188 results on '"Bouhassira, Eric E."'
Search Results
152. Increased Global Gene Promoter Methylation after Relapse (Rel) of Acute Promyelocytic Leukemia (APL) from All-transRetinoic Acid (ATRA)-Containing Treatment Is Dissociated from Concurrent Gene Expression Changes.
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Zhou, Da-Cheng, Cui, Qingping, Kim, Haesook, Thompson, Reid, Greally, John, Bouhassira, Eric E., and Gallagher, Robert E.
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Increased methylation of gene-regulatory DNA sequences has been associated with the progression of malignancies, and, in APL, the pathogenetic PML-RARα fusion gene has been associated with DNA methyltransferase activity, which might contribute to this process. We, thus, hypothesized that promoter methylation levels would be increased in APL samples at disease Rel and that this might be contributory to gene expression changes related to Rel. We investigated this hypothesis using matched pretreatment (PTx) and Rel DNA and RNA samples from 5 patients of whom 3 had received combined chemotherapy(CT)-ATRA (A-set) and 2 had received only CT (C-set); one additional RNA matched pair was available from another CT-only patient. The specimens used for DNA/RNA extraction had ≥ 85% blasts. The HELP assay (Khulan, et al, Genome Res. 16, 1046, 2006), which is based on comparative hybridization of HpaII(methylation-sensitive) versus MspI(methylation-insensitive) digested genomic DNA to a microarray representing the promoters of ∼24,000 human genes, was used to distinguish hypermethylated loci (HR) from hypomethylated loci (HO). Gene expression analysis utilized the Affymetrix Human Genome U-133 Plus 2.0 chip. Unsupervised cluster analysis, performed on subsets of HELP gene probes (hgp), selected for variation of ≥1 sample by ≥2-fold from the mean methylation value of all samples, demonstrated segregation by patient in 4/5 of the paired DNA sets but by treatment type (A-set vs C-set) at Rel. A subset of 6,220 Affymetrix gene probe sets (gps), selected for a mean expression level of ≥100 and a mean absolute change from PTx to Rel of ≥100 among the 3 pairs of A-set and C-set RNA samples, also segregated by treatment type at Rel. In an overall analysis of methylation based on >23,000 hgp, the average HR increased by 5% in A-set and decreased by 6% in C-set patients. Conversely, the average HO decreased by 4.3% in the A-set and increased by 7% in the C-set patients. In order to more critically evaluate the methylation changes and their possible relationship to gene expression changes, we selected a subset of hgp within −500 to +200nt of the transcription start site (TSS) in each gene promoter. Further selecting these hgp for an ≥1.5-fold change from PTx to Rel for each matched sample pair from the A- or C-sets and performing a scatter plot analysis of this selected hgp subset versus the change in expression of all 6,220 Affymetrix gps, there was a predominant increase in the HR hgp in the A-set and of HO hgp in the C-set, paralleling the overall hgp results. However, the distribution of methylation changes was random with respect to changes in gene expression. Similar results were observed by a reverse selection analysis starting from Affymetrix gps that were changed by ≥1.5-fold in all samples from each set (559, A-set; 361, C-set) and plotting versus all near-TSS hgp (15,782 hgp). Within the limits of the small sample size analyzed, we conclude that treatment type affects both gene promoter methylation and gene expression at Rel, and, most confidently, that Rel from ATRA-containing treatment was associated with increased gene promoter methylation that was not broadly correlated with gene expression changes. Further studies are required to assess whether coordinated methylation/ expression changes occur in key genes with pathogenetic potential.
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- 2007
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153. Generation of Transgenic Mice Expressing Human Hemoglobin E.
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Chen, Qiuying, Bouhassira, Eric E., Besse, Arnaud, Suzuka, Sandra M., Fabry, Mary E., Nagel, Ronald L., and Elison-Hirsch, Rhoda
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HbE (β26 Glu → Lys) is the most common pathogenic Hb variant in the world and is found with the greatest frequency in Southeast Asia. The HbE mutation at codon 26 of the βE-globin leads to an alternative splicing site, making this globin both a structural as well as a thalassemic mutation; furthermore, βEglobin chains are unstable. HbE trait is asymptomatic and the HbEE genotype has only mild clinical manifestations. On the other hand, HbE/thalassemia presents a panoply of phenotypes from very severe to a mild beta/+ thalassemia. To date, there is no animal model of HbE disease. An animal model of this disorder could lead to better understanding of the effects of modifier genes and some of the pathogenic features of this disease. In addition, an animal model can facilitate the development of gene therapy. We report here the creation of four lines of transgenic mice that are PCR positive for HbE: two with founders that express high levels of HbE (32% human α, 26% human βE), one with low expression of HbE (9% human α, 8% βE), and one without detectable expression, but PCR positive. Knockouts (KOs) for mouse α and β globins have been bred into the founders. To date, the most severe mouse generated has a full KO of mouse α and a partial KO of mouse β. RBCs from all of the partial KOs generated to date have normal MCHC and minimally elevated reticulocyte counts (less than 1% greater than the C57Bl background). We previously demonstrated that HbEE RBCs are microcytic, but have normal MCHC. We found that HbE mice with full α-KO and partial β-KO, are microcytic (MCH 10.9 vs 14.2 for C57Bl and MCV 35.4 vs 47.5 for C57Bl), but have normal MCHC (MCHC 30.0 vs 30.5 for C57Bl). Smears from these mice exhibit numerous target cells that are not necessarily associated with low MCHC. As reported for human red cells containing HbE, elevated levels of hemoglobin oxidation products were detected in founder mice expressing HbE, but not in negative litter mates, and were found, at an even higher level, in partial KO mice expressing HbE. The next step will be to generate a mouse similar to the HbE/thalassemia phenotype by generating mice expressing exclusively HbE from either the lines expressing high levels of HbE or the line expressing low levels of HbE. These models will help elucidate several of the challenging features of the phenotypes described in humans and allow for the development of the potential cure or amelioration of HbE/thalassemia.
