Publications (30)216.01 Total impact
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Article: Mi2b Is Required for c-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in b-YAC Transgenic Mice
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ABSTRACT: Activation of c-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the 2566 GATA motif of the A c-globin gene. We show that Mi2b is essential for c-globin gene silencing using Mi2b conditional knockout b-YAC transgenic mice. In addition, increased expression of A c-globin was detected in adult blood from b-YAC transgenic mice containing a T.G HPFH point mutation at the 2566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type b-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the 2566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of c-globin gene expression and that either a trans-acting Mi2b knockout deletion mutation or the cis-acting 2566 A c-globin HPFH point mutation disrupts establishment of repression, resulting in continued c-globin gene transcription during adult definitive erythropoiesis.PLoS Genetics 12/2012; · 8.69 Impact Factor -
Article: Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice.
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ABSTRACT: Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.PLoS Genetics 12/2012; 8(12):e1003155. · 8.69 Impact Factor -
Article: LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.
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ABSTRACT: The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c). The 5'ΔHS3c mice expressed β(m)-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5'HS3 core was not required for β(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5'HS complete deletion mice, except β(m)-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.Nucleic Acids Research 10/2012; · 8.03 Impact Factor -
Article: Induction of Fetal Hemoglobin In Vivo Mediated by a Synthetic γ-Globin Zinc Finger Activator.
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ABSTRACT: Sickle cell disease (SCD) and β-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF, γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation of γ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the (A)γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34(+) erythroid progenitor cells from normal donors and β-thalassemia patients. Herein, we report that gg1-VP64 increased γ-globin gene expression in vivo, in peripheral blood samples from gg1-VP64 β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.Anemia 01/2012; 2012:507894. -
Article: メタデータマイクロアトリビューションアプローチを使った異常ヘモグロビン症におけるヒトの遺伝的多様性の系統的な考証と解析
Nature Genetics 03/2011; 43(4):295-301. · 35.53 Impact Factor -
Article: Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach.
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ABSTRACT: We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.Nature Genetics 03/2011; 43(4):295-301. · 35.53 Impact Factor -
Article: Transgenic expression of human LAMA5 suppresses murine Lama5 mRNA and laminin α5 protein deposition.
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ABSTRACT: Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ∼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1-α5 isoform switch, or that for mouse laminin β1-β2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.PLoS ONE 01/2011; 6(9):e23926. · 4.09 Impact Factor -
Article: Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production.
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ABSTRACT: Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TF(ZF)s). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TF(ZF) encoding gene. Using this strategy, we successfully selected specific TF(ZF)s that upregulate the expression of the gamma-globin promoter. Selected transcription factors induced the expression of gamma-globin when coupled to an activation domain and reduced expression when linked to a repression domain. This new retroviral approach might be used to select other TF(ZF)s but might also be generalized for the selection of other protein and small-molecule interactions.Nature Structural & Molecular Biology 11/2009; 16(11):1195-9. · 12.71 Impact Factor -
Article: Minor histocompatibility antigens on transfused leukoreduced units of red blood cells induce bone marrow transplant rejection in a mouse model.
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ABSTRACT: When successful, human leukocyte antigen (HLA)-matched bone marrow transplantation with reduced-intensity conditioning is a cure for several nonmalignant hematologic disorders that require chronic transfusion, such as sickle cell disease and aplastic anemia. However, there are unusually high bone marrow transplant (BMT) rejection rates in these patients. Rejection correlates with the number of transfusions before bone marrow transplantation, and it has been hypothesized that preimmunization to antigens on transfused blood may prime BMT rejection. Using a novel mouse model of red blood cell (RBC) transfusion and major histocompatibility complex-matched bone marrow transplantation, we report that transfusion of RBC products induced BMT rejection across minor histocompatibility antigen (mHA) barriers. It has been proposed that contaminating leukocytes are responsible for transfusion-induced BMT rejection; however, filter leukoreduction did not prevent rejection in the current studies. Moreover, we generated a novel transgenic mouse with RBC-specific expression of a model mHA and demonstrated that transfusion of RBCs induced a CD8(+) T-cell response. Together, these data suggest that mHAs on RBCs themselves are capable of inducing BMT rejection. Cellular immunization to mHAs is neither monitored nor managed by current transfusion medicine practice; however, the current data suggest that mHAs on RBCs may represent an unappreciated and significant consequence of RBC transfusion.Blood 07/2009; 114(11):2315-22. · 9.90 Impact Factor -
Article: Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site.
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ABSTRACT: Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.Molecular and cellular biology 06/2008; 28(10):3101-13. · 6.06 Impact Factor -
Article: Preparation of intact yeast artificial chromosome DNA for transgenesis of mice.
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ABSTRACT: Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.Nature Protocol 02/2007; 2(11):3009-15. · 8.36 Impact Factor -
Article: Investigations of a human embryonic globin gene silencing element using YAC transgenic mice.
