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GATA1 is an essential master regulator of erythropoiesis and megakaryopoiesis. Accumulating lines of evidence have shown that dynamic changes in GATA1 gene expression levels during erythropoiesis are crucial for proper erythroid differentiation. Since GATA1 is an X-chromosome gene, GATA1 knockout leads to embryonic lethal dyserythropoiesis in male mice, while heterozygous female mice can survive. In the past decade, it has become clear that germline GATA1 gene mutations leading to structural changes in the GATA1 protein are involved in congenital dyserythropoiesis in males. In contrast, decreased GATA1 expression levels, which cause embryonic lethal dyserythropoiesis in male mice, increase the risk of erythroleukemia development in female mice, while female GATA1-knockout mice do not show substantial phenotypic alterations in erythroid or megakaryocyte lineages. In this review, we summarize the recent progress in elucidating the roles of GATA1 in normal and pathogenetic erythropoiesis and discuss the possible mechanisms of pathogenesis of dyserythropoiesis and erythroleukemia.

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.

Space flight produces an extreme environment with unique stressors, but little is known about how our body responds to these stresses. While there are many intractable limitations for in-flight space research, some can be overcome by utilizing gene knockout-disease model mice. Here, we report how deletion of Nrf2, a master regulator of stress defense pathways, affects the health of mice transported for a stay in the International Space Station (ISS). After 31 days in the ISS, all flight mice returned safely to Earth. Transcriptome and metabolome analyses revealed that the stresses of space travel evoked ageing-like changes of plasma metabolites and activated the Nrf2 signaling pathway. Especially, Nrf2 was found to be important for maintaining homeostasis of white adipose tissues. This study opens approaches for future space research utilizing murine gene knockout-disease models, and provides insights into mitigating space-induced stresses that limit the further exploration of space by humans., Using Nrf2 knockout mice, Suzuki, Uruno, Yumoto et al. show that space travel activates Nrf2 signaling, which contributes to the weight gain of mice by regulating fat metabolism of white adipose tissues. This study provides insights into potential interventions to mitigate stresses that accompany space travels.

In order to assess the long-term impact of the Great East Japan Earthquake on the oral health of disaster victims and to evaluate gene-environmental interactions in the development of major oral diseases and oral-systemic associations, the oral part of two large-scale genome cohort studies by the Tohoku Medical Megabank Organization (ToMMo), including the Community-based cohort (CommCohort) study and the Birth and Three-Generation cohort (BirThree) study, have been conducted. The study population comprised 32,185 subjects, including 16,886 participants in the CommCohort study and 15,299 participants in the BirThree cohort study, recruited from 2013 to 2017. The oral studies consist of a questionnaire regarding oral hygiene behavior, clinical examinations by dentists, and oral plaque and saliva sampling for microbiome analyses, which were carried out at seven community support centers in Miyagi prefecture. The median age of all participants was 55.0 years, and 66.1% of participants were women. Almost all participants reported that they brushed their teeth more than once a day. The median number of present teeth was 27.0, and the decayed, missing and filled tooth number was 16.0, with a significant difference according to age and sex. The median periodontal pocket and clinical attachment level was 2.48 mm and 4.00 mm, respectively. Periodontal parameters increased significantly according to age, except for the accumulation of dental calculus. The oral part of these extensive cross-sectional studies provides a unique and important platform for future studies on oral health and diseases that elicit through interactions with systemic diseases, lifestyles, life events and genetic backgrounds, and contributes to researches clarifying the long-term effects of disasters on oral health.

A germ line copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP, was identified in families with myeloproliferative neoplasm (MPN). Here, we show that mice lacking both Atg2b and Gskip, but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP, the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double-knockout fetal livers was significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

Bone morphogenetic protein-9 (BMP9), a member of the transforming growth factor β (TGFβ) superfamily, plays important roles in the development and maintenance of various cell lineages via complexes of type I and type II TGFβ receptors. Endoglin is a coreceptor for several TGFβ family members, including BMP9, which is highly expressed in a particular stage of differentiation in erythroid cells as well as in endothelial cells. Although the importance of the interaction between BMP9 and endoglin for endothelial development has been reported, the contribution of BMP9 to endoglin-expressing erythroid cells remains to be clarified. To address this point, we prepared an anti-BMP9 antibody that blocks the BMP9-endoglin interaction. Of note, challenge with the antibody promotes erythropoiesis in wild-type mice but not in a mouse model of renal anemia in which erythropoietin (EPO) production in the kidneys is genetically ablated. While endoglin-positive erythroid progenitors are mainly maintained as progenitors when bone marrow-derived lineage-negative and cKit-positive cells are cultured in the presence of EPO and stem cell factor, the erythroid-biased accumulation of progenitors is impeded by the presence of BMP9. Our findings uncover an unrecognized role for BMP9 in attenuating erythroid differentiation via its interaction with endoglin on erythroid progenitors.