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- 2004
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154. Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.
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Twayana, Shyam, Bacolla, Albino, Barreto-Galvez, Angelica, De-Paula, Ruth B., Drosopoulos, William C., Kosiyatrakul, Settapong T., Bouhassira, Eric E., Tainer, John A., Madireddy, Advaitha, and Schildkraut, Carl L.
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HUMAN genetic variation , *DNA replication , *CHROMOSOME replication , *DNA structure , *DNA polymerases , *RAPESEED - Abstract
Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites. [ABSTRACT FROM AUTHOR]
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- 2021
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155. GenPlay Multi-Genome, a tool to compare and analyze multiple human genomes in a graphical interface.
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Lajugie, Julien, Fourel, Nicolas, and Bouhassira, Eric E.
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GENOTYPES , *CANCER genetics , *GENOMES , *HAPLOIDY , *PLOIDY - Abstract
Summary: Parallel visualization of multiple individual human genomes is a complex endeavor that is rapidly gaining importance with the increasing number of personal, phased and cancer genomes that are being generated. It requires the display of variants such as SNPs, indels and structural variants that are unique to specific genomes and the introduction of multiple overlapping gaps in the reference sequence. Here, we describe GenPlay Multi-Genome, an application specifically written to visualize and analyze multiple human genomes in parallel. GenPlay Multi-Genome is ideally suited for the comparison of allele-specific expression and functional genomic data obtained from multiple phased genomes in a graphical interface with access to multiple-track operation. It also allows the analysis of data that have been aligned to custom genomes rather than to a standard reference and can be used as a variant calling format file browser and as a tool to compare different genome assembly, such as hg19 and hg38.Availability and implementation: GenPlay is available under the GNU public license (GPL-3) from http://genplay.einstein.yu.edu. The source code is available at https://github.com/JulienLajugie/GenPlayContact: eric.bouhassira@einstein.yu.edu or julien.lajugie@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM PUBLISHER]
- Published
- 2015
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156. Hi stone H1.3 Suppresses H19 Non coding RNA Expression and Cell Growth of Ovarian Cancer Cells.
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Medrzycki, Magdalena, Zhang, Yunzhe, Zhang, Weijia, Cao, Kaixiang, Pan, Chenyi, Lailler, Nathalie, McDonald, John F., Bouhassira, Eric E., and Fan, Yuhong
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OVARIAN cancer diagnosis , *CANCER treatment , *BIOMARKERS , *CANCER genetics , *CELL lines - Abstract
Ovarian cancer is a deadly gynecologic malignancy for which novel bio markers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker hi stone variants as potential bio markers for ovarian cancer. To investigate the role of hi stone H1 in ovarian cancer cells, we characterize individual H1 variants and over express one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line.We find that over expression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify hi stone H1.3 as a specific repressor for the non coding oncogene H19. Over expression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that hi stone H1.3 over expression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 over expression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis. [ABSTRACT FROM AUTHOR]
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- 2014
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157. The exosome complex establishes a barricade to erythroid maturation.
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Mclver, Skye C., Kang, Yoon-A., DeVilbiss, Andrew W., O'Driscoll, Chelsea A., Ouellette, Jonathan N., Pope, Nathaniel J., Camprecios, Genis, Chan-Jung Chang, David Yang, Bouhassira, Eric E., Ghaffari, Saghi, and Bresnick, Emery H.
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EXOSOMES , *HEMATOPOIETIC stem cells , *CELL differentiation , *PROGENITOR cells , *FORKHEAD transcription factors - Abstract
Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor Foxo3 amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating ExoscB or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program. [ABSTRACT FROM AUTHOR]
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- 2014
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158. A transgenic mouse model expressing exclusively human hemoglobin E: Indications of a mild oxidative stress
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Chen, Qiuying, Fabry, Mary E., Rybicki, Anne C., Suzuka, Sandra M., Balazs, Tatiana C., Etzion, Zipora, de Jong, Kitty, Akoto, Edna K., Canterino, Joseph E., Kaul, Dhananjay K., Kuypers, Frans A., Lefer, David, Bouhassira, Eric E., and Hirsch, Rhoda Elison
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HEMOGLOBINS , *LABORATORY mice , *OXIDATIVE stress , *GENETIC mutation , *ERYTHROCYTES , *PATHOLOGICAL physiology , *THALASSEMIA - Abstract
Abstract: Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid βE-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of βE globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation. [Copyright &y& Elsevier]
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- 2012
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159. Decreased replication origin activity in temporal transition regions.
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Guan, Zeqiang, Hughes, Christina M., Kosiyatrakul, Settapong, Norio, Paolo, Sen, Ranjan, Fiering, Steven, Allis, C. David, Bouhassira, Eric E., and Schildkraut, Carl L.
