Mikiko Suzuki

Mouse Spermatogenic Stem Cells Continually Interconvert between Equipotent Singly Isolated and Syncytial States

Summary The identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing “As model,” stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFRα1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial spermatogonia also contribute to stem cell function in homeostasis. We use live imaging and pulse labeling to quantitatively determine the fates of individual GFRα1+ cells and find that, during steady-state spermatogenesis, the entire GFRα1+ population comprises a single stem cell pool, in which cells continually interconvert between As and syncytial states. A minimal biophysical model, relying only on the rates of incomplete cell division and syncytial fragmentation, precisely predicts the stochastic fates of GFRα1+ cells during steady state and postinsult regeneration. Thus, our results define an alternative and dynamic model for spermatogenic stem cell function in the mouse testis.

A Remote GATA2 Hematopoietic Enhancer Drives Leukemogenesis in inv(3)(q21;q26) by Activating EVI1 Expression

Chromosomal 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.

Amelioration of inflammation and tissue damage in sickle cell model mice by Nrf2 activation

Significance Sickle cell disease (SCD) is one of the most common inherited disorders. A mutation in the β-globin gene causes deformation of red blood cells into a sickle shape, which in turn causes intravascular hemolysis and vaso-occlusion resulting in damage to multiple organs. Most studies that propose to develop new SCD therapies include the induction of fetal γ-globin expression to inhibit sickle cell formation as their ultimate goal. In contrast, we demonstrate here that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) ameliorates the development of inflammation and tissue damage that strongly affect the morbidity of SCD patients. Notably, several compounds that serve as Nrf2 inducers have been developed or are under development. The data indicate that Nrf2 activation could improve the prognosis for SCD patients. Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene, leading to the production of abnormally shaped red blood cells. Sickle cells are prone to hemolysis and thereby release free heme into plasma, causing oxidative stress and inflammation that in turn result in damage to multiple organs. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of the antioxidant cell-defense system. Here we show that constitutive Nrf2 activation by ablation of its negative regulator Keap1 (kelch-like ECH-associated protein 1) significantly improves symptoms in SCD model mice. SCD mice exhibit severe liver damage and lung inflammation associated with high expression levels of proinflammatory cytokines and adhesion molecules compared with normal mice. Importantly, these symptoms subsided after Nrf2 activation. Although hemolysis and stress erythropoiesis did not change substantially in the Nrf2-activated SCD mice, Nrf2 promoted the elimination of plasma heme released by sickle cells’ hemolysis and thereby reduced oxidative stress and inflammation, demonstrating that Nrf2 activation reduces organ damage and segregates inflammation from prevention of hemolysis in SCD mice. Furthermore, administration of the Nrf2 inducer CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) also relieved inflammation and organ failure in SCD mice. These results support the contention that Nrf2 induction may be an important means to protect organs from the pathophysiology of sickle cell-induced damage.

Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation.

ABSTRACT GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.

Fetal Globin Gene Repressors as Drug Targets for Molecular Therapies To Treat the β-Globinopathies

ABSTRACT The human β-globin locus is comprised of embryonic, fetal, and adult globin genes that are expressed in a developmental stage-specific manner. Mutations in the globin locus give rise to the β-globinopathies, β-thalassemia and sickle cell disease, which begin to manifest symptoms around the time of birth. Although the fetal globin genes are autonomously silenced in adult-stage erythroid cells, mutations lying both within and outside the locus lead to natural variations in the level of fetal globin gene expression, and some of these significantly ameliorate the clinical symptoms of the β-globinopathies. Multiple reports have now identified several transcription factors that are involved in fetal globin gene repression in definitive (adult)-stage erythroid cells (the TR2/TR4 heterodimer, MYB, KLFs, BCL11A, and SOX6). To carry out their repression functions, chromatin-modifying enzymes (such as DNA methyltransferase, histone deacetylases, and lysine-specific histone demethylase 1) are additionally involved as a consequence of forming large macromolecular complexes with the DNA-binding subunits of these cellular machines. This review focuses on the molecular mechanisms underlying fetal globin gene silencing and possible near-future molecularly targeted therapies for treating the β-globinopathies.

GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation

Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the ‘GATA factor switching’. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1‐knockdown embryos that concomitantly express Gata2‐GFP reporter, high‐level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1‐binding sites widely overlap with the sites pre‐occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient‐phase genes.