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ABSTRACT: A silencing element has been previously located upstream of the human epsilon-globin gene promoter using transient assays and transgenic mice carrying plasmid constructs in which the element has been deleted or its transcriptional motifs have been mutated. To investigate whether this element functions in the context of the whole beta-globin locus, we analyzed epsilon-globin gene expression in transgenic mice carrying a deletion of the silencing element in the context of a 213-kilobase human beta-globin yeast artificial chromosome (beta-YAC). epsilon-Globin gene expression was measured during embryonic and fetal development and in adult mice. epsilon-mRNA levels in embryonic cells in Day 12 blood were as high as those measured in wild-type beta-YAC controls, indicating that the deletion does not affect epsilon gene promoter function. epsilon-Globin gene expression was confined to the embryonic cells, indicating that deletion of this silencing element did not affect epsilon-globin developmental expression in the context of the beta-YAC. These results suggest that in the context of the whole beta-globin locus, other proximal and upstream epsilon gene promoter elements as well as competition by the downstream globin genes contribute to the silencing of the epsilon-globin gene in the cells of definitive erythropoiesis.Experimental Biology and Medicine 04/2006; 231(3):328-34. · 2.64 Impact Factor -
Article: Establishment of cell lines that exhibit correct ontogenic stage-specific gene expression profiles from tissues of yeast artificial chromosome transgenic mice using chemically induced growth signals.
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ABSTRACT: Transgenic mice produced with human yeast artificial chromosomes (YACs) generally display transgene expression patterns that reflect those of the normal human host. Because mice are expensive and time-consuming to generate and maintain, extensive mutation-phenotype correlation studies cannot be readily carried out. Cell lines are better suited for analysis of a plethora of mutations. However, these types of gene regulatory studies have been complicated by the lack of suitable cell lines, most of which do not exactly replicate the gene expression patterns observed in vivo. We reasoned that cells established from tissues of YAC transgenic mice might express the transgenes in the correct tissue and developmental stage-specific pattern from which they were derived because YAC transgenic mice display correct regulation of gene expression during ontogeny. We used our human beta-globin locus YAC (beta-YAC) transgenic mice to demonstrate this approach. All existing erythroid cell lines coexpress beta-like globins from different developmental stages or express them inappropriately based on the developmental stage from which they were obtained. Cell populations were established from the adult bone marrow (BM) of beta-YAC transgenic mice, which express exclusively adult beta-globin, using dimerizer technology. A derivative of the thrombopoietin receptor (mpl) was used to bring the proliferative status of primary BM marrow cells under the control of a small molecule drug called a chemical inducer of dimerization (CID). Cells generated in this manner can be expanded to extremely large numbers, remain strictly CID-dependent, and retain megakaryocytic, erythroid, and granulocytic potential. Marrow cells transduced with a retrovirus vector encoding the mpl derivative proliferated extensively in the presence of the CID, AP20187. RNAse protection assays demonstrated that the transcripts for human beta-globin and mouse alpha-globin were present, while gamma-globin transcripts were absent, thus, these cells had the predicted expression phenotype. Exposure to 5-azacytidine or introduction of a hereditary persistence of fetal hemoglobin mutation activated gamma-globin, which was expressed in addition to beta-globin, again consistent with the predicted expression profile of these cells. This approach extends the usefulness of YAC transgenic mice for the generation of cell lines amenable to more detailed studies regarding gene regulation.Methods in molecular biology (Clifton, N.J.) 02/2006; 349:163-73. -
Article: {gamma}-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human {beta}-globin locus yeast artificial chromosome ({beta}-YAC) transgenic mice.
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ABSTRACT: Identification of trans-acting factors or drugs capable of reactivating gamma-globin gene expression is complicated by the lack of suitable cell lines. Human K562 cells co-express epsilon- and gamma-globin but not beta-globin; transgenic mouse erythroleukemia 585 cells express predominantly human beta-globin but also gamma-globin; and transgenic murine GM979 cells co-express human gamma-and beta-globin. Human beta-globin locus yeast artificial chromosome transgenic mice display correct developmental regulation of beta-like globin gene expression. We rationalized that cells established from the adult bone marrow of these mice might express exclusively beta-globin and therefore could be employed to select or screen inducers of gamma-globin expression. A thrombopoietin receptor derivative that brings the proliferative status of primary mouse bone marrow cells under control of a chemical inducer of dimerization was employed to institute and maintain these cell populations. Human beta-globin was expressed, but gamma-globin was not; a similar expression pattern was observed in cells derived from fetal liver. gamma-Globin expression was induced upon exposure to 5-azacytidine, in cells derived from -117 Greek hereditary persistence of fetal hemoglobin human beta-globin locus yeast artificial chromosome (beta-YAC) mice, showing that the hereditary persistence of fetal hemoglobin (HPFH) phenotype was maintained in these cells or was reactivated by an artificial zinc finger-gamma-globin transcription factor and the previously identified fetal globin transactivators fetal Krüppel-like factor (FKLF) and fetal globin-increasing factor (FGIF). These cells may be useful for identifying transcription factors that reactivate gamma-globin synthesis or screening gamma-globin inducers for the treatment of sickle cell disease or beta-thalassemia.Journal of Biological Chemistry 12/2005; 280(44):36642-7. · 4.77 Impact Factor -
Article: Genome architecture of the human beta-globin locus affects developmental regulation of gene expression.