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MAMMAL genetics , *IMMUNOGLOBULINS , *CELL growth , *DNA replication , *LYMPHOCYTES - Abstract
In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication. Introduction of a transcription unit into the Igh TTR, activation of gene transcription, and enhancement of local histone modifications characteristic of active chromatin did not lead to origin activation. Moreover, very few replication initiation events were observed when two ectopic replication origin sequences were inserted into the TTR. These findings indicate that the Igh TTR represents a repressive compartment that inhibits replication initiation, thus maintaining the boundaries between early and late replication domains. [ABSTRACT FROM AUTHOR]
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- 2009
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160. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery.
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Kiprilov, Enko N., Awan, Aashir, Desprat, Romain, Velho, Michelle, Clement, Christian A., Byskov, Anne Grete, Andersen, Claus Y., Satir, Peter, Bouhassira, Eric E., Christensen, Søren T., and Hirsch, Rhoda Elison
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EMBRYONIC stem cells , *CELL culture , *CILIA & ciliary motion , *HEDGEHOG signaling proteins , *TISSUE differentiation , *CELLULAR control mechanisms - Abstract
Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery. [ABSTRACT FROM AUTHOR]
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- 2008
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161. Enzymatically Active ADAMTS13 Variants Are Not Inhibited by Anti-ADAMTS13 Autoantibodies.
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Wenhua Zhou, Lingli Dong, Ginsburg, David, Bouhassira, Eric E., and Han-Mou Tsai
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IMMUNOGLOBULINS , *BLOOD proteins , *GLOBULINS , *PLASMA cells , *HEMOLYTIC anemia , *ANEMIA , *HEMOLYSIS & hemolysins , *THROMBOTIC thrombocytopenic purpura , *IMMUNOGLOBULIN G - Abstract
ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing yon Willebrand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP. [ABSTRACT FROM AUTHOR]
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- 2005
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162. Methylation protects cytidines from AID-mediated deamination
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Larijani, Mani, Frieder, Darina, Sonbuchner, Timothy M., Bransteitter, Ronda, Goodman, Myron F., Bouhassira, Eric E., Scharff, Matthew D., and Martin, Alberto
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GENES , *METHYLATION , *DEAMINATION , *NUCLEIC acids - Abstract
Abstract: Somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion of immunoglobulin genes require activation-induced cytidine deaminase (AID). AID initiates these events by deaminating cytidines within antibody variable and switch regions. The mechanism that restricts mutation to antibody genes is not known. Although genes other than antibody genes have been found to mutate, not all highly transcribed genes mutate. Thus, somatic hypermutation does not target all genes and suggests a mechanism that either recruits AID to genes for mutation, and/or one that protects genes from promiscuous AID activity. Recent evidence suggests that AID deaminates methyl cytidines inefficiently. Methylation of cytidines could thus represent a means to protect the genome from potentially harmful AID activity that occurs outside of the immunoglobulin loci. To test this premise, we examined whether AID could deaminate methylated-CpG motifs in different sequence contexts. In agreement with a report that suggests that AID has processive-like properties in vitro, we found that AID could completely deaminate single-stranded DNA tracks in plasmid substrates that were greater than 300 nucleotides in length. In addition, methylated-CpG motifs, but not their unmethylated counterparts, were protected from AID-mediated deamination. However, methylation did not protect cytidines that neighbored CpG motifs indicating that methylation per se does not provide a more global safeguard against AID-mediated activity. These data also suggest that AID, and possibly other related cytidine deaminases, might represent a more rapid alternative to bisulfite sequencing for identifying methylated-CpG motifs. [Copyright &y& Elsevier]
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- 2005
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163. Mammalian linker-histone subtypes differentially affect gene expression in vivo.
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Alami, Raouf, Yuhong Fan, Pack, Stephanie, Sonbuchner, Timothy M., Besse, Arnaud, Qingcong Lin, Greally, John M., Skoultchi, Arthur I., and Bouhassira, Eric E.
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HISTONES , *GENE expression , *CHROMATIN , *SOMATIC cells , *LABORATORY mice - Abstract
Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linkerhistone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels. [ABSTRACT FROM AUTHOR]
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- 2003
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164. Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells.
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Imren, Suzan, Payen, Emmanuel, Westerman, Karen A., Pawliuk, Robert, Fabry, Mary E., Eaves, Connie J., Cavilla, Benjamin, Wadsworth, Louis D., Beuzard, Yves, Bouhassira, Eric E., Russell, Robert, London, Irving M., Nagel, Ronald L., Leboulch, Philippe, and Humphries, R. Keith
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THALASSEMIA , *LENTIVIRUSES , *HEMATOPOIETIC stem cells - Abstract
Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid β globin gene expression has not yet been achieved in β thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe β thalassemia in mice, resulting from a homozygous deletion of the β major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-l-derived [β globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time ≈95% of the red blood cells in all mice contained human β globin contributing to 32 ± 4% of all β-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free α globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral... [ABSTRACT FROM AUTHOR]
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- 2002
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165. Correction of Sickle Cell Disease in Transgenic Mouse Models by Gene Therapy.
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Pawliuk, Robert, Westerman, Karen A., Fabry, Mary E., Payen, Emmanuel, Tighe, Robert, Bouhassira, Eric E., Acharya, Seetharama A., Ellis, James, London, Irving M., Eaves, Connie J., Humphries, R. Keith, Beuzard, Yves, Nagel, Ronald L., and Leboulch, Philippe
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SICKLE cell anemia , *GENE therapy , *LABORATORY mice - Abstract
Reports the correction of sickle cell disease (SCD) in transgenic mouse models by gene therapy. Design of a beta[sup A] globin gene variant preventing the abnormal hemoglobin polymerization; Inhibition of red blood cell dehydration and sickling in SCD models; Prevention of the characteristic urine concentration defect in SCD.
