Iron and Heme Coordinate Erythropoiesis through HRI-Mediated Regulation of Protein Translation and Gene Expression

Iron and heme play central roles in red blood cell production. However, the mechanisms by which iron and heme levels coordinate erythropoiesis remain incompletely understood. HRI is a heme-regulated kinase that controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. By defining the underlying changes in translation during iron and HRI deficiencies, we validate known regulators of this process, including Atf4, and identify novel pathways such as co-regulation of ribosomal protein mRNA translation. Surprisingly, we found that heme and HRI pathways, but not iron-regulated pathways, mediate the major protein translational and transcriptional responses to iron deficiency in erythroblasts in vivo and thereby identify previously unappreciated regulators of erythropoiesis. Our genome-wide study uncovers the major impact of the HRI-mediated integrated stress response for the adaptation to iron deficiency anemia.


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Iron deficiency anemia is estimated to affect one-third of the global population (Lopez 36 et al. 2016). In addition to being key components of hemoglobin, the primary oxygen 37 transport molecule, cellular iron and heme levels impact globin synthesis and red blood 38 cell production. Specifically, globin is transcriptionally regulated by BACH1 (BTB Domain 39 and CNC homolog 1) (Igarashi and Sun 2006) and regulated at the level of protein

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The following table supplement is available for Figure 1:

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Since Hri and Atf4 are among the most highly expressed and efficiently translated 156 mRNAs in Wt EBs (top 3%, Table 1), we investigated whether Atf4 mRNA was poised for

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Among the four eIF2α kinases, Hri (Eif2αk1) is most highly expressed in EBs, about 2 orders of the 163 magnitude of Pkr (Eif2αk2) and Perk (Eif2αk3). Eif2αk4 (Gcn2) was expressed at a level lower than 164 the cut-off.

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The following table supplement is available for

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HRI-ATF4 mediated mRNA expression are most highly upregulated in ID

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We next investigated the transcriptome impacts of ID and the role of HRI in mediating 217 the cellular response to this stress. Analysis of mRNA-seq data revealed that there were 218 substantially more genes that displayed significant differential expression between Wt-Fe 219 and Wt+Fe EBs than between Hri -/--Fe and Hri -/-+Fe EBs (232 vs 37, Figure 4A and Table   220 supplement 5), demonstrating the near-absolute requirement for HRI in regulating the

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We employed ex vivo FL differentiation to interrogate the function of Grb10 in 302 erythropoiesis.

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First, we validated that Grb10 and Atf5, but not Atf4, RNA expression was increased 304 in Wt EBs, but not Hri -/-EBs, during ID ( Figure 5C). In addition, Grb10 expression in Wt

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EBs was increased during ex vivo erythroid differentiation from 20 to 30 hours, and was 306 greatly reduced in Hri -/-, AA and Atf4 -/erythroblasts ( Figure 5D). We prepared five shRNA 307 recombinant retroviruses, all of which were able to knockdown Grb10 RNA expression 308 greater than 80% during the expansion phase. However, only one, shRNA_G3, was able 309 to maintain persistent knockdown of Grb10 RNA during differentiation ( Figure 6A (Table supplement 2 and Table supplement 5). The exact role of HRI in HbF production 463 remains to be clarified.

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In summary, our genome-wide study reveals the prominent contribution of HRI-ISR

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Gene expression was performed by RT-qPCR and Western blot analyses as 536 previously described (Zhang et al. 2018). Primers were listed in Table supplement 7.

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Gapdh was used as internal control for RT-qPCR. Antibodies used in Western blot were 538 described in           Figure   865 6.

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Differentially expressed genes from mRNA-seq data were determined using DESeq2 969 package of R/Bioconductor, and genes with the mean of reads of all conditions greater 970 than 100 were used for further analysis (Table supplement 5