To elucidate the differences in ocular biometric parameters by generation and gender and to identify axial length (AL)-associated genetic variants in Japanese individuals, we analyzed Tohoku Medical Megabank Organization (ToMMo) Eye Study data.We designed the ToMMo Eye Study, examined AL variations, and conducted genome-wide association studies (GWASs).In total, 33 483 participants aged18 years who were recruited into the community-based cohort (CommCohort) and the birth and three-generation cohort (BirThree Cohort) of the ToMMo Eye Study were examined.Each participant was screened with an interview, ophthalmic examinations, and a microarray analysis. The GWASs were performed in 22 379 participants in the CommCohort (discovery stage) and 11 104 participants in the BirThree Cohort (replication stage). We evaluated the associations of single nucleotide polymorphisms (SNPs) with AL using a genome-wide significance threshold (5 × 10We identified the association of SNPs with AL and distributions of AL in right and left eyes and individuals of different sexes and ages.In the discovery stage, the mean AL of the right eye (23.99 mm) was significantly greater than that of the left eye (23.95 mm). This difference was reproducible across sexes and ages. The GWASs revealed 703 and 215 AL-associated SNPs with genome-wide significance in the discovery and validation stages, respectively, and many of the SNPs in the discovery stage were replicated in the validation stage. Validated SNPs and their associated loci were meta-analyzed for statistical significance (Our large-scale GWASs exploiting ToMMo Eye Study data identified 31 loci linked to variations in AL, 7 of which are newly reported in this article. The results revealed genetic heterogeneity and similarity in SNPs related to ethnic variations in AL.

The Tohoku Medical Megabank (TMM) project was established to provide creative reconstruction of the Tohoku area that suffered from a huge earthquake and ensuing tsunami (the Great East Japan Earthquake, GEJE). TMM aims to establish two large-scale genome cohorts and an integrated biobank managing biospecimen and related information. It supports community medicine by establishing next-generation medical systems through a combination of the prospective genome cohort studies with a total of 150,000 participants and genomic medicine. The strategies for genome analyses in TMM are to develop an elaborate genome reference panel by means of high-fidelity Japanese whole-genome sequence, to design custom single nucleotide polymorphism (SNP) arrays based on the reference panel, and to obtain genotype data for all the TMM cohort participants subsequently. Disease-associated genomic information and omics data, including metabolomics and microbiome analysis, provide an essential platform for precision medicine and personalized healthcare (PHC). Ethical, legal, and social issues (ELSI) and education are important for implementing genomic medicine. The major considerations of ELSI regarding each participant of the cohort studies are the respect for the autonomy and the protection of privacies. Moreover, developing and provide human resources not only for the TMM project but also for the social implementation of precision medicine and PHC is required. We started a pilot study of the return of genomic results for familial hypercholesterolemia (FH) as a target disease. TMM aims to establish solid platforms that support precision medicine and PHC based on the genomic and omics information and environmental and lifestyle factors of the individuals, which is one of the most advanced medical care beyond the evidenced-based medicine in the near future.

The transcription factor GATA2 regulates normal hematopoiesis, particularly in‐ stem cell maintenance and myeloid differentiation. Various heteroallelic GATA2 gene mutations are associated with a variety of hematological neoplasms, including myelodysplastic syndromes and leukemias. Here, we report that impaired GATA2 expression induces myelodysplastic and myeloproliferative neoplasm development in elderly animals, and this neoplasm resembles chronic myelomonocytic leukemia in humans. GATA2 hypomorphic mutant (G2 f GN / fGN) mice that were generated by the germline insertion of a neocassette into the Gata2 gene locus avoided the early embryonic lethality observed in Gata2‐null mice. However, adult G2 f GN / fGN mice suffered from exacerbated leukocytosis concomitant with progressive anemia and thrombocytopenia and eventually developed massive granulomonocytosis accompanied by trilineage dysplasia. The reconstitution activity of G2 f GN / fGN mouse stem cells was impaired. Furthermore, G2 f GN / fGN progenitors showed myeloid lineage‐biased proliferation and differentiation. Myeloid progenitor accumulation started at a younger age in G2 f GN / fGN mice and appeared to worsen with age. G2 f GN / fGN mice showed increased expression of transcripts encoding cytokine receptors, such as macrophage colony‐stimulating factor receptor and interleukin‐6 receptor, in granulocyte‐monocyte progenitors. This increased expression could be correlated with the hypersensitive granulomonocytic proliferation reaction when the mice were exposed to lipopolysaccharide. Taken together, these observations indicate that GATA2 hypomorphism leads to a hyperreactive defense response to infections, and this reaction is attributed to a unique intrinsic cell defect in the regulation of myeloid expansion that increases the risk of hematological neoplasm transformation.

