Osamu Tanabe

FIP200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells

Little is known about whether autophagic mechanisms are active in hematopoietic stem cells (HSCs) or how they are regulated. FIP200 (200-kDa FAK-family interacting protein) plays important roles in mammalian autophagy and other cellular functions, but its role in hematopoietic cells has not been examined. Here we show that conditional deletion of FIP200 in hematopoietic cells leads to perinatal lethality and severe anemia. FIP200 was cell-autonomously required for the maintenance and function of fetal HSCs. FIP200-deficient HSC were unable to reconstitute lethally irradiated recipients. FIP200 ablation did not result in increased HSC apoptosis, but it did increase the rate of HSC proliferation. Consistent with an essential role for FIP200 in autophagy, FIP200-null fetal HSCs exhibited both increased mitochondrial mass and reactive oxygen species. These data identify FIP200 as a key intrinsic regulator of fetal HSCs and implicate a potential role for autophagy in the maintenance of fetal hematopoiesis and HSCs.

Lysine-specific demethylase 1 is a therapeutic target for fetal hemoglobin induction.

Enhanced fetal γ-globin synthesis alleviates symptoms of β-globinopathies such as sickle cell disease and β-thalassemia, but current γ-globin–inducing drugs offer limited beneficial effects. We show here that lysine-specific demethylase 1 (LSD1) inhibition by RNAi in human erythroid cells or by the monoamine oxidase inhibitor tranylcypromine in human erythroid cells or β-type globin–transgenic mice enhances γ-globin expression. LSD1 is thus a promising therapeutic target for γ-globin induction, and tranylcypromine may serve as a lead compound for the development of a new γ-globin inducer.

Embryonic and fetal β‐globin gene repression by the orphan nuclear receptors, TR2 and TR4

The TR2 and TR4 orphan nuclear receptors comprise the DNA‐binding core of direct repeat erythroid definitive, a protein complex that binds to direct repeat elements in the embryonic and fetal β‐type globin gene promoters. Silencing of both the embryonic and fetal β‐type globin genes is delayed in definitive erythroid cells of Tr2 and Tr4 null mutant mice, whereas in transgenic mice that express dominant‐negative TR4 (dnTR4), human embryonic ε‐globin is activated in primitive and definitive erythroid cells. In contrast, human fetal γ‐globin is activated by dnTR4 only in definitive, but not in primitive, erythroid cells, implicating TR2/TR4 as a stage‐selective repressor. Forced expression of wild‐type TR2 and TR4 leads to precocious repression of ε‐globin, but in contrast to induction of γ‐globin in definitive erythroid cells. These temporally specific, gene‐selective alterations in ε‐ and γ‐globin gene expression by gain and loss of TR2/TR4 function provide the first genetic evidence for a role for these nuclear receptors in sequential, gene‐autonomous silencing of the ε‐ and γ‐globin genes during development, and suggest that their differential utilization controls stage‐specific repression of the human ε‐ and γ‐globin genes.

Nuclear Receptors TR2 and TR4 Recruit Multiple Epigenetic Transcriptional Corepressors That Associate Specifically with the Embryonic β-Type Globin Promoters in Differentiated Adult Erythroid Cells

ABSTRACT Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal β-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic β-type globin promoters but not to the adult β-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1β, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult β-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult β-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic β-type globin genes through coordinated epigenetic chromatin modifications.

The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43xa0kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.

The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

Forced TR2/TR4 expression in sickle cell disease mice confers enhanced fetal hemoglobin synthesis and alleviated disease phenotypes

Sickle cell disease (SCD) is a hematologic disorder caused by a missense mutation in the adult β-globin gene. Higher fetal hemoglobin (HbF) levels in red blood cells of SCD patients have been shown to improve morbidity and mortality. We previously found that nuclear receptors TR2 and TR4 repress expression of the human embryonic ε-globin and fetal γ-globin genes in definitive erythroid cells. Because forced expression of TR2/TR4 in murine adult erythroid cells paradoxically enhanced fetal γ-globin gene expression in transgenic mice, we wished to determine if forced TR2/TR4 expression in a SCD model mouse would result in elevated HbF synthesis and thereby alleviate the disease phenotype. In a “humanized” sickle cell model mouse, forced TR2/TR4 expression increased HbF abundance from 7.6% of total hemoglobin to 18.6%, accompanied by increased hematocrit from 23% to 34% and reticulocyte reduction from 61% to 18%, indicating a significant reduction in hemolysis. Moreover, forced TR2/TR4 expression reduced hepatosplenomegaly and liver parenchymal necrosis and inflammation in SCD mice, indicating alleviation of usual pathophysiological characteristics. This article shows that genetic manipulation of nonglobin proteins, or transcription factors regulating globin gene expression, can ameliorate the disease phenotype in a SCD model animal. This proof-of-concept study demonstrates that modulating TR2/TR4 activity in SCD patients may be a promising therapeutic approach to induce persistent HbF accumulation and for treatment of the disease.

Adult Stage γ-Globin Silencing Is Mediated by a Promoter Direct Repeat Element

ABSTRACT The human β-like globin genes (5′-ε-Gγ-Aγ-δ-β-3′) are temporally expressed in sequential order from the 5′ to 3′ end of the locus, but the nonadult ε- and γ-globin genes are autonomously silenced in adult erythroid cells. Two cis elements have been proposed to regulate definitive erythroid γ-globin repression: the DR (direct repeat) and CCTTG elements. Since these two elements partially overlap, and since a well-characterized HPFH point mutation maps to an overlapping nucleotide, it is not clear if both or only one of the two participate in γ-globin silencing. To evaluate the contribution of these hypothetical silencers to γ-globin regulation, we generated point mutations that individually disrupted either the single DR or all four CCTTG elements. These two were separately incorporated into human β-globin yeast artificial chromosomes, which were then used to generate γ-globin mutant transgenic mice. While DR element mutation led to a dramatic increase in Aγ-globin expression only during definitive erythropoiesis, the CCTTG mutation did not affect adult stage transcription. These results demonstrate that the DR sequence element autonomously mediates definitive stage-specific γ-globin gene silencing.

Disruption of the Hbs1l-Myb Locus Causes Hereditary Persistence of Fetal Hemoglobin in a Mouse Model

ABSTRACT The human β-globin locus is comprised of embryonic, fetal, and adult globin genes, each of which is expressed at distinct stages of pre- and postnatal development. Functional defects in globin proteins or expression results in mild to severe anemia, such as in sickle-cell disease or β-thalassemia, but the clinical symptoms of both disorders are ameliorated by persistent expression of the fetal globin genes. Recent genome-wide association studies (GWAS) identified the intergenic region between the HBS1L and MYB loci as a candidate modifier of fetal hemoglobin expression in adults. However, it remains to be clarified whether the enhancer activity within the HBS1L-MYB regulatory domain contributes to the production of fetal hemoglobin in adults. Here we report a new mouse model of hereditary persistence of fetal hemoglobin (HPFH) in which a transgene was randomly inserted into the orthologous murine Hbs1l-Myb locus. This mutant mouse exhibited typically elevated expression of embryonic globins and hematopoietic parameters similar to those observed in human HPFH. These results support the contention that mutation of the HBS1L-MYB genomic domain is responsible for elevated expression of the fetal globin genes, and this model serves as an important means for the analysis of networks that regulate fetal globin gene expression.

Genome-wide binding of the orphan nuclear receptor TR4 suggests its general role in fundamental biological processes

BackgroundThe orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear.ResultsWe report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors.ConclusionsOur data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases.