[Return of Individual Genomic Results to Germline Pathogenic Variant Carriers of Hereditary Cancer in Population Based Cohort Study].

Return of individual genomic results(ROGR)to participants in population-based biobank has been rarely conducted in research settings, and the procedure of ROGR performed in foreign countries may not be simply applied to Japanese participants, because of the difference in social background. The Tohoku Medical Megabank Project, which was launched in 2012 aiming to build a foundation of personalized genomic medicine, obtained the consent from research participants by explaining the future possibility of ROGR. After careful consideration of appropriate procedure for ROGR, individual genomic results were returned to 111 pathogenic variant(PV)carriers of hereditary breast and ovarian cancer syndrome(HBOC)or Lynch syndrome(LS)based on 50,000 whole genome sequencing(WGS)data in FY 2022. Since majority of the participants has no cancer diagnosis, participants' right to not know was carefully considered. In addition, the variants to be returned were carefully evaluated by using multiple databases, and the WGS results of the participants were further confirmed by the single- site analysis. When the genomic results were returned, the participants were informed about clinical risk surveillance at the hospital. Seventy-eight and 33 PV carriers of HBOC and LS, respectively, participated in the study. Most participants were in their 30s or 40s. Unexpectedly, validation testing results of 12 LS participants were found to be negative or variant of uncertain significance, because detecting these variants by WGS were technically challenging. Twenty-eight participants (HBOC 20, LS 8)had been diagnosed as cancer, and 6 females who had breast cancer were genetically diagnosed as HBOC and underwent or planned risk-reducing surgery. Eighteen participants refused to undergo clinical risk surveillance because of the medical expense that is not covered by health insurance and the burden of visiting the hospital. The opportunity to undergo medical surveillance should be provided to population-based cohort participants who carry actionable pathogenic variants, and ROGR to general population should be promoted to create the base of personalized genomic medicine.

The return of individual genomic results to research participants: design and pilot study of Tohoku Medical Megabank Project.

Certain large genome cohort studies attempt to return the individual genomic results to the participants; however, the implementation process and psychosocial impacts remain largely unknown. The Tohoku Medical Megabank Project has conducted large genome cohort studies of general residents. To implement the disclosure of individual genomic results, we extracted the potential challenges and obstacles. Major challenges include the determination of genes/disorders based on the current medical system in Japan, the storage of results, prevention of misunderstanding, and collaboration of medical professionals. To overcome these challenges, we plan to conduct multilayer pilot studies, which deal with different disorders/genes. We finally chose familial hypercholesterolemia (FH) as a target disease for the first pilot study. Of the 665 eligible candidates, 33.5% were interested in the pilot study and provided consent after an educational "genetics workshop" on the basic genetics and medical facts of FH. The genetics professionals disclosed the results to the participants. All positive participants were referred to medical care, and a serial questionnaire revealed no significant psychosocial distress after the disclosure. Return of genomic results to research participants was implemented using a well-prepared protocol. To further elucidate the impact of different disorders, we will perform multilayer pilot studies with different disorders, including actionable pharmacogenomics and hereditary tumor syndromes.

Mouse Tryptase Gene Expression is Coordinately Regulated by GATA1 and GATA2 in Bone Marrow-Derived Mast Cells.

Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the Tpsb2 and Tpsg1 genes, which reside at the 3'-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other's DNA binding activity to these regions. Deletion of the -72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the -72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs.

GATA2 is critical for the maintenance of cellular identity in differentiated mast cells derived from mouse bone marrow.

GATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage.

Microvesicles enhance the mobility of human diabetic adipose tissue-derived mesenchymal stem cells in vitro and improve wound healing in vivo.

Microvesicles (MVs) derived from mesenchymal stem cells showed the ability to alter the cell phenotype and function. We previously demonstrated that type 2 diabetic adipose tissue-derived mesenchymal stem cells (dAT-MSCs) increase in cell aggregation and adhesion in vitro and impair wound healing in vivo. However, the characterization and function of MVs derived from human non-diabetic AT-MSCs (nAT-MSCs) remain unknown. In this study, we characterized nAT-MSC-derived MVs and their function after the transfection of dAT-MSCs with MVs using the scratch assay and a flap mouse model. We found that human nAT-MSC-derived MVs expressed MSC-surface markers and improved dAT-MSC functions by altering the expression of genes associated with cell migration, survival, inflammation, and angiogenesis as well as miR29c and miR150. Remarkably, the transfection of dAT-MSCs with nAT-MSC-derived MVs improved their migration ability in vitro and wound healing ability in a flap mouse model. These results demonstrate a promising opportunity to modify the function of dAT-MSCs for therapeutic stem cell application in diabetic patients.

Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs.

Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites.

Hypoxia-inducible factor-3α promotes angiogenic activity of pulmonary endothelial cells by repressing the expression of the VE-cadherin gene.

The variants of the hypoxia-inducible factor-3α gene HIF-3α and NEPAS are known to repress the transcriptional activities driven by HIF-1α and HIF-2α. Although NEPAS has been shown to play an important role in vascular remodeling during lung development, little is known about the roles of HIF-3α in adult lung function. Here, we examined pulmonary endothelial cells (ECs) isolated from wild-type (WT) and HIF-3α functional knockout (KO) mice. The expression levels of angiogenic factors (Flk1, Ang2 and Tie2) were significantly greater in the HIF-3α KO ECs than those in the WT ECs irrespective of oxygen tension. However, the HIF-3α KO ECs showed impaired proliferative and angiogenic activities. The impaired EC function was likely due to the excess vascular endothelial (VE)-cadherin, an inhibitor of Flk1/PI3 kinase/Akt signaling, as treatment of the cells to a neutralizing antibody partly restored the phenotype of the HIF-3α KO ECs. Importantly, we found that the mRNA levels of HIF-2α and Ets-1 were significantly increased by HIF-3α ablation. Given that both factors are known to activate the VE-cadherin gene, the transcriptional repression of these factors by HIF-3α might be important for silencing the irrelevant expression of the VE-cadherin gene. Collectively, these data show novel and unique roles of HIF-3α for angiogenic gene regulation in pulmonary ECs.

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.

Establishment of erythroleukemic GAK14 cells and characterization of GATA1 N-terminal domain.

GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1(G1.05/+)) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1(G1.05/+) mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr-1-, Mac1-, B220-, CD3e- or CD49b-positive hematopoietic cells when co-cultured with DAS104-8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full-length GATA1 and co-cultured with fetal liver-derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N-terminal domain was complemented. The N-terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N-terminal domain is essential for the erythroid differentiation of GAK14 cells.

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.

Whole blood transcriptome analysis for age- and gender-specific gene expression profiling in Japanese individuals.

Whole blood transcriptome analysis is a valuable approachin medical research, primarily due to the ease of sample collection and the richness of the information obtained. Since the expression profile of individual genes in the analysis is influenced by medical traits and demographic attributes such as age and gender, there has been a growing demand for a comprehensive database for blood transcriptome analysis. Here, we performed whole blood RNA sequencing (RNA-seq) analysis on 576 participants stratified by age (20-30s and 60-70s) and gender from cohorts of the Tohoku Medical Megabank (TMM). A part of female segment included pregnant women. We did not exclude the globin gene family in our RNA-seq study, which enabled us to identify instances of hereditary persistence of fetal hemoglobin based on the HBG1 and HBG2 expression information. Comparing stratified populations allowed us to identify groups of genes associated with age-related changes and gender differences. We also found that the immune response status, particularly measured by neutrophil-to-lymphocyte ratio (NLR), strongly influences the diversity of individual gene expression profiles in whole blood transcriptome analysis. This stratification has resulted in a data set that will be highly beneficial for future whole blood transcriptome analysis in the Japanese population.

Ablation of Mina53 in mice reduces allergic response in the airways.

The mina53 (myc-induced nuclear antigen with a 53 kDa molecular mass; also known as mina) was identified as a direct transcriptional target of the oncoprotein Myc and encodes a conserved protein in vertebrates. While Mina53 is known to be associated with tumorigenesis, it is not clear what role Mina53 plays in non-neoplastic tissues. To directly address the roles of Mina53 in non-neoplastic tissues, we created mina53-deficient mice. Both male and female mina53-deficient mice reached adulthood and were fertile, suggesting that Mina53 is dispensable for the basic developmental processes. Since we found that Mina53 was expressed in cells responsible for immune responses, we investigated whether Mina53 was involved in immune responses. When mice were exposed intranasally to house dust mites as an allergen, the airway tract showed hyperresponsiveness to methacholine in wild-type mice but not in mina53-deficient mice. The mina53-deficient mice also showed a significantly reduced migration of immune cells, including eosinophils, into bronchoalveolar lavage fluid compared with wild-type mice. The levels of Th2 cytokines, IL-4 and IL-5, produced in response to house dust mites were lower in the mina53-deficient mice than in wild-type mice. The level of IFN-γ in bronchoalveolar lavage fluid was significantly decreased by exposure to house dust mites in wild-type mice but not in the mina53-deficient mice. These results suggest that Mina53 plays a role in the allergic response to inhaled allergens, possibly through controlling IL-4 production.