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ABSTRACT: To test the role of gene order in globin gene expression, mutant human beta-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a "marked" transcript (epsilon(m), gamma(m), or beta(m)) integrated at different locations within the locus. When a beta(m)-globin gene was placed between the locus control region (LCR) and the epsilon-globin gene, beta(m)-globin expression dominated primitive and definitive erythropoiesis; only beta(m)-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an (A)gamma(m)-globin gene was placed at the same location, (A)gamma(m)-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type gamma-globin genes were not expressed. When an epsilon(m)-globin gene was placed between the delta- and beta-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type epsilon-globin gene was expressed. Placement of a beta(m)-globin gene upstream of the (G)gamma-globin gene resulted in expression of beta(m)-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type beta-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.Molecular and Cellular Biology 11/2005; 25(20):8765-78. · 5.53 Impact Factor -
Article: A FTZ-F1-containing yeast artificial chromosome recapitulates expression of steroidogenic factor 1 in vivo.
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ABSTRACT: Steroidogenic factor 1 (SF-1/Nr5a1) is an orphan nuclear receptor encoded by the Ftz-F1 gene and is required for gonad and adrenal development and regulation of hormone production within the reproductive and adrenal axes. To extend our understanding of Ftz-F1 and its role in SF-1 expression, we identified and characterized a yeast artificial chromosome (YAC) containing Ftz-F1. Within this YAC, Ftz-F1 is centrally located and flanked by genes encoding a second orphan nuclear receptor, germ cell nuclear factor, and proteasome (prosome, macropain) subunit beta type 7. Three lines of transgenic mice carrying the YAC were generated and in two lines (lines 7 and 14), RT-PCR and ribonuclease protection analysis showed that expression of transgenic SF-1 mimicked that of endogenous SF-1, both spatially and quantitatively. In the third line (line 15), pituitary and hypothalamic expression were absent. Comparison of the integrated transgenes revealed that line 15 was truncated at the end of intron 4 and revealed a region within the locus that is responsible for SF-1 expression in the pituitary and hypothalamus. The line 14 transgene was introduced into a mouse strain lacking functional SF-1. Examination of SF-1-deficient, transgene-positive mice revealed that the YAC was able to rescue adrenal and gonad development, which normally arrests in the SF-1-null embryos and showed that the 153-kb transgene integrated in line 14 is sufficient to properly direct SF-1 expression and support its biological activity. Thus, the study defines a region of Ftz-F1 that contains the requisite set of regulatory elements to direct SF-1 cell-specific expression and all temporal and quantitative changes need for its biological activity.Molecular Endocrinology 11/2005; 19(10):2549-63. · 4.54 Impact Factor -
Article: Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture.
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ABSTRACT: Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.Molecular and Cellular Biology 09/2005; 25(16):7033-41. · 5.53 Impact Factor -
Article: Transgenic Cre expression mice for generation of erythroid-specific gene alterations.
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ABSTRACT: Transgenic mice that express Cre recombinase in erythroid cell lineages were developed so that genes affecting erythropoiesis/hematopoiesis may be altered without necessarily affecting fetus viability. A micro-LCR cassette-beta-globin promoter-Cre recombinase gene (microLCR-betapr-Cre) construct was synthesized and used to generate transgenic mice. Concurrently, we produced mice containing a microLCR-loxP-flanked beta sickle gene (microLCR-loxP-beta(S)-loxP) construct. microLCR-betapr-Cre mice with intact transgenes in variable copy number were identified. Cre expression was assessed by RNAse protection and RT-PCR. Cre function was ascertained by breeding to microLCR-loxP-beta(S)-loxP mice. We demonstrate that beta(S) expression was not detected in the blood of bigenics, but the gene was present in nonerythroid cells. Thus, excision of the loxP-flanked beta(S) gene was restricted to erythroid cell lineages.genesis 06/2004; 39(1):1-9. · 2.53 Impact Factor -
Article: Rapid isolation of yeast genomic DNA: Bust n' Grab.
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ABSTRACT: Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng--3 microg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase cocktail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human beta-globin locus YAC. An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.BMC Biotechnology 05/2004; 4:8. · 2.35 Impact Factor -
Article: Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes.
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ABSTRACT: High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.Human Molecular Genetics 12/2003; 12(22):2941-8. · 7.64 Impact Factor
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Institutions
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2002–2012
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Kansas City VA Medical Center
Kansas City, MO, USA
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1998–2006
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University of Washington Seattle
- • Division of Hematology
- • Division of Medical Genetics
Seattle, WA, USA
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