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- 2001
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166. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.
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Levy, Galia G., Nichols, William C., Lian, Eric C., Foroud, Tatiana, McClintick, Jeanette N., McGee, Beth M., Yang, Angela Y., Siemieniak, David R., Stark, Kenneth R., Gruppo, Ralph, Sarode, Ravinda, Shurin, Susan B., Chandrasekaran, Visalam, Stabler, Sally P., Sabio, Hernan, Bouhassira, Eric E., Upshaw Jr., Jefferson D., Ginsburg, David, and Han-Mou Tsai
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THROMBOTIC thrombocytopenic purpura , *PROTEOLYTIC enzyme genes , *METALLOPROTEINASES , *MUTAGENESIS , *GENETICS - Abstract
Shows that deficiency of ADAMTS13, a member of the ADAMTS family of zinc metalloproteinase genes, is the molecular mechanism responsible for thrombotic thrombocytopenic purpura (TTP). Role of the proteolysis of von Willebrande and/or other ADAMTS13 substrates in maintaining the balance between bleeding and clotting; Genetic mutations related to TTP.
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- 2001
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167. HbF Levels in Sickle Cell Disease Are Associated with Proportion of Circulating Hematopoietic Stem and Progenitor Cells and CC-Chemokines.
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Minniti, Caterina P., Tolu, Seda S., Wang, Kai, Yan, Zi, Robert, Karl, Zhang, Shouping, Crouch, Andrew S., Uehlinger, Joan, Manwani, Deepa, and Bouhassira, Eric E.
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SICKLE cell anemia , *HEMATOPOIETIC stem cells , *PROGENITOR cells , *FETAL hemoglobin , *CHEMOKINES - Abstract
The concentration of circulating hematopoietic stem and progenitor cells has not been studied longitudinally. Here, we report that the proportions of Lin-CD34+38- hematopoietic multipotent cells (HMCs) and of Lin-CD34+CD38+ hematopoietic progenitors cells (HPCs) are highly variable between individuals but stable over long periods of time, in both healthy individuals and sickle cell disease (SCD) patients. This suggests that these proportions are regulated by genetic polymorphisms or by epigenetic mechanisms. We also report that in SCD patients treated with hydroxyurea, the proportions of circulating HMCs and HPCs show a strong positive and negative correlation with fetal hemoglobin (HbF) levels, respectively. Titration of 65 cytokines revealed that the plasma concentration of chemokines CCL2, CCL11, CCL17, CCL24, CCL27, and PDGF-BB were highly correlated with the proportion of HMCs and HPCs and that a subset of these cytokines were also correlated with HbF levels. A linear model based on four of these chemokines could explain 80% of the variability in the proportion of circulating HMCs between individuals. The proportion of circulating HMCs and HPCs and the concentration of these chemokines might therefore become useful biomarkers for HbF response to HU in SCD patients. Such markers might become increasingly clinically relevant, as alternative treatment modalities for SCD are becoming available. [ABSTRACT FROM AUTHOR]
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- 2020
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168. Characterization of Hematopoiesis in Sickle Cell Disease by Prospective Isolation of Stem and Progenitor Cells.
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Tolu, Seda S., Wang, Kai, Yan, Zi, Zhang, Shouping, Roberts, Karl, Crouch, Andrew S., Sebastian, Gracy, Chaitowitz, Mark, Fornari, Eric D., Schwechter, Evan M., Uehlinger, Joan, Manwani, Deepa, Minniti, Caterina P., and Bouhassira, Eric E.
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SICKLE cell anemia , *PROGENITOR cells , *STEM cells , *HEMATOPOIETIC stem cells , *HEMATOPOIESIS , *PREMATURE aging (Medicine) , *TUMOR lysis syndrome - Abstract
The consequences of sickle cell disease (SCD) include ongoing hematopoietic stress, hemolysis, vascular damage, and effect of chronic therapies, such as blood transfusions and hydroxyurea, on hematopoietic stem and progenitor cell (HSPC) have been poorly characterized. We have quantified the frequencies of nine HSPC populations by flow cytometry in the peripheral blood of pediatric and adult patients, stratified by treatment and control cohorts. We observed broad differences between SCD patients and healthy controls. SCD is associated with 10 to 20-fold increase in CD34dim cells, a two to five-fold increase in CD34bright cells, a depletion in Megakaryocyte-Erythroid Progenitors, and an increase in hematopoietic stem cells, when compared to controls. SCD is also associated with abnormal expression of CD235a as well as high levels CD49f antigen expression. These findings were present to varying degrees in all patients with SCD, including those on chronic therapy and those who were therapy naive. HU treatment appeared to normalize many of these parameters. Chronic stress erythropoiesis and inflammation incited by SCD and HU therapy have long been suspected of causing premature aging of the hematopoietic system, and potentially increasing the risk of hematological malignancies. An important finding of this study was that the observed concentration of CD34bright cells and of all the HSPCs decreased logarithmically with time of treatment with HU. This correlation was independent of age and specific to HU treatment. Although the number of circulating HSPCs is influenced by many parameters, our findings suggest that HU treatment may decrease premature aging and hematologic malignancy risk compared to the other therapeutic modalities in SCD. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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169. Clonal origin in normal adults of all blood lineages and circulating hematopoietic stem cells.
