TMEM14C is required for erythroid mitochondrial heme metabolism.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Histones to the cytosol: exportin 7 is essential for normal terminal erythroid nuclear maturation.

Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7), is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly,many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus, terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by Xpo7.

Snx3 regulates recycling of the transferrin receptor and iron assimilation.

Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.

Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts.

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications.

This article reviews the regulation of production of RBCs at several levels. We focus on the regulated expansion of burst-forming unit-erythroid erythroid progenitors by glucocorticoids and other factors that occur during chronic anemia, inflammation, and other conditions of stress. We also highlight the rapid production of RBCs by the coordinated regulation of terminal proliferation and differentiation of committed erythroid colony-forming unit-erythroid progenitors by external signals, such as erythropoietin and adhesion to a fibronectin matrix. We discuss the complex intracellular networks of coordinated gene regulation by transcription factors, chromatin modifiers, and miRNAs that regulate the different stages of erythropoiesis.

Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes.

It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA polymerase II (Pol II) occupancy, and multiple posttranslational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac, and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels, in particular, H3K79me2 and H4K16ac, were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, whereas gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data map the epigenetic landscape of terminal erythropoiesis and suggest that control of transcription elongation regulates gene expression during terminal erythroid differentiation.

Mir193b-365 is essential for brown fat differentiation.

Mammals have two principal types of fat. White adipose tissue primarily serves to store extra energy as triglycerides, whereas brown adipose tissue is specialized to burn lipids for heat generation and energy expenditure as a defence against cold and obesity. Recent studies have demonstrated that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of Prdm16 (PR domain containing 16). Here, we identified a brown-fat-enriched miRNA cluster, MiR-193b-365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes markedly impaired brown adipocyte adipogenesis by enhancing Runx1t1 (runt-related transcription factor 1; translocated to, 1) expression, whereas myogenic markers were significantly induced. Forced expression of Mir193b and/or Mir365 in C2C12 myoblasts blocked the entire programme of myogenesis, and, in adipogenic conditions, miR-193b induced myoblasts to differentiate into brown adipocytes. Mir193b-365 was upregulated by Prdm16 partially through Pparα. Our results demonstrate that Mir193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis.

Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation.

Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as alpha-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

Regulation of erythrocyte lifespan: do reactive oxygen species set the clock?

The forkhead box O (Foxo) subfamily of transcription factors regulates expression of genes important for many cellular processes, ranging from initiation of cell cycle arrest and apoptosis to induction of DNA damage repair. Invertebrate Foxo orthologs such as DAF-16 also regulate longevity. Cellular responses inducing resistance to ROS are important for cellular survival and organism lifespan, but until recently, mammalian factors regulating resistance to oxidative stress have not been well characterized. Marinkovic and colleagues demonstrate in this issue of the JCI that Foxo3 is specifically required for induction of proteins that regulate the in vivo oxidative stress response in murine erythrocytes (see the related article beginning on page 2133). Their work offers the interesting hypothesis that in so doing, Foxo3 may regulate the lifespan of red blood cells, and underlies the importance of understanding the direct targets of this transcription factor and its regulation.