GATA transcription factors are involved in IgE-dependent mast cell degranulation by enhancing the expression of phospholipase C-γ1.

Mast cell degranulation is a dynamic, highly organized process involving numerous signaling molecules and enzymes. Although the molecular mechanisms underlying antigen-mediated mast cell degranulation have been studied intensively, little is known about the transcriptional control of this process. Here, we show that the hematopoietic transcription factors GATA1 and GATA2 are involved in mast cell degranulation through the control of phospholipase C-γ1 (PLC-γ1) expression. Knockdown of GATA1 and/or GATA2 by specific siRNA significantly reduced antigen-induced degranulation and Ca(2+) mobilization in the rat basophilic leukemia cell line RBL-2H3. RT-PCR analyses showed that PLC-γ1 expression was significantly decreased by this GATA factor repression. Other GATA factor targets, such as the previously reported α and ß subunits of the high-affinity IgE receptor (FcεRI), were unaffected. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that GATA factors directly activate PLC-γ1 gene transcription through a conserved GATA-binding motif that resides in the 5'-upstream sequence. Furthermore, we show evidence that the PLC-γ1 expression is regulated by GATA2 in mast cells derived from mouse bone marrow. These data indicate that PLC-γ1 is a target gene of GATA factors in mast cells and provide evidence that GATA1 and GATA2 control antigen-mediated mast cell degranulation by regulating the expression of PLC-γ1.

Effect of Nicotinamide Mononucleotide Concentration in Human Milk on Neurodevelopmental Outcome: The Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study.

(1) Background: Breast milk is the only source of nutrition for breastfed infants, but few studies have examined the relationship between breast milk micronutrients and infant neurodevelopmental outcome in exclusively breastfed infants. The aim of this study was to characterize the association between nicotinamide adenine dinucleotide (NAD)-related compounds in the breast milk of Japanese subjects and infant neurodevelopmental outcome. (2) Methods: A total of 150 mother-child pairs were randomly selected from the three-generation cohort of the Tohoku Medical Megabank in Japan. Infants were exclusively breastfed for up to 6 months. Breast milk was collected at 1 month postpartum, and the quantity of NAD-related substances in the breast milk was quantified. The mothers also completed developmental questionnaires at 6, 12, and 24 months. The relationship between the concentration of NAD-related substances in breast milk and developmental indicators was evaluated via ordinal logistic regression analysis. (3) Results: Nicotinamide mononucleotide (NMN) was quantified as the major NAD precursor in breast milk. The median amount of NMN in the breast milk was 9.2 µM. The NMN concentration in breast milk was the only NAD-related substance in breast milk that showed a significant positive correlation with neurodevelopmental outcome in infants at 24 months. (4) Conclusions: The results suggest that NMN in human milk may be an important nutrient for early childhood development.

Returning individual genomic results to population-based cohort study participants with BRCA1/2 pathogenic variants.

BACKGROUND: Recent advances in human genome research have provided evidence for genotype-phenotype associations, pathogenicity, and clinical actionability of variants and genomic risk prediction of disease. However, the return of individual genomic results to healthy individuals is fraught with ethical and practical complexity. METHODS: Individual genomic results were returned to BRCA1/2 pathogenic variant (PV) carriers of the Tohoku Medical Megabank cohort study participants with an information on hereditary breast and ovarian cancer syndrome (HBOC). One hundred and eighty participants, including 9 BRCA1/2 PV carriers, were asked about their willingness to receive individual genomic results, without revealing the gene name and related disorders, prior to the study. Of the 142 participants who responded, 103 showed willingness to know their genomic information. Each of the six BRCA1/2 PV carriers who consented to participate in the study received information about HBOC in person and underwent validation testing with blood resampling. RESULTS: All participants were in their 60s or 70s; of the four females and two males, two had a history of breast cancer and five had a family history of HBOC-related cancers. All participants appreciated the information, without remarkable negative psychological impact of the return, and intended to undergo clinical risk surveillance. Five participants were accompanied by family members while receiving the results, and three first-degree female relatives wished to undergo genomic testing at the hospital. CONCLUSIONS: Our results suggest that returning actionable genomic information to participants in a population-based genome cohort study is beneficial for preventing or providing early-stage intervention for associated diseases.