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Wang, Kai, Yan, Zi, Zhang, Shouping, Bartholdy, Boris, Eaves, Connie J., and Bouhassira, Eric E.
- Subjects
- *
HEMATOPOIETIC stem cells , *SINGLE nucleotide polymorphisms , *BLOOD cells , *BLOOD groups , *ADULTS - Abstract
• Lineage tracing of somatically acquired DNA transversions enables a sensitive method for tracking hematopoietic clones. • This approach has revealed multilineage clones persisting in two normal individuals up to 53 years of age. Characterization of human cells that sustain blood cell production lifelong has historically been inferred from phenotypically defined subsets of cells assayed in vitro , in transplanted immunodeficient mice, or in patients transplanted with genetically marked cells. These approaches have led to the concept of a persistent complex hierarchical process of differentiation divisions originating from a rare population of CD34+CD38−CD45RA−CD90+CD49f+ cells with an average self-renewal potential of >0.5 and an ability to produce some or all blood cell types for >1 year. However, the role of these "49f" cells in the unperturbed adult has remained poorly understood. To address this gap, somatic single-nucleotide polymorphisms (SNVs) have recently been exploited as lineage tracing markers to enumerate and characterize active hematopoietic clones in normal adults using a capture and recapture approach. We show here that the use of somatic transversions to identify somatically acquired variant alleles enabled their detection in bulk populations at frequencies of approximately 1 in 80,000 cells. We then applied this method to blood cells isolated from two normal adults (aged 31 and 53 years) over a 1- to 3-year period. The results revealed in both donors a continued clonal output of both T- and B-lymphoid cells as well as myeloid cells identified by the same unique transversions found to distinguish single 49f cells isolated from the same donors' initial blood samples. These findings provide the first evidence of a continuing hematopoietic stem cell–derived source of all mature blood cell types in normal (unperturbed) adult humans. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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170. PSC-RED and MNC-RED: Albumin-free and low-transferrin robust erythroid differentiation protocols to produce human enucleated red blood cells.
- Author
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Olivier, Emmanuel N., Zhang, Shouping, Yan, Zi, Suzuka, Sandra, Roberts, Karl, Wang, Kai, and Bouhassira, Eric E.
- Subjects
- *
ERYTHROCYTES , *IRON chelates , *BLOOD cells , *STEM cells , *INDUSTRIAL costs - Abstract
• Albumin-free, low-transferrin, chemically defined protocols were used to produce cultured red blood cells. • FeIII-EDTA, an iron chelator, allows transferrin recycling to take place in cell culture. • The long-PSC-RED protocol yields iPSC-derived erythroid cells that enucleate at rates as high as 50%. • Low costs, high rates of enucleation, and high yields improve the economic viability of cultured RBCs. Many methods have been developed to produce cultured red blood cells (cRBCs) in vitro but translational applications have been hampered by high costs of production and by low rates of enucleation. We have developed R6 and IMIT, two chemically defined culture media and combined them into robust erythroid differentiation (RED) protocols to differentiate induced-pluripotent stem cells (iPSCs) and peripheral blood mononuclear cells (MNCs) into enucleated erythroid cells. The RED protocols do not require any albumin or animal components and require ten- to twentyfold less transferrin (Tf) than previously, because iron is provided to the differentiating erythroblasts by small amounts of recombinant Tf supplemented with FeIII-EDTA, an iron chelator that allows Tf recycling to take place in cell culture. Importantly, cRBCs produced by iPSC differentiation using the long PSC-RED protocol enucleate at much higher rates than with previous protocols, eliminating one of the impediments to the use of these cells to produce clinically useful cRBCs. The absence of albumin, the reduced amounts of Tf, the improved reproducibility associated with the elimination of all animal components, and the high yield on the RED protocols decrease the cost of production of cultured red blood cells. RED protocols should therefore help to make translational applications of cultured RBCs more economically realistic. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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171. Stem cell factor and erythropoietin-independent production of cultured reticulocytes.
- Author
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Olivier E, Zhang S, Yan Z, and Bouhassira EE
- Abstract
Cultured reticulocytes can supplement transfusion needs and offer promise for drug delivery and immune tolerization. They can be produced from induced pluripotent stem cells (iPSCs), but the 45-day culture time and cytokine costs make large-scale production prohibitive. To overcome these limitations, we have generated IPSCs that express constitutive SCF receptor and jak2 adaptor alleles. We show that iPSC lines carrying these alleles can differentiate into self-renewing erythroblast (SRE) that can proliferate for up to 70 cell-doubling in a cost-effective, chemically-defined, albumin- and cytokine-free medium. These kitjak2 SREs retain the ability to enucleate at a high rate up to senescence. Kitjak2 derived cultured reticulocytes should be safe for transfusion because they can be irradiated to eliminate residual nucleated cells. The kitjak2 cells express blood group 0 and test negative for RhD and other clinically significant RBCs antigens and have sufficient proliferation capacity to meet global RBC needs.
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- 2024
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172. Epigenetic control of chromosome-associated lncRNA genes essential for replication and stability.
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Heskett MB, Vouzas AE, Smith LG, Yates PA, Boniface C, Bouhassira EE, Spellman PT, Gilbert DM, and Thayer MJ
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- Animals, Humans, Epigenesis, Genetic, DNA Replication Timing, Chromosomes metabolism, RNA, Untranslated, Mammals genetics, RNA, Long Noncoding genetics, RNA, Long Noncoding metabolism
- Abstract
ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilize a set of distinctive ASAR characteristics that allow for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci result in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome., (© 2022. The Author(s).)