Accumulating lines of clinical evidence support the emerging hypothesis that loss-of-function mutations of GATA2 cause inherited hematopoietic diseases, including Emberger syndrome; dendritic cell, monocyte B and NK lymphoid (DCML) deficiency; and MonoMAC syndrome. Here, we show that mice heterozygous for an arginine-to-tryptophan substitution mutation in GATA2 (G2R398W/+), which was found in a patient with DCML deficiency, substantially phenocopy human DCML deficiency. Mice heterozygous for the GATA2-null mutation (G2-/+) do not show such phenotypes. The G2R398W protein possesses a decreased DNA-binding affinity but obstructs the function of coexpressed wild-type GATA2 through specific cis-regulatory regions, which contain two GATA motifs in direct-repeat arrangements. In contrast, G2R398W is innocuous in mice containing single GATA motifs. We conclude that the dominant-negative effect of mutant GATA2 on wild-type GATA2 through specific enhancer/silencer of GATA2 target genes perturbs the GATA2 transcriptional network, leading to the development of the DCML-like phenotype. The present mouse model provides an avenue for the understanding of molecular mechanisms underlying the pathogenesis of GATA2-related hematopoietic diseases.

GATA1-deficient mice die in utero on 12.5 embryonic day (E12.5) due to a complete block of primitive erythropoiesis in the yolk sac, while GATA2-deficient mice die on E10.5 due to severe anemia and hemorrhage, since GATA2 is essential for the development of hemangioblasts, which are common precursor cells of hematopoietic stem cells and endothelial cells. In contrast, GATA3 is critical to the development of Th2 cells. However, GATA3-deficient mice die in utero before the particular phenotype of hematopoietic system emerges, which is caused by a defect in the development of nervous and renal urinary systems. It has been well elucidated that defects in the hematopoietic GATA factors disturb hematopoietic homeostasis. However, details on how GATA factor dysfunction leads to human hematopoietic diseases remain to be clarified. At the end of the twentieth century, several mutations in GATA1 gene were identified as the cause of familial thrombocytopenia. Since then, various types of hematopoietic diseases elicited by GATA1 and GATA2 dysfunctions have been reported. This review summarizes recent topics of GATA factor-related hematopoietic diseases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we “clone” the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz (“TcMAC21”). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

Down syndrome (DS) is a complex human condition, and animal models trisomic for human chromosome 21 (HSA21) genes or orthologs provide insights into better understanding and treating DS. However, HSA21 orthologs are distributed into three mouse chromosomes, preventing us from generating mouse models trisomy of a complete set of HSA21 orthologs. The only existing humanized mouse DS model, Tc1, carries a HSA21 with over 20% of protein coding genes (PCGs) disrupted. More importantly, due to the human centromere, Tc1 is mosaic (a mix of euploid and trisomic cells), which makes every mouse unique and compromises interpretation of results. Here, we used mouse artificial chromosome (MAC) technology to “clone” the 34 MB long arm of HSA21 (HSA21q). Through multiple steps of microcell-mediated chromosome transfer we created a new humanized DS mouse model, Tc(HSA21q;MAC)1Yakaz (“TcMAC21”). Constitutive EGFP expression from the transchromosome and fluorescent in situ hybridization validate that TcMAC21, containing a hybrid chromosome of HSA21q and mouse centromere, is not mosaic. Whole genome sequencing shows that TcMAC21 contains a nearly complete copy of HSA21q with 93% of intact PCGs, while RNA-seq and additional mRNA/protein expression analyses confirm that PCGs are transcribed and regulated. A battery of tests show that TcMAC21 recapitulates many DS phenotypes including morphological anomalies in heart, craniofacial skeleton and brain, pathologies at molecular and cellular level, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.Significance StatementIn the last 25 years, mouse models of trisomy 21 have supported research into Down syndrome, from defining the basis for developmental effects up to support for clinical trials. However, existing models have significant shortfalls, especially for preclinical studies. These deficiencies include incomplete or inappropriate representation of trisomic genes, absence of an extra chromosome, and mosaicism.Using cutting edge technologies we produced a mouse artificial chromosome containing the entire 34Mb long arm of human chromosome 21 and, with assisted reproductive technologies, established it in the germ line of mice. This trisomic mouse manifests developmental and functional features of Down syndrome, including hippocampal-based learning and memory deficits. This is the most complete model of Down syndrome produced to date.

