Biomedical subjects

George Stamatoyannopoulos

Publications and source records attributed to George Stamatoyannopoulos.

At least 19 recordsLinked to original sources

PubMed2007

Protein kinase Calpha is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the (A)gamma globin-promoter in cellular models of hemoglobin switching.

PKCalpha was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

Acetophenones↗

PubMed2007

The transcription factor KLF11 can induce gamma-globin gene expression in the setting of in vivo adult erythropoiesis.

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Animals↗

PubMed2006

Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation.

Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of gamma globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation.

Cell Differentiation↗

PubMed2006

Purification and identification of proteins that bind to the hereditary persistence of fetal hemoglobin -198 mutation in the gamma-globin gene promoter.

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Adult↗

PubMed2006

Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Animals↗

PubMed2006

Transcriptional potential of the gamma-globin gene is dependent on the CACCC box in a developmental stage-specific manner.

To test the role of CACCC box on gamma-globin gene activation, the CACCC box was deleted or mutated and gamma-gene expression was monitored in transgenic mice. Disruption of the CACCC box had no effect on gamma-gene expression in the cells of embryonic erythropoiesis but it strikingly reduced gamma-gene expression in fetal erythropoiesis, and abolished gamma-gene expression in adult erythroid cells. The CACCC mutation diminished HS formation, as well as TBP and polII recruitment at the gamma-gene promoter; however, it only resulted in slight or no effects on histone H3 and H4 acetylation in adult erythropoiesis. Our findings indicate that each basic cis element of the proximal gamma-gene promoter, i.e. CACCC, CCAAT or TATA box, can be disrupted without affecting the activation of gamma gene in embryonic erythroid cells. We propose that the trans factors recruited by the three boxes interact with each other to form a 'promoter complex'. In embryonic erythropoiesis the locus control region enhancer is able to interact with the complex even when components normally binding to one of the motifs are missing, but it can only activate an intact 'promoter complex' in adult erythroid cells.

Animals↗

PubMed2006

Integration bias of gammaretrovirus vectors following transduction and growth of primary mouse hematopoietic progenitor cells with and without selection.

The recent recognition that recombinant retrovirus vectors can induce oncogenic transformation has stimulated much interest in the pattern of vector integration sites. We report here on the integration pattern of a gammaretrovirus reporter vector following transduction and ex vivo culture of primary mouse bone marrow progenitor cells in the absence and presence of drug selection. Using a novel method of cloning junction fragments, we observed no bias for integrations within genes, but did observe a bias for integrations within gene-dense regions and especially near transcriptional start sites of highly active genes, similar to previous reports in other cell types. We also document a novel bias for integrations within or near a class of genes that encode nuclear-localized proteins. We found that drug selection resulted in an increase in the frequency of recovered integration events that were located within the beginning of genes, integration events that were located in less gene-dense regions, and integration events that were oriented in an antisense direction relative to flanking gene transcription. Taken together, these studies provide new insights into the nature of retrovirus vector integration patterns in primary cells and demonstrate that selection based on vector expression can bias the integration site repertoire.

Animals↗

PubMed2006

Histone deacetylase inhibitor FK228 is a potent inducer of human fetal hemoglobin.

We investigated the induction of the human fetal globin gene using five potent histone deacetylase (HDAC) inhibitors: FK-228, HC-Toxin, Trichostatin, MS-275, and Apicidin, using in vitro assays and cultures of primary human erythroblasts. The results showed that FK228 is the most potent inducer of fetal hemoglobin and exhibits its effects in picomolar concentrations. FK228 should be considered as a potential therapeutic for induction of fetal hemoglobin in patients with beta chain hemoglobinopathies.

Antibiotics, Antineoplastic↗

PubMed2006

Effects of human gamma-globin in murine beta-thalassaemia.

Murine models of beta-thalassaemia have been used to test therapeutic globin gene vectors. However, the level of gamma-globin expression necessary to achieve full phenotypic correction in these models is unclear. In order to address this issue, we carried out breeding and transplantation studies in murine models of beta-thalassaemia intermedia (Hbb(th-3)/+) and severe beta-thalassaemia major (Hbb(th-3)/Hbb(th-3)) using transgenic lines expressing various levels of human gamma-globin. Expression of gamma-globin RNA at a modest 7-14% of total alpha-globin RNA resulted in the selective survival of HbF(+) erythrocytes, a fivefold increase in total HbF, and a phenotypic improvement in the beta-thalassaemia intermedia model. Full normalisation of erythrocyte indices in this model required gamma-globin RNA expression at 27% of alpha-globin, resulting in an average 40% (6.8 g/dl) HbF. Studies using the homozygous Hbb(th-3) model of lethal beta-thalassaemia major demonstrated that even this high level of gamma-globin expression, for reasons related to the function of the hybrid globin tetramers, could only prolong, but not fully support, survival. Taken together, these results indicate that only the heterozygous Hbb(th-3) model of beta-thalassaemia intermedia can be reliably used for the pre-clinical assessment of gamma-globin gene therapy vectors, as well as other means of gamma-globin gene induction.