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- 2022
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173. Hemoglobin F mitigation of sickle cell complications decreases with aging.
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Tolu SS, Reyes-Gil M, Ogu UO, Thomas M, Bouhassira EE, and Minniti CP
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- Adult, Age Factors, Aged, Aging, Female, Humans, Male, Middle Aged, Young Adult, Anemia, Sickle Cell complications, Fetal Hemoglobin metabolism
- Published
- 2020
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174. The exosome complex establishes a barricade to erythroid maturation.
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McIver SC, Kang YA, DeVilbiss AW, O'Driscoll CA, Ouellette JN, Pope NJ, Camprecios G, Chang CJ, Yang D, Bouhassira EE, Ghaffari S, and Bresnick EH
- Subjects
- Animals, Autophagy, Cell Differentiation, Epigenesis, Genetic, Erythroblasts cytology, Erythroid Cells metabolism, Erythropoiesis genetics, Forkhead Box Protein O3, Gene Expression Regulation, Mice, RNA metabolism, Transcriptional Activation, Erythrocytes cytology, Exosomes physiology, Forkhead Transcription Factors metabolism, GATA1 Transcription Factor metabolism
- Abstract
Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program., (© 2014 by The American Society of Hematology.)
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- 2014
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175. Erythropoiesis from human embryonic stem cells through erythropoietin-independent AKT signaling.
- Author
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Kim WS, Zhu Y, Deng Q, Chin CJ, He CB, Grieco AJ, Dravid GG, Parekh C, Hollis RP, Lane TF, Bouhassira EE, Kohn DB, and Crooks GM
- Subjects
- Cell Cycle, Cell Differentiation, Cell Line, Cell Proliferation, Cell Survival, Erythroid Precursor Cells cytology, Erythroid Precursor Cells metabolism, Humans, Megakaryocytes cytology, Megakaryocytes metabolism, Protein Multimerization, Protein Structure, Tertiary, Receptors, Thrombopoietin chemistry, Receptors, Thrombopoietin metabolism, Embryonic Stem Cells cytology, Embryonic Stem Cells enzymology, Erythropoiesis, Erythropoietin metabolism, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction
- Abstract
Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBCs) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homodimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of this study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESCs) and to identify the signaling pathways activated by this strategy. We present here the evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis, and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle, and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC., (© 2014 AlphaMed Press.)
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- 2014
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176. Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function.
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Kim HS, Mukhopadhyay R, Rothbart SB, Silva AC, Vanoosthuyse V, Radovani E, Kislinger T, Roguev A, Ryan CJ, Xu J, Jahari H, Hardwick KG, Greenblatt JF, Krogan NJ, Fillingham JS, Strahl BD, Bouhassira EE, Edelmann W, and Keogh MC
- Subjects
- Acetylation, Centromere metabolism, Histone Acetyltransferases metabolism, Histones metabolism, Mitosis physiology, Yeasts metabolism, Adenosine Triphosphatases metabolism, Casein Kinase II metabolism, Chromatin metabolism, DNA-Binding Proteins metabolism, Multiprotein Complexes metabolism, Nuclear Proteins metabolism, RNA-Binding Proteins metabolism
- Abstract
Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
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177. GenPlay, a multipurpose genome analyzer and browser.
- Author
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Lajugie J and Bouhassira EE
- Subjects
- Base Sequence, Gene Expression, Humans, Internet, Genome, Sequence Analysis, DNA methods, Software
- Abstract
Motivation: Rapidly decreasing sequencing cost due to the emergence and improvement of massively parallel sequencing technologies has resulted in a dramatic increase in the quantity of data that needs to be analyzed. Therefore, software tools to process, visualize, analyze and integrate data produced on multiple platforms and using multiple methods are needed., Results: GenPlay is a fast, easy to use and stable tool for rapid analysis and data processing. It is written in Java and runs on all major operating systems. GenPlay recognizes a wide variety of common genomic data formats from microarray- or sequencing-based platforms and offers a library of operations (normalization, binning, smoothing) to process raw data into visualizable tracks. GenPlay displays tracks adapted to summarize gene structure, gene expression, repeat families, CPG islands, etc. as well as custom tracks to show the results of RNA-Seq, ChIP-Seq, TimEX-Seq and single nucleotide polymorphism (SNP) analysis. GenPlay can generate statistics (minimum, maximum, SD, correlation, etc.). The tools provided include Gaussian filter, peak finders, signal saturation, island finders. The software also offers graphical features such as scatter plots and bar charts to depict signal repartition. The library of operations is continuously growing based on the emerging needs., Availability: GenPlay is an open-source project available from http://www.genplay.net. The code source of the software is available at https://genplay.einstein.yu.edu/svn/GenPlay.
- Published
- 2011
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178. Differentiation of human embryonic stem cells into mesenchymal stem cells by the "raclure" method.