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.

Genetic Analysis of Hierarchical Regulation for Gata1 and NF-E2 p45 Gene Expression in Megakaryopoiesis

ABSTRACT GATA1 and NF-E2 p45 are two important regulators of megakaryopoiesis. Whereas GATA1 is known to regulate the p45 gene, details of the GATA1 contribution to the spatiotemporal expression of the p45 gene remain to be elucidated. To clarify the relationship between GATA1 and p45, we performed genetic complementation rescue analysis of p45 function in megakaryocytes utilizing the hematopoietic regulatory domain of the Gata1 gene (G1HRD). We established transgenic mouse lines expressing p45 under G1HRD regulation and crossed the mice with p45-null mice. Compound mutant mice displayed normal platelet counts and no sign of hemorrhage, indicating that G1HRD has the ability to express p45 in a spatiotemporally correct manner. However, deletion of 38 amino acids from the N-terminal region of p45 abrogated the p45 rescue function, suggesting the presence of an essential transactivation activity in the region. We then crossed the G1HRD-p45 transgenic mice with megakaryocyte-specific Gata1 gene knockdown (Gata1ΔneoΔHS) mice. The G1HRD-p45 transgene was insufficient for complete rescue of the Gata1ΔneoΔHS megakaryocytes, suggesting that GATA1 or other factors regulated by GATA1 are required to cooperate with p45 for normal megakaryopoiesis. This study thus provides a unique in vivo validation of the hierarchical relationship between GATA1 and p45 in megakaryocytes.

Transcriptional regulation by GATA1 and GATA2 during erythropoiesis

The transcription factor GATA1 regulates multiple genes in erythroid lineage cells. However, the means by which GATA1 regulates the expression of target genes during erythropoiesis remains to be elucidated. Three mechanisms have been postulated for the regulation of genes by GATA1. First, individual target genes may have multiple discrete thresholds for cellular GATA1. GATA1 has a dynamic expression profile during erythropoiesis, thus the expression of a set of GATA1 target genes may be triggered at a given stage of differentiation by cellular GATA1. Second, the expression of GATA1 target genes may be modified, at least in part, by GATA2 occupying the GATA-binding motifs. GATA2 is expressed earlier in erythropoiesis than GATA1, and prior GATA2 binding may afford GATA1 access to GATA motifs through epigenetic remodeling and thus facilitate target gene expression. Third, other regulatory molecules specific to each target gene may function cooperatively with GATA1. If GATA1 is required for the expression of such cofactors, a regulatory network will be formed and relevant gene expression will be delayed. We propose that the stage-specific regulation of erythroid genes by GATA1 is tightly controlled through a combination of these mechanisms in vivo.

The Gata1 5′ region harbors distinct cis-regulatory modules that direct gene activation in erythroid cells and gene inactivation in HSCs

GATA1 is a master regulator of hematopoietic differentiation, but Gata1 expression is inactivated in hematopoietic stem cells (HSCs). Using a bacterial artificial chromosome containing the Gata1 gene modified with green fluorescent protein (GFP) reporter, we explored the function of the 3.7-kb Gata1 upstream region (GdC region) that harbors 3 core cis-elements: Gata1 hematopoietic enhancer, double GATA-motif, and CACCC-motif. Transgenic GFP expression directed by the Gata1-BAC faithfully recapitulated the endogenous Gata1 expression pattern. However, deletion of the GdC-region eliminated reporter expression in all hematopoietic cells. To test whether the combination of the core cis-elements represents the regulatory function of the GdC-region, we replaced the region with a 659-bp minigene that linked the three cis-elements (MG-GFP). The GFP reporter expression directed by the MG-GFP BAC fully recapitulated the erythroid-megakaryocytic Gata1 expression. However, the GFP expression was aberrantly increased in the HSCs and was associated with decreases in DNA methylation and abundant GATA2 binding to the transgenic MG-GFP allele. The 3.2-kb sequences interspaced between the Gata1 hematopoietic enhancer and the double GATA-motif were able to recruit DNA methyltransferase 1, thereby exerting a cis-repressive function in the HSC-like cell line. These results indicate that the 3.2-kb interspacing sequences inactivate Gata1 by maintaining DNA-methylation in the HSCs.