Here we show that Pin1, a peptidyl-prolyl cis/trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro, is a regulatory molecule of thrombopoiesis. We found that mice lacking the Pin1 gene (Pin1-⁄- mice) formed more megakaryocytes (MKs) than wild type mice (WT mice), and that the proplatelet formation of MKs was poorer in Pin1-⁄- mice than WT mice. Treatment of Meg-01 cells, a megakaryoblastic floating cell line, with shRNA against Pin1 suppressed the proplatelet formation. Expression of tau, a microtubule associated protein was induced in MKs during proplatelet formation. Pin1 bound tau and promoted microtubule polymerization. Our results show that Pin1 serves as a positive regulatory molecule of proplatelet formation of MKs by enhancing the function of phosphorylated tau.

Erythropoietin (EPO) is a hormone that promotes proliferation, differentiation and survival of erythroid progenitors. EPO gene expression is regulated in a tissue-specific and hypoxia-inducible manner and is mainly restricted to renal EPO-producing cells after birth. Chronic kidney disease (CKD) confers high risk for renal anemia due to lower EPO production from injured kidneys. In transgenic reporter lines of mice, disruption of a GATA-binding motif within the Epo gene promoter-proximal region restores constitutive reporter expression in epithelial cells. Here, mitoxantrone and its analogues, identified as GATA factor inhibitors through high-throughput chemical library screenings, markedly induce EPO/Epo gene expression in epithelium-derived cell lines and mice regardless of oxygen levels. In contrast, mitoxantrone interferes with hypoxia-induced EPO gene expression in Hep3B cells. Cryptic promoters are created for the EPO/Epo gene expression in epithelial cells upon mitoxantrone treatment, and consequently, unique 5'-untranslated regions are generated. The mitoxantrone-induced aberrant transcripts contribute to the reporter protein production in epithelial cells that carry the reporter gene in the proper reading frame of mouse Epo gene. Thus, EPO production in uninjured adult epithelial cells may be a therapeutic approach for renal anemia in patients with CKD.

GATA2 is a key transcription factor critical for hematopoietic cell development. During the past decade, it became clear that heterozygous germline mutations in the GATA2 gene cause bone marrow failure and primary immunodeficiency syndrome, conditions that lead to a predisposition toward myeloid neoplasms, such as myelodysplastic syndrome, acute myeloid leukemia, and chronic myelomonocytic leukemia. Somatic mutations of the GATA2 gene are also involved in the pathogenesis of myeloid malignancies. Cases with GATA2 gene mutations are divided into two groups, resulting in either a quantitative deficiency or a qualitative defect in the GATA2 protein depending on the mutation position and type. In the former case, GATA2 mRNA expression from the mutant allele is markedly reduced or completely abrogated, and reduced GATA2 protein expression is involved in the pathogenesis. In the latter case, almost equal amounts of structurally abnormal and wildtype GATA2 proteins are predicted to be present and contribute to the pathogenesis. The development of mouse models of these human GATA2-related diseases has been undertaken, which naturally develop myeloid neoplasms.