- Author
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Olivier EN and Bouhassira EE
- Subjects
- Adipogenesis, Animals, Biological Assay, Biomarkers metabolism, Cell Line, Cell Shape, Flow Cytometry, Humans, Mice, Osteogenesis, Cell Culture Techniques methods, Cell Differentiation, Embryonic Stem Cells cytology, Mesenchymal Stem Cells cytology
- Abstract
Mesenchymal stem cells also called mesenchymal stromal cells (MSCs) are multipotent progenitors that can be found in many connective tissues including fat, bone, cartilage, and muscle. We report here a simple method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layers or exogenous cytokines. The cells obtained with this procedure have a normal karyotype, are morphologically similar to bone marrow MSCs, are contact-inhibited, can be grown in culture for about 20-25 passages, exhibit an immuno-phenotype similar to bone marrow MSCs (negative for CD34 and CD45, but positive for CD44, CD71, CD73, CD105, CD166, HLA ABC, and SSEA-4), and can differentiate into osteocytes and adipocytes. They are also a very useful source of autogenic feeder cells to support the growth of undifferentiated hESCs. The ability to produce MSCs from hESCs should prove useful in obtaining large amounts of genetically identical and genetically modifiable MSCs that can be subsequently used to study the biology of MSCs as well as possible therapeutic applications.
- Published
- 2011
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179. Transferrin therapy ameliorates disease in beta-thalassemic mice.
- Author
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Li H, Rybicki AC, Suzuka SM, von Bonsdorff L, Breuer W, Hall CB, Cabantchik ZI, Bouhassira EE, Fabry ME, and Ginzburg YZ
- Subjects
- Animals, Apoptosis, Cell Survival, Disease Models, Animal, Erythropoietin blood, Hematopoiesis, Extramedullary, Mice, Splenomegaly drug therapy, Transferrin administration & dosage, beta-Thalassemia blood, Transferrin therapeutic use, beta-Thalassemia drug therapy
- Abstract
Individuals with beta-thalassemia develop progressive systemic iron overload, resulting in high morbidity and mortality. These complications are caused by labile plasma iron, which is taken up by parenchymal cells in a dysregulated manner; in contrast, erythropoiesis depends on transferrin-bound iron uptake via the transferrin receptor. We hypothesized that the ineffective erythropoiesis and anemia observed in beta-thalassemia might be ameliorated by increasing the amount of circulating transferrin. We tested the ability of transferrin injections to modulate iron metabolism and erythropoiesis in Hbb(th1/th1) mice, an experimental model of beta-thalassemia. Injected transferrin reversed or markedly improved the thalassemia phenotype in these mice. Specifically, transferrin injections normalized labile plasma iron concentrations, increased hepcidin expression, normalized red blood cell survival and increased hemoglobin production; this treatment concomitantly decreased reticulocytosis, erythropoietin abundance and splenomegaly. These results indicate that transferrin is a limiting factor contributing to anemia in these mice and suggest that transferrin therapy might be beneficial in human beta-thalassemia.
- Published
- 2010
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180. High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers.
- Author
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Oda M, Glass JL, Thompson RF, Mo Y, Olivier EN, Figueroa ME, Selzer RR, Richmond TA, Zhang X, Dannenberg L, Green RD, Melnick A, Hatchwell E, Bouhassira EE, Verma A, Suzuki M, and Greally JM
- Subjects
- Cells, Cultured, DNA chemistry, Deoxyribonuclease HpaII, Genome, Human, Humans, Cytosine metabolism, DNA analysis, DNA Methylation, Polymerase Chain Reaction methods
- Abstract
Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.
- Published
- 2009
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181. Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells.
- Author
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Qiu C, Olivier EN, Velho M, and Bouhassira EE
- Subjects
- Antigens, CD analysis, Antigens, CD34 analysis, Cell Differentiation, Cell Line, Embryonic Stem Cells cytology, Erythrocytes cytology, Fetus, Hemoglobins, Abnormal physiology, Humans, Embryonic Stem Cells physiology, Erythrocytes physiology, Globins physiology, Hemoglobins physiology, Hepatocytes physiology, Yolk Sac physiology
- Abstract
We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.
- Published
- 2008
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182. An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers.
- Author
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Zhou W, Bouhassira EE, and Tsai HM
- Subjects
- ADAM Proteins genetics, ADAM Proteins metabolism, ADAMTS13 Protein, Alleles, Animals, Cloning, Molecular, Humans, Metalloendopeptidases genetics, Mice, Peptides metabolism, Protein Structure, Tertiary genetics, Purpura, Thrombotic Thrombocytopenic enzymology, Purpura, Thrombotic Thrombocytopenic genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity genetics, Genes, Intracisternal A-Particle genetics, Metalloendopeptidases metabolism, Multiprotein Complexes metabolism, Mutagenesis, Insertional, RNA Splicing, von Willebrand Factor metabolism
- Abstract
Severe deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated into a high and a low group that differed by approximately 10 fold. Low ADAMTS13 activity was detected in mice containing 2 alleles of intracisternal A-type particle (IAP) retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified 2 truncated variants (IAP-a and IAP-b) in the low-activity mice. Both of the IAP variants lacked the 2 carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared with full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.
- Published
- 2007
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183. Transcriptional interference among the murine beta-like globin genes.
