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Eric E Bouhassira

Publications and source records attributed to Eric E Bouhassira.

18 recordsLinked to original sources
PubMed2006

Transcriptional interference among the murine beta-like globin genes.

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.

Animals↗
PubMed2006

DNA methylation supports intrinsic epigenetic memory in mammalian cells.

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.

Animals↗
PubMed2006

Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can be found in many connective tissues, including fat, bone, cartilage, and muscle. We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layer. The cells obtained with this procedure are morphologically similar to bone marrow MSCs, are contact-inhibited, can be grown in culture for about 20 to 25 passages, have an immunophenotype similar to bone marrow MSCs (negative for CD34 and CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, human leukocyte antigen [HLA]-ABC, and stage-specific embryonic antigen [SSEA]-4), can differentiate into osteocytes and adipocytes, and can be used as feeder cells to support the growth of undifferentiated hESCs. The ability to produce MSCs from hESCs should prove useful to produce large amounts of genetically identical and genetically modifiable MSCs that can be used to study the biology of MSCs and for therapeutic applications.

Cell Differentiation↗
PubMed2006

Preventing gene silencing with human replicators.

Transcriptional silencing, one of the major impediments to gene therapy in humans, is often accompanied by replication during late S-phase. We report that transcriptional silencing and late replication were prevented by DNA sequences that can initiate DNA replication (replicators). When replicators were included in silencing-prone transgenes, they did not undergo transcriptional silencing, replicated early and maintained histone acetylation patterns characteristic of euchromatin. A mutant replicator, which could not initiate replication, could not prevent gene silencing and replicated late when included in identical transgenes and inserted at identical locations. These observations suggest that replicators introduce epigenetic chromatin changes that facilitate initiation of DNA replication and affect gene silencing. Inclusion of functional replicators in gene therapy vectors may provide a tool for stabilizing gene expression patterns.

Animals↗
PubMed2006

Large-scale production of embryonic red blood cells from human embryonic stem cells.

OBJECTIVE: To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. MATERIALS AND METHODS: Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. RESULTS: CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. CONCLUSIONS: We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Animals↗
PubMed2005

Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation.

Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.

Animals↗
PubMed2005

Enzymatically active ADAMTS13 variants are not inhibited by anti-ADAMTS13 autoantibodies: a novel therapeutic strategy?

ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing von 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.

ADAM Proteins↗
PubMed2005

Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development.

OBJECTIVE: To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the beta-like globin genes in hESC-derived erythroid cells. MATERIALS AND METHODS: FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction. RESULTS: Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34(+) cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of beta-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined. CONCLUSIONS: FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.

Cell Culture Techniques↗
PubMed2005

Methylation protects cytidines from AID-mediated deamination.

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.

Animals↗
PubMed2005

ADAMTS13 is expressed in hepatic stellate cells.

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.

ADAMTS13 Protein↗
PubMed2005

The human beta-globin locus control region can silence as well as activate gene expression.

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.

Animals↗
PubMed2003

Gene length and proximity to neighbors affect genome-wide expression levels.

Steady-state levels of mRNA in cells theoretically depend on the rate and efficiency of transcription and posttranscriptional processing, on mRNA stability, on transcriptional interference from other genes, and on poorly defined long-range chromatin effects. Although each of these cellular processes has been studied in detail for a few genes, it is not possible to predict expression levels by simply examining gene sequences. In this report, we have used a bioinformatics approach to identify critical factors that influence expression levels. To simplify the problem, we have limited our analysis to the collection of genes expressed in all tissues, because such genes provide a unique opportunity to distinguish the role of general genomic features that constrain gene expression from the effect of tissue-specific factors. Using correlation and regression techniques, we have investigated the dependence between expression level and morphological parameters (distance to neighbors, gene, mRNA or 3'-UTR length, number of exons, etc.) that can be directly related to transcription, posttranscriptional processing, mRNA stability, or transcriptional interference. We found that, on a genome-wide scale, highly expressed genes are significantly farther from their closest neighboring genes, are smaller, contain a moderate number of exons, and produce shorter mRNAs with shorter 3'-UTRs. This confirms that transcriptional and posttranscriptional processes are highly interrelated and implies that transcriptional interference plays a role in determining steady-state levels of mRNA in cells.

Computational Biology↗
PubMed2003

Mammalian linker-histone subtypes differentially affect gene expression in vivo.

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 linker-histone 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.

Anemia, Hemolytic↗
PubMed2003

Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region.

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.

Alleles↗
PubMed2002

Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells.

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 beta globin gene expression has not yet been achieved in beta 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 beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta 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 approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-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 alpha 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 vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.

3T3 Cells↗
PubMed2002

Transcriptional interference by independently regulated genes occurs in any relative arrangement of the genes and is influenced by chromosomal integration position.

Transcriptional interference is the influence, generally suppressive, of one active transcriptional unit on another unit linked in cis. Its wide occurrence in experimental systems suggests that it may also influence transcription in many loci, but little is known about its precise nature or underlying mechanisms. Here we report a study of the interaction of two nearly identical transcription units juxtaposed in various arrangements. Each reporter gene in the constructs has its own promoter and enhancer and a strong polyadenylation signal. We used recombinase-mediated cassette exchange (RMCE) to insert the constructs into previously tagged genomic sites in cultured cells. This strategy also allows the constructs to be assessed in both orientations with respect to flanking chromatin. In each of the possible arrangements (tandem, divergent, and convergent), the presence of two genes strongly suppresses expression of both genes compared to that of an identical single gene at the same integration site. The suppression is most severe with the convergent arrangement and least severe in total with the divergent arrangement, while the tandem arrangement is most strongly influenced by the integration site and the genes' orientation within the site. These results suggest that transcriptional interference could underlie some position effects and contribute to the regulation of genes in complex loci.

Animals↗
PubMedDate not supplied

Generation of transgenic mice expressing human hemoglobin E.

Hemoglobin E (HbE, beta26 Glu-->Lys) is the most common abnormal Hb variant in the world, and found in greatest frequency in Southeast (SE) Asia. In the United States, HbE is the third most prevalent variant (after HbS and HbC); and its now increasing frequency is due to immigration from SE Asia. HbE homozygotes present a benign clinical picture, but when HbE is coupled with beta0-thalassemia or HbS, variably severe hemoglobinopathies arise. To date, there are no transgenic animal models of HbE-related diseases. We report here the creation of transgenic mice expressing human HbE as a step toward creating animal models for HbE-related diseases. The betaE mice exhibit red blood cell hypochromia and target cells consistent with those observed in human patients exhibiting HbE trait. Furthermore, the transgenic HbE hemolysates contain increased amounts of Hb oxidation products.

Animals↗