WTAP were all identified by the screen, suggesting that m6A is involved in erythroid lineage determination.

The next step of the investigation was technically challenging, said Dr. Daniel Kuppers, the first author of the study: “Drs. Wang and Hansen He developed a refined version of the m6A IP-seq mapping technique2, enabling us to partially map the m6A transcriptome across primary human hematopoietic cells for the first time. The original method was published in 2012 by two independent groups3, 4. However, this method requires 100-300ug of RNA per sample, which had largely precluded its use to map m6A outside of cell lines and mouse tissue. It’s the first published m6A mapping in primary hematopoietic progenitor cell populations made possible by the refined protocol.” The researchers demonstrated that m6A modification was found on mRNA of many hematopoiesis and erythropoisesis regulators such as GATA1, FLI1, KLF1 and MPL. In addition, mRNA from genes involved in anemia and myelodysplasia were also found to be m6A-methylated, suggesting a role of this post-transcriptional modification in these diseases. Dr. Kuppers explained: “In our data, key genes associated with impaired erythropoiesis, including drivers of Diamond–Blackfan Anemia (DBA) and Myelodysplastic Syndrome (MDS), are m6A targets and require m6A-MTase activity for their protein expression in HEL cells. Further studies need to be done to investigate whether m6A levels are altered in these diseases and if mutations arise that alter m6A marking of specific transcripts.”

Importantly, inhibition of the MTase complex by genetic deletion of either WTAP, METTL3 or METTL14 resulted in decreased m6A abundance and eythroid differentiation defects. “We’ve shown for the first time that developing red blood cells use m6A epitranscriptomic regulation to control translation of essential erythroid proteins. Eliminating the m6A methyltransferase complex blocks stem cells’ ability to differentiate into red blood cells, but not the development of other non-lymphoid blood cell types,” said Dr. Kuppers.

m6A methylation of specific genes by the MTase complex leads to enhanced translation of epigenetic regulators which in turn control transcription of heme biosynthesis genes, anemia associated genes and novel regulators all responsible for erythroid gene expression and complete erythropoiesis.
m6A methylation of specific genes by the MTase complex leads to enhanced translation of epigenetic regulators which in turn control transcription of heme biosynthesis genes, anemia associated genes and novel regulators all responsible for erythroid gene expression and complete erythropoiesis. Illustration from publication.

Subsequent transcriptomic analysis allowed Kuppers et al. to demonstrate that m6A-mediated enhanced translation set up a transcription program involved in heme biosynthesis, anemia associated genes and other novel regulators all responsible for erythroid gene expression and complete erythropoiesis. The authors are already seeking to understand better the novel mechanism they identified: “While we established the requirement for m6A during erythroid differentiation in primary cells, most of the work in the paper was done in an erythroleukemic cell line,” said Kuppers. “We’re now focusing on investigating the role of m6A during erythropoiesis in primary cells. A few of the initial questions we’re focusing on are whether m6A is required for all stages of erythropoiesis, how m6A marking changes across the various erythroid progenitor populations, and the role of m6A in regulating hematopoietic transcription factors. We’ve also begun collaborating with Anthony Rongvaux in the Clinical Research Division, utilizing his MISTRG humanized mouse model for in vivo studies of m6A in human hematopoiesis.”

This work was supported by the AACR NextGen Grant for Transformative Cancer Research and the National Institutes of Health.

Fred Hutch/UW Cancer Consortium members Drs. Torok-Storb, Hsieh, and Paddison contributed to this research.

1.       Kuppers DA, Arora S, Lim Y, Lim AR, Carter LM, Corrin PD, Plaisier CL, Basom R, Delrow JJ, Wang S, Hansen He H, Torok-Storb B, Hsieh AC, Paddison PJ. 2019. N6-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis. Nat Commun. 10(1):4596. doi: 10.1038/s41467-019-12518-6.

2.       Zeng et al. 2018. Refined RIP-seq protocol for epitranscriptome analysis with low input materials. PLoS Biol. 16(9):e2006092. doi: 10.1371/journal.pbio.2006092.

3.       Dominissini et al. 2012. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 485(7397):201-6. doi: 10.1038/nature11112.

4.       Meyer et al. 2012 Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 149(7):1635-46. doi: 10.1016/j.cell.2012.05.003.

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