- Author
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Hu X, Eszterhas S, Pallazzi N, Bouhassira EE, Fields J, Tanabe O, Gerber SA, Bulger M, Engel JD, Groudine M, and Fiering S
- Subjects
- Animals, Erythropoiesis, Eye Proteins genetics, Eye Proteins metabolism, Gene Deletion, Mice, Promoter Regions, Genetic genetics, Globins genetics, Transcription, Genetic genetics
- Abstract
Mammalian beta-globin loci contain multiple genes that are activated at different developmental stages. Studies have suggested that the transcription of one gene in a locus can influence the expression of the other locus genes. The prevalent model to explain this transcriptional interference is that all potentially active genes compete for locus control region (LCR) activity. To investigate the influence of transcription by the murine embryonic genes on transcription of the other beta-like genes, we generated mice with deletions of the promoter regions of Ey and betah1 and measured transcription of the remaining genes. Deletion of the Ey and betah1 promoters increased transcription of betamajor and betaminor 2-fold to 3-fold during primitive erythropoiesis. Deletion of Ey did not affect betah1 nor did deletion of betah1 affect Ey, but Ey deletion uniquely activated transcription from betah0, a beta-like globin gene immediately downstream of Ey. Protein analysis showed that betah0 encodes a translatable beta-like globin protein that can pair with alpha globin. The lack of transcriptional interference between Ey and betah1 and the gene-specific repression of betah0 did not support LCR competition among the embryonic genes and suggested that direct transcriptional interference from Ey suppressed betah0.
- Published
- 2007
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184. CG dinucleotide clustering is a species-specific property of the genome.
- Author
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Glass JL, Thompson RF, Khulan B, Figueroa ME, Olivier EN, Oakley EJ, Van Zant G, Bouhassira EE, Melnick A, Golden A, Fazzari MJ, and Greally JM
- Subjects
- Animals, CpG Islands, DNA Methylation, Humans, Mice, Species Specificity, Takifugu genetics, Dinucleoside Phosphates analysis, Genome
- Abstract
Cytosines at cytosine-guanine (CG) dinucleotides are the near-exclusive target of DNA methyltransferases in mammalian genomes. Spontaneous deamination of methylcytosine to thymine makes methylated cytosines unusually susceptible to mutation and consequent depletion. The loci where CG dinucleotides remain relatively enriched, presumably due to their unmethylated status during the germ cell cycle, have been referred to as CpG islands. Currently, CpG islands are solely defined by base compositional criteria, allowing annotation of any sequenced genome. Using a novel bioinformatic approach, we show that CG clusters can be identified as an inherent property of genomic sequence without imposing a base compositional a priori assumption. We also show that the CG clusters co-localize in the human genome with hypomethylated loci and annotated transcription start sites to a greater extent than annotations produced by prior CpG island definitions. Moreover, this new approach allows CG clusters to be identified in a species-specific manner, revealing a degree of orthologous conservation that is not revealed by current base compositional approaches. Finally, our approach is able to identify methylating genomes (such as Takifugu rubripes) that lack CG clustering entirely, in which it is inappropriate to annotate CpG islands or CG clusters.
- Published
- 2007
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185. ADAMTS13 is expressed in hepatic stellate cells.
- Author
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Zhou W, Inada M, Lee TP, Benten D, Lyubsky S, Bouhassira EE, Gupta S, and Tsai HM
- Subjects
- ADAMTS13 Protein, Animals, Cell Line, Cells, Cultured, Cloning, Molecular, Culture Media, Gene Expression Regulation, Enzymologic, In Situ Hybridization, Mice, Mice, Inbred Strains, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Liver cytology, Liver enzymology, Metalloendopeptidases genetics
- Abstract
ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression.
- Published
- 2005
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186. The human beta-globin locus control region can silence as well as activate gene expression.
- Author
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Feng YQ, Warin R, Li T, Olivier E, Besse A, Lobell A, Fu H, Lin CM, Aladjem MI, and Bouhassira EE
- Subjects
- Animals, Cell Line, Chromatin chemistry, Chromatin genetics, Chromatin metabolism, Chromosomes, Mammalian genetics, DNA Methylation, DNA Replication genetics, DNA, Intergenic genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Humans, Mice, Models, Genetic, Promoter Regions, Genetic genetics, Transcription, Genetic genetics, Transgenes genetics, Gene Silencing, Globins genetics, Locus Control Region genetics, Transcriptional Activation genetics
- Abstract
Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.
- Published
- 2005
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- View/download PDF
187. Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region.
- Author
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Hu X, Bulger M, Roach JN, Eszterhas SK, Olivier E, Bouhassira EE, Groudine MT, and Fiering S
- Subjects
- Alleles, Animals, Blotting, Southern, Chromatography, High Pressure Liquid, Gene Deletion, Genetic Vectors, Humans, Mice, Models, Genetic, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Transgenes, Globins genetics, Promoter Regions, Genetic
- Abstract
Mammalian beta-globin loci contain multiple beta-like genes that are expressed at different times during development. The murine beta-globin locus contains two genes expressed during the embryo stage, Ey and betah1, and two genes expressed at both the fetal and postnatal stages, beta-major and beta-minor. Studies of transgenic human beta-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or betah1 promoter in the endogenous murine beta-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal-adult beta-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic beta-globin gene promoters and other genes in the locus. The implication of these findings for models of beta-globin gene expression are discussed.
- Published
- 2003
- Full Text
- View/download PDF
188. Transgenic mice and hemoglobinopathies.
- Author
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Fabry ME, Bouhassira EE, Suzuka SM, and Nagel RL
- Subjects
- Anemia, Sickle Cell diagnosis, Anemia, Sickle Cell genetics, Animals, Chromatography, High Pressure Liquid, Disease Models, Animal, Epistasis, Genetic, Genetic Predisposition to Disease, Genotype, Hemoglobin C genetics, Humans, Immunity, Innate, Isoelectric Focusing, Lentivirus genetics, Malaria genetics, Mice, Mice, Knockout, Phenotype, Hemoglobinopathies diagnosis, Hemoglobinopathies genetics, Mice, Transgenic
- Published
- 2003
- Full Text
- View/download PDF
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