The Il6 -39 kb enhancer containing clustered GATA2- and PU.1-binding sites is essential for Il6 expression in murine mast cells.

Mast cells serve as a first-line defense of innate immunity. Interleukin-6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1-clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.

A Pilot Study for Return of Individual Pharmacogenomic Results to Population-Based Cohort Study Participants.

Introduction: Pharmacogenomic (PGx) testing results provide valuable information on drug selection and appropriate dosing, maximization of efficacy, and minimization of adverse effects. Although the number of large-scale, next-generation-sequencing-based PGx studies has recently increased, little is known about the risks and benefits of returning PGx results to ostensibly healthy individuals in research settings. Methods: Single-nucleotide variants of three actionable PGx genes, namely, MT-RNR1, CYP2C19, and NUDT15, were returned to 161 participants in a population-based Tohoku Medical Megabank project. Informed consent was obtained from the participants after a seminar on the outline of this study. The results were sent by mail alongside sealed information letter intended for clinicians. As an exception, genetic counseling was performed for the MT-RNR1 m.1555A > G variant carriers by a medical geneticist, and consultation with an otolaryngologist was encouraged. Questionnaire surveys (QSs) were conducted five times to evaluate the participants' understanding of the topic, psychological impact, and attitude toward the study. Results: Whereas the majority of participants were unfamiliar with the term PGx, and none had undergone PGx testing before the study, more than 80% of the participants felt that they could acquire basic PGx knowledge sufficient to understand their genomic results and were satisfied with their potential benefit and use in future prescriptions. On the other hand, some felt that the PGx concepts or terminology was difficult to fully understand and suggested that in-person return of the results was desirable. Conclusions: These results collectively suggest possible benefits of returning preemptive PGx information to ostensibly healthy cohort participants in a research setting.

Identification of human placenta-derived mesenchymal stem cells involved in re-endothelialization.

Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled-9, a receptor for Wnt ligands, was positive in placenta-derived MSC. So, we isolated CD349-negative and -positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349-negative and -positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349-negative, but not CD349-positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFß and bFGF mRNAs by hypoxia was greater in CD349-negative cells than in CD349-positive cells while the expression of VEGF was not significantly different in CD349-negative and CD349-positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re-endothelialization.

Characterization of a functional ZBP-89 binding site that mediates Gata1 gene expression during hematopoietic development.

GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5' region of the enhancer. However, the importance of the GC-rich region adjacent to the 3'-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G(5) string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G(5) string (G(4) mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G(5) string sequence. Finally, transgenic mouse studies reveal that the G(4) mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G(5) string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.

The microenvironment for erythropoiesis is regulated by HIF-2alpha through VCAM-1 in endothelial cells.

Erythropoiesis is a dynamic process regulated by oxygen in vertebrates. Recent evidence has indicated that erythropoietin (Epo) expression is regulated by hypoxia-inducible transcription factors (HIFs), HIF-2alpha in particular. In this study, we report that knockdown mutation of HIF-2alpha in mice (kd/kd) results in normocytic anemia, despite Epo induction in response to hypoxia not being severely affected. Transplantation analyses clearly demonstrated that the hematopoietic microenvironment, but not the hematopoietic cells, was altered in kd/kd. Furthermore, cell-type specific recovery of HIF-2alpha expression in endothelial cells (ECs) abrogated the anemic condition of the kd/kd mice, indicating that HIF-2alpha in EC plays an essential role in supporting erythropoiesis. In the absence of HIF-2alpha, the expression of vascular adhesion molecule-1 (VCAM-1) was reduced significantly and restoration of VCAM-1 expression in kd/kd ECs enhanced the development of erythroid progenitors. Finally, a chromatin immunoprecipitation assay and a reporter assay indicated that VCAM-1 gene transcription is directly regulated by HIF-2alpha. These data suggest that the hematopoietic microenvironment required for erythropoiesis is dynamically regulated by oxygen through the functions of HIF-2alpha in ECs.