Animals↗

PubMed2006

Autonomous silencing as well as competition controls gamma-globin gene expression during development.

To investigate the control of the gamma-globin gene during development, we produced transgenic mice in which sequences of the beta-gene promoter were replaced by equivalent sequences of the gamma-gene promoter in the context of a human beta-globin locus yeast artificial chromosome (betaYAC) and analyzed the effects on globin gene expression during development. Replacement of 1,077 nucleotides (nt) of the beta-gene promoter by 1,359 nt of the gamma promoter resulted in striking inhibition of the gamma-promoter/beta-gene expression in the adult stage of development, providing direct evidence that the expression of the gamma gene in the adult is mainly controlled by autonomous silencing. Measurements of the expression of the gamma promoter/beta-globin gene as well as the wild gamma genes showed that gene competition is also involved in the control of gamma-gene expression in the fetal stage of development. We conclude that autonomous silencing is the main mechanism controlling gamma-gene expression in the adult, while autonomous silencing as well as competition between gamma and beta genes contributes to the control of gamma to beta switching during fetal development.

Animals↗

PubMed2006

Investigations of a human embryonic globin gene silencing element using YAC transgenic mice.

A silencing element has been previously located upstream of the human epsilon-globin gene promoter using transient assays and transgenic mice carrying plasmid constructs in which the element has been deleted or its transcriptional motifs have been mutated. To investigate whether this element functions in the context of the whole beta-globin locus, we analyzed epsilon-globin gene expression in transgenic mice carrying a deletion of the silencing element in the context of a 213-kilobase human beta-globin yeast artificial chromosome (beta-YAC). epsilon-Globin gene expression was measured during embryonic and fetal development and in adult mice. epsilon-mRNA levels in embryonic cells in Day 12 blood were as high as those measured in wild-type beta-YAC controls, indicating that the deletion does not affect epsilon gene promoter function. epsilon-Globin gene expression was confined to the embryonic cells, indicating that deletion of this silencing element did not affect epsilon-globin developmental expression in the context of the beta-YAC. These results suggest that in the context of the whole beta-globin locus, other proximal and upstream epsilon gene promoter elements as well as competition by the downstream globin genes contribute to the silencing of the epsilon-globin gene in the cells of definitive erythropoiesis.

Animals↗

PubMed2005

Investigations of the induction of the goat beta(C) globin gene by erythropoietin: studies in transgenic mice.

The genes of the beta globin locus of sheep and goats undergo three developmental switches, from embryonic epsilon to fetal gamma to juvenile beta(C) and from beta(C) to adult beta(A). The juvenile beta(C) gene of adult goats and sheep are strikingly induced by erythropoietin (Epo). To obtain insights on the mechanism of beta(C) induction by Epo, we produced transgenic mice carrying various beta(C) gene constructs and examined the inducibility of the beta(C) gene following administration of high doses of erythropoietin. None of the treatments resulted in reproducible induction of the beta(C) gene by erythropoietin. We conclude that the Epo inducibility elements are not contained in the 4.7 kb (which included 0.88 kb of the beta(C) promoter and 2.3 kb downstream sequence) beta(C) gene we used or that a trans-acting environment specific to the goat and sheep erythroid cell lineage is required for induction of the beta(C) globin gene by erythropoietin.

Animals↗

PubMed2005

Juxtaposition of the HPFH2 enhancer is not sufficient to reactivate the gamma-globin gene in adult erythropoiesis.

Previous studies have suggested that juxtaposition of a downstream enhancer to the fetal gamma-globin gene results in reactivation of the gamma-gene in adult erythrocytes of individuals with hereditary persistence of fetal hemoglobin (HPFH). To test the hypothesis in a much stricter basis, we produced beta locus YAC transgenic mice carrying an exact beta locus replicate of a deletional HPFH mutation, HPFH 2. Although the gamma-globin gene was expressed in the HPFH 2/beta locus YAC (HPFH2/YAC) transgenic mice in the early stage of development, it was completely silenced in the adult mice. The failure of gamma-gene reactivation by the juxtaposed HPFH2 enhancer contradicts the results of previous studies. We speculate that the discrepant results reflect differences in the distance between the locus of region (LCR) and the gamma-globin gene characteristic of the plasmid, cosmid or YAC constructs used for production of transgenic mice. The difference in the phenotype of the HPFH2/YAC transgenic mice and the humans with HPFH2 mutation suggests that in addition to juxtaposition of HPFH enhancers, the upstream region that is absent in the beta-YAC construct might be involved in gamma-gene reactivation in HPFH individuals. The DNase I hypersensitive sites of the LCR were well formed and the chromatin histones were acetylated. A moderate level of pol II binding was detected in the LCR, despite the fact that no transcription occurred in the globin-genes of the adult HPFH2/YAC transgenic mice. The results suggest that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.

