SummaryAs the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.

Summary Human trisomies can alter cellular phenotypes and produce congenital abnormalities such as Down syndrome (DS). Here we have generated induced pluripotent stem cells (iPSCs) from DS fibroblasts and introduced a TKNEO transgene into one copy of chromosome 21 by gene targeting. When selecting against TKNEO, spontaneous chromosome loss was the most common cause for survival, with a frequency of ∼10−4, while point mutations, epigenetic silencing, and TKNEO deletions occurred at lower frequencies in this unbiased comparison of inactivating mutations. Mitotic recombination events resulting in extended loss of heterozygosity were not observed in DS iPSCs. The derived, disomic cells proliferated faster and produced more endothelia in vivo than their otherwise isogenic trisomic counterparts, but in vitro hematopoietic differentiation was not consistently altered. Our study describes a targeted removal of a human trisomy, which could prove useful in both clinical and research applications.

Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45 ؉ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45 ؊ erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (⑀) and fetal (␥) globins, with little or no adult globin (␤). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms. IntroductionDuring human development, hematopoietic cells sequentially recruit new anatomic sites for their development, from the yolk sac, to the fetal liver, to the bone marrow (BM) in adults. Erythroid cells developing at these sites are distinguished morphologically, and they display distinct transcriptional factor and growth factor requirements, proliferative kinetics, and globin patterns. 1 Thus, erythroid cells maturing in yolk sac (primitive erythroid cells) have a characteristic morphology: they remain mostly nucleated at terminal maturation and synthesize mainly embryonic globins (⑀, , and ␣). Fetal cells have a macrocytic cell morphology and synthesize more than 80% fetal globins (␣2␥2), in contrast to adult cells that synthesize more than 90% adult globins (␣2␤2). Fetal and adult cells in circulation are enucleated, and both are considered of definitive type.Due to the transient nature of primitive erythropoiesis and because of ethical concerns in conducting experiments in human embryos, the regulation of primitive erythropoiesis has remained inadequately explored. Extensive research with murine embryonic stem (ES) cells differentiated through embryoid body (EB) formation and directed hematopoietic differentiation has shown that it recapitulates the earliest stages of murine hematopoietic development, as the appearance of primitive hematopoietic cells was followed by the emergence of definitive cells expressing the appropriate globin phenotypes. 2,3 Similar studies with human ES cells have been conducted only recently. [4][5][6][7][8][9][10][11] However, there are discrepancies among the studies published regarding the kinetics as well as the morphology and globin patterns of erythroid cell...

Regulatory regions harbor multiple transcription factor recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe a facile approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

Inappropriately low reticulocytosis may exacerbate malarial anemia, but the underlying mechanism is not clear. In this study, naive and infected mice were treated with recombinant murine erythropoietin (EPO), and the upstream events of erythropoiesis affected by blood-stage Plasmodium chabaudi AS were investigated. Malaria infection, with or without EPO treatment, led to a suboptimal increase in TER119 ؉ erythroblasts compared with EPO-treated naive mice. Furthermore, a lower percentage of TER119 ؉ erythroblasts in infected mice were undergoing terminal differentiation to become mature hemoglobinproducing erythroblasts. The impaired maturation of erythroblasts during infection was associated with a shift in the transferrin receptor (CD71) expression from the TER119 ؉ population to B220 ؉ population. Moreover, the suboptimal increase in TER119 ؉ erythroblasts during infection coincided with a blunted proliferative response by splenocytes to EPO stimulation in vitro, although a high frequency of these splenocytes expressed EPO receptor (EPOR). Taken together, these data suggest that during malaria, EPO-induced proliferation of early EPORpositive erythroid progenitors is suppressed, which may lead to a suboptimal generation of TER119 ؉ erythroblasts. The shift in CD71 expression may result in impaired terminal maturation of these erythroblasts. Thus, inadequate reticulocytosis during malaria is associated with suppressed proliferation, differentiation, and maturation of erythroid precursors. IntroductionMalaria is an important etiologic factor for severe anemia among children and pregnant women in malaria-endemic areas, especially in sub-Saharan Africa. [1][2][3] The mortality rate of malaria-related anemia is between 5.6% and 16% for children [4][5][6] and 6% for pregnant women, especially in primigravidae. 7,8 The cause of severe malarial anemia is considered to be multifactorial. It has been proposed that increased destruction of red blood cells (RBCs), including the rupture of infected RBCs by schizonts and increased clearance of infected and uninfected RBCs due to hemolysis and/or phagocytosis, 9-11 is a major contributing factor. 12 In addition, insufficient erythropoiesis, as evidenced by inappropriately low numbers of reticulocytes in the peripheral blood of some malaria patients with anemia, 13,14 may fail to alleviate malarial anemia and aggravate the severity of the disease.When anemia occurs, tissue hypoxia results in increased renal erythropoietin (EPO) production. 15 Subsequently, EPO travels systemically to hematopoietic tissues, including bone marrow and spleen, to stimulate erythropoiesis via binding to the EPO receptor (EPOR), which is expressed on erythroid progenitors, especially the erythroid colony-forming unit (CFU-E) and proerythroblasts. [16][17][18] As a result, an increased number of reticulocytes is released into the bloodstream to alleviate anemia. Therefore, inappropriately low reticulocytosis during malaria infection may be the result of inadequate EPO production and/or a suboptimal ...

Severe anemia is a major life-threatening complication of malaria. The roles of erythropoietin (Epo) and erythropoiesis during blood-stage malaria were investigated. By treating Plasmodium chabaudi AS-infected C57BL/6 (B6) mice, which are resistant to malaria, with polyclonal anti-human Epo neutralizing antibody, we demonstrated that Epo-induced reticulocytosis was important for alleviating malarial anemia and for host survival. By inducing erythropoiesis in A/J mice, which are susceptible to malaria, and in B6 mice at various periods during infection, by use of exogenous recombinant murine Epo, untimely onset of reticulocytosis was shown to augment multiplication of parasites and result in lethal infection. However, timely inducement of reticulocytosis with Epo treatment alleviated malarial anemia and increased survival. Our data reveal the important role of Epo-induced reticulocytosis in modulating the course and outcome of blood-stage malaria. However, the mechanisms underlying the increased mortality associated with untimely treatment with Epo and the increased protection associated with timely treatment with Epo remain to be investigated.

The important contributions of the a4 integrin VLA-4 and the CXCR4/SDF-1 axis in mobilization have been demonstrated and thereby, these pathways can be suggested as rational targets for clinical stem cell mobilization in the absence of cytokine use. a4-blockade alone (in humans, macaques and mice), or genetic ablation of a4-integrin in mice, provides reproducible, but modest mobilization. Similarly, CXCR4 blockade with small-molecule antagonists mobilizes hematopoietic stem cells in all three species, but at least with the established single-injection schedule, the mobilization efficiency is marginally sufficient for clinical purposes. Hypothesizing that the different molecular targets (a4-integrin vs. CXCR4) might allow for additive mobilization effects, we therefore tested the efficacy of the combination of a4-integrin blockade with antifunctional antibodies and CXCR4 blockade with the small-molecule inhibitor AMD3100 in macaques, or the combination of conditional a4-integrin ablation and AMD3100 in mice. Mobilization was at least additive. While the prolonged effects of a4-blocking antibodies may not be suitable for clinical mobilization, future availability of small-molecule a4-antagonists in combination with AMD3100 could provide an alternative to granulocyte colony-stimulating factor. STEM CELLS 2009;27:836-837