Ring sideroblasts are a hallmark of sideroblastic anemia, although little is known about their characteristics. Here, we first generated mutant mice by disrupting the GATA-1 binding motif at the intron 1 enhancer of the ALAS2 gene, a gene responsible for X-linked sideroblastic anemia (XLSA). Although heterozygous female mice showed an anemic phenotype, ring sideroblasts were not observed in their bone marrow. We next established human induced pluripotent stem cell-derived proerythroblast clones harboring the same ALAS2 gene mutation. Through coculture with sodium ferrous citrate, mutant clones differentiated into mature erythroblasts and became ring sideroblasts with upregulation of metal transporters (MFRN1, ZIP8, and DMT1), suggesting a key role for ferrous iron in erythroid differentiation. Interestingly, holo-transferrin (holo-Tf) did not induce erythroid differentiation as well as ring sideroblast formation, and mutant cells underwent apoptosis. Despite massive iron granule content, ring sideroblasts were less apoptotic than holo-Tf-treated undifferentiated cells. Microarray analysis revealed upregulation of antiapoptotic genes in ring sideroblasts, a profile partly shared with erythroblasts from a patient with XLSA. These results suggest that ring sideroblasts exert a reaction to avoid cell death by activating antiapoptotic programs. Our model may become an important tool to clarify the pathophysiology of sideroblastic anemia.

Transcription factor GATA3 plays vital roles in inner ear development, while regulatory mechanisms controlling its inner ear-specific expression are undefined. We demonstrate that a cis-regulatory element lying 571 kb 3′ to the Gata3 gene directs inner ear-specific Gata3 expression, which we refer to as the Gata3 otic vesicle enhancer (OVE). In transgenic murine embryos, a 1.5-kb OVE-directed lacZ reporter (Tg(OVE-LacZ)) exhibited robust lacZ expression specifically in the otic vesicle (OV), an inner ear primordial tissue, and its derivative semicircular canal. To further define the regulatory activity of this OVE, we generated Cre transgenic mice in which Cre expression was directed by a 246-bp core sequence within the OVE element (Tg(coreOVE-Cre)). Tg(coreOVE-Cre) successfully marked the OV-derived inner ear tissues, including cochlea, semicircular canal and spiral ganglion, when crossed with ROSA26 lacZ reporter mice. Furthermore, Gata3 conditionally mutant mice, when crossed with the Tg(coreOVE-Cre), showed hypoplasia throughout the inner ear tissues. These results demonstrate that OVE has a sufficient regulatory activity to direct Gata3 expression specifically in the otic vesicle and semicircular canal and that Gata3 expression driven by the OVE is crucial for normal inner ear development.

Personalized healthcare (PHC) based on an individual's genetic make-up is one of the most advanced, yet feasible, forms of medical care. The Tohoku Medical Megabank (TMM) Project aims to combine population genomics, medical genetics and prospective cohort studies to develop a critical infrastructure for the establishment of PHC. To date, a TMM CommCohort (adult general population) and a TMM BirThree Cohort (birth+three-generation families) have conducted recruitments and baseline surveys. Genome analyses as part of the TMM Project will aid in the development of a high-fidelity whole-genome Japanese reference panel, in designing custom single-nucleotide polymorphism (SNP) arrays specific to Japanese, and in estimation of the biological significance of genetic variations through linked investigations of the cohorts. Whole-genome sequencing from >3,500 unrelated Japanese and establishment of a Japanese reference genome sequence from long-read data have been done. We next aim to obtain genotype data for all TMM cohort participants (>150,000) using our custom SNP arrays. These data will help identify disease-associated genomic signatures in the Japanese population, while genomic data from TMM BirThree Cohort participants will be used to improve the reference genome panel. Follow-up of the cohort participants will allow us to test the genetic markers and, consequently, contribute to the realization of PHC.

Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia reperfusion injury (IRI). Notably, G2CKO mice exhibited less severe kidney damage, with reduced granulomacrophagic infiltration upon IRI. Transcriptome analysis revealed that a series of inflammatory cytokine genes were downregulated in GATA2-deficient CD cells, suggesting that GATA2 induces inflammatory cytokine expression in diseased kidney CD cells. Through high-throughput chemical library screening, we identified a potent GATA inhibitor. The chemical reduces cytokine production in CD cells and protects the mouse kidney from IRI. These results revealed a novel pathological mechanism of renal IRI, namely, that CD cells produce inflammatory cytokines and promote IRI progression. In injured kidney CD cells, GATA2 exerts a proinflammatory function by upregulating inflammatory cytokine gene expression. GATA2 can therefore be considered a therapeutic target for AKI.