Acetylation↗

PubMed2005

Transcriptional regulation of FKLF-2 (KLF13) gene in erythroid cells.

FKLF-2 (KLF13) was cloned from fetal globin-expressing tissues and has been shown to be abundantly expressed in erythroid cells. In this study we examined the transcriptional regulation of the KLF13 gene. A 5.5 kb 5' flanking region cloned from mouse erythroleukemia (MEL) cell genomic DNA showed that major cis regulatory activities exist in the 550 bp sequence to the unique transcription start site, and that the promoter is more active in K562 cells than in COS-7 cells. The promoter was trans-activated by co-expressed GATA-1 through the sequence containing two CCAAT motifs, suggesting that GATA-1 is involved in the abundant expression of KLF13 mRNA in the erythroid tissue. Dual action, i.e. activating effect in COS-7 and repressive effect in K562 cell, was observed on its own promoter, suggesting a feedback mechanism for the transcriptional control of the KLF13 gene in the erythroid environment. These findings provide an insight on the mechanism of inducible mRNA expression of the KLF13 gene in erythroid cells.

Animals↗

PubMed2005

Control of globin gene expression during development and erythroid differentiation.

Extensive studies during the last 30 years have led to considerable understanding of cellular and molecular control of hemoglobin switching. Cell biology studies in the 1970s defined the control of globin genes during erythroid differentiation and led to development of therapies for sickle cell disease. Molecular investigations of the last 20 years have delineated the two basic mechanisms that control globin gene activity during development--autonomous silencing and gene competition. Studies of hemoglobin switching have provided major insights on the control of gene loci by remote regulatory elements. Research in this field has an impact on understanding regulatory mechanisms in general and is of particular importance for eventual development of molecular cures for sickle cell disease and beta thalassemia.

Anemia, Sickle Cell↗

PubMed2005

Hydroxamide derivatives of short-chain fatty acid have erythropoietic activity and induce gamma gene expression in vivo.

OBJECTIVE: The hydroxamic acid derivatives of short-chain fatty acids, butyryl and propionyl hydroxamate, subericbishydoxamic acid, and suberoylanilide hydroxamic acid, are potent inhibitors of histone deacetylase (HDAC) and have been shown to induce fetal hemoglobin in vitro. In this study we examined whether these compounds have erythropoietic activity and can induce gamma globin gene expression in vivo. MATERIALS AND METHODS: Transgenic mice heterozygous for a deletion beta thalassemia and hemizygous for a human gamma globin transgene were treated with these compounds and hematologic responses as well as the induction of gamma gene expression were evaluated. Hematologic studies included measurement of reticulocytes, hematocrit, and the in vivo levels of BFU-E. Effects on globin gene expression were assessed by measuring F reticulocytes and the human gamma/murine alpha globin mRNA ratios by RNAse protection assay. RESULTS: Propionyl and butyryl hydroxamate increased reticulocytes by 71% and 139%, the in vivo BFU-E counts by 75% and 51%, and the in vivo gamma gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. CONCLUSION: Hydroxamic acid derivatives can stimulate the in vivo erythropoiesis and fetal globin production in a thalassemic murine model. The combined effect of certain histone deacetylase inhibitors on erythropoiesis and on gamma gene expression make these compounds desirable targets for development of therapeutics for beta chain hemoglobinopathies.

Animals↗

PubMed2005

Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture.

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.

Acetylation↗

PubMed2005

Genome architecture of the human beta-globin locus affects developmental regulation of gene expression.

To test the role of gene order in globin gene expression, mutant human beta-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a "marked" transcript (epsilon(m), gamma(m), or beta(m)) integrated at different locations within the locus. When a beta(m)-globin gene was placed between the locus control region (LCR) and the epsilon-globin gene, beta(m)-globin expression dominated primitive and definitive erythropoiesis; only beta(m)-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an (A)gamma(m)-globin gene was placed at the same location, (A)gamma(m)-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type gamma-globin genes were not expressed. When an epsilon(m)-globin gene was placed between the delta- and beta-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type epsilon-globin gene was expressed. Placement of a beta(m)-globin gene upstream of the (G)gamma-globin gene resulted in expression of beta(m)-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type beta-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.

Animals↗