The erythropoietin (Epo) gene is under tissue-specific inducible regulation. Because the kidney is the primary EPO-producing tissue in adults, impaired EPO production in chronic kidney disorders results in serious renal anemia. The Epo gene contains a liver-specific enhancer in the 3′ region, but the kidney-specific enhancer for gene expression in renal EPO-producing (REP) cells remains elusive. Here, we examined a conserved upstream element for renal Epo regulation (CURE) region that spans 17.4 kb to 3.6 kb upstream of the Epo gene and harbors several phylogenetically conserved elements. We prepared various Epo gene-reporter constructs utilizing a bacterial artificial chromosome and generated a number of transgenic-mouse lines. We observed that deletion of the CURE region (δCURE) abrogated Epo gene expression in REP cells. Although transgenic expression of the δCURE construct rescued Epo-deficient mice from embryonic lethality, the rescued mice had severe EPO-dependent anemia. These mouse lines serve as an elaborate model for the search for erythroid stimulatory activity and are referred to as AnRED (anemic model with renal EPO deficiency) mice. We also dissected the CURE region by exploiting a minigene harboring four phylogenetically conserved elements in reporter transgenic-mouse analyses. Our analyses revealed that Epo gene regulation in REP cells is a complex process that utilizes multiple regulatory influences.

The transcription factor GATA1 regulates the expression of essential erythroid and megakaryocytic differentiation genes through binding to the DNA consensus sequence WGATAR. The GATA1 protein has four functional domains, including two centrally located zinc-finger domains and two transactivation domains at the N- and C-termini. These functional domains play characteristic roles in the elaborate regulation of diversified GATA1 target genes, each of which exhibits a unique expression profile. Three types of GATA1-related hematological malignancies have been reported. One is a structural mutation in the GATA1 gene, resulting in production of a short form of GATA1 that lacks the N-terminal transactivation domain and is found in Down syndrome-related acute megakaryocytic leukemias. The other two are cis-acting regulatory mutations affecting expression of the Gata1 gene, which are described as causes of acute erythroblastic leukemias and myelofibrosis in mice. Therefore, imbalanced gene regulation caused by qualitative and quantitative changes in GATA1 is thought to be involved in specific hematological disease pathogenesis. In the present review, we discuss recent advances in understanding the mechanisms of differential transcriptional regulation by GATA1 during erythroid differentiation, with special reference to the binding kinetics of GATA1 at conformation-specific binding sites.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.

SummaryChromosomal inversion between 3q21 and 3q26 results in high-risk acute myeloid leukemia (AML). In this study, we identified a mechanism whereby a GATA2 distal hematopoietic enhancer (G2DHE or −77-kb enhancer) is brought into close proximity to the EVI1 gene in inv(3)(q21;q26) inversions, leading to leukemogenesis. We examined the contribution of G2DHE to leukemogenesis by creating a bacterial artificial chromosome (BAC) transgenic model that recapitulates the inv(3)(q21;q26) allele. Transgenic mice harboring a linked BAC developed leukemia accompanied by EVI1 overexpression—neoplasia that was not detected in mice bearing the same transgene but that was missing the GATA2 enhancer. These results establish the mechanistic basis underlying the pathogenesis of a severe form of leukemia through aberrant expression of the EVI1 proto-oncogene.

Nrf2 is a major transcriptional activator of cytoprotective genes against oxidative/electrophilic stress, and Keap1 negatively regulates Nrf2. Emerging works have also suggested a role for Nrf2 as a regulator of differentiation in various cells, but the contribution of Nrf2 to the differentiation of hematopoietic stem cells (HSCs) remains elusive. Clarifying this point is important to understand Nrf2 functions in the development and/or resolution of inflammation. Here, we established two transgenic reporter mouse lines that allowed us to examine Nrf2 expression precisely in HSCs. Nrf2 was abundantly transcribed in HSCs, but its activity was maintained at low levels due to the Keap1-mediated degradation of Nrf2 protein. When we characterized Keap1-deficient mice, their bone marrow cells showed enhanced granulocyte-monocyte differentiation at the expense of erythroid and lymphoid differentiation. Importantly, Keap1-null HSCs showed lower expression of erythroid and lymphoid genes than did control HSCs, suggesting granulocyte-monocyte lineage priming in Keap1-null HSCs. This abnormal lineage commitment was restored by a concomitant deletion of Nrf2, demonstrating the Nrf2-dependency of the skewing. Analysis of Nrf2-deficient mice revealed that the physiological level of Nrf2 is sufficient to contribute to the lineage commitment. This study unequivocally shows that the Keap1-Nrf2 system regulates the cell fate determination of HSCs.

Hes1 is required for the development of hematopoietic stem cells in the mouse embryo through repression of Gata2., Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs.