scholarly journals Role of gene order in developmental control of human gamma- and beta-globin gene expression.

1993 ◽  
Vol 13 (8) ◽  
pp. 4836-4843 ◽  
Author(s):  
K R Peterson ◽  
G Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Globin ◽  
Globin Gene Expression ◽  
Beta Gene

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.

Role of gene order in developmental control of human gamma- and beta-globin gene expression

1993 ◽  
Vol 13 (8) ◽  
pp. 4836-4843
Author(s):  
K R Peterson ◽  
G Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Globin ◽  
Globin Gene Expression ◽  
Beta Gene

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.

scholarly journals Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

1992 ◽  
Vol 12 (4) ◽  
pp. 1561-1567 ◽  
Author(s):  
J A Lloyd ◽  
J M Krakowsky ◽  
S C Crable ◽  
J B Lingrel
Keyword(s):  
Transgenic Mice ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
High Level ◽  
Gene Switching ◽  
Globin Gene Expression

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice

1992 ◽  
Vol 12 (4) ◽  
pp. 1561-1567
Author(s):  
J A Lloyd ◽  
J M Krakowsky ◽  
S C Crable ◽  
J B Lingrel
Keyword(s):  
Transgenic Mice ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
High Level ◽  
Gene Switching ◽  
Globin Gene Expression

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3632-3632
Author(s):  
Benjamin L. Ebert ◽  
Raymond Mak ◽  
Jennifer L. Pretz ◽  
David Peck ◽  
Stephen Haggerty ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Diversity Oriented Synthesis ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.

Methyl Binding Domain Protein 2 Mediates gamma-Globin Silencing in Adult beta-YAC Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3626-3626
Author(s):  
Jeremy W. Rupon ◽  
ShouZhen Wang ◽  
Karin Gaensler ◽  
Joyce Lloyd ◽  
Gordon D. Ginder
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Quantitative Pcr ◽  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Rnase Protection ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract The genes of the vertebrate beta-globin locus undergo a switch in expression during development whereby embryonic/fetal genes of the cluster are sequentially silenced and the adult genes are activated during erythroid development. DNA methylation has been shown to be associated with developmentally silenced globin genes, and compounds that inhibit cytosine methylation have been shown to activate transcription from developmentally silenced globin genes in several species, including humans. Previously, we have shown that the methyl domain binding protein 2 (MBD2) is involved in maintaining embryonic rho-globin gene silencing in adult avian erythroid cells. We describe here a role for MBD2 in the DNA methylation mediated silencing and maintenance of silencing of the human fetal gamma-globin gene in a transgenic mouse model. We confirmed the previously published report by Pace et al that the gamma-globin gene is reactivated, upon treatment with the DNA methyltransferase inhibitor, 5-azacytidine, of mice containing the entire beta-globin locus as a yeast artificial chromosome (BetaYAC) transgene. In order to elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2−/− mice were generated by breeding BetaYAC mice with MBD2−/− mice. Anemic adult betaYAC/MBD2−/− mice continue to express the gamma-globin gene at a level commensurate with animals treated with 5-azacytidine, which is10–20 fold over those treated with 1-acetyl-2-phenylhydrazine alone, as measured by both quantitative PCR and by RNase protection assays. In addition, the level of gamma-globin gene expression is consistently several fold higher in MBD2−/− compared to wild type BetaYAC mice in 14.5 and 16.5 dpc fetal liver erythroblasts. Furthermore, transcriptional activation of the gamma-globin gene in adult erythroblasts is associated with a modest decrease in DNA methylation around the gamma-globin promoters and a ~4-fold enrichment of histone H3 trimethylated at lysine 4 (TriK4), as measured by chromatin immunoprecipitation assay using quantitative PCR. Finally, treatment of MBD2 null mice with 5-azacytidine induces only a small, non-additive induction of gamma-globin expression indicating that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells. These results suggest that MBD2 is a potential target for therapeutic induction of gamma-globin gene expression in the settings of sickle cell anemia and beta-thalassemia.

Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 365-365 ◽  
Author(s):  
Valerie M. Jansen ◽  
Shaji Ramachandran ◽  
Aurelie Desgardin ◽  
Jin He ◽  
Vishwas Parekh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Globin Genes ◽  
Context Specific ◽  
High Level ◽  
Kinetics Of ◽  
Globin Gene Expression

Abstract Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.

scholarly journals The Corfu delta beta zero thalassemia: a small deletion acts at a distance to selectively abolish beta globin gene expression [published erratum appears in Blood 1988 May;71(5):1509]

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 457-462 ◽  
Author(s):  
AE Kulozik ◽  
N Yarwood ◽  
RW Jones
Keyword(s):  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Promoter Regions ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Exon 1 ◽  
Normal Gene ◽  
High Level

Abstract The Corfu delta beta zero thalassemia is characterized by the clinical picture of thalassemia intermedia. In the homozygous state there is a complete absence of hemoglobin (Hb) A and Hb A2 and a high level of Hb F. A DNA fragment containing the gamma and beta globin genes has been cosmid cloned, and the deletion breakpoint region, the beta globin gene and the promoter regions of the gamma globin genes sequenced. The deletion removes 7,201 base pairs (bp) containing part of the delta globin gene and sequences upstream. The beta globin gene contains a G--- -A mutation at IVS 1 position 5. The gamma globin gene promoters are normal. Analysis of the transcription of the mutated beta globin gene in transfected HeLa cells shows that normal message is produced at a level of approximately 20% compared with a normal gene, the remaining 80% being spliced at cryptic sites in exon 1 and intron 1. This indicates that the mutation in the beta globin gene is not the sole cause of the absence of Hb A in Corfu delta beta zero thalassemia. It is concluded that the 7.2 kilobase (kb) of deleted DNA contains sequences necessary for the normal activation of the beta globin gene. Possible mechanisms for the effect of the deletion on the expression of beta and gamma globin genes are discussed.

Changes in Globin Gene Methylation and Covalent Histone Modifications of Chromatin Associated with the ε-, γ-, and β-Globin Promoters of the Baboon (P. Anubis) during Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1206-1206
Author(s):  
Donald Lavelle ◽  
Kestas Vaitkus ◽  
Maria Hankewych ◽  
Mahipal Singh ◽  
Joseph DeSimone
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Rna Pol Ii ◽  
Cpg Sites ◽  
Globin Promoter ◽  
Globin Gene Expression ◽  
Covalent Histone Modifications

Abstract The pattern of globin gene expression during development is conserved in all simian primates, but not in prosimians or other species. Therefore knowledge of the mechanisms regulating globin gene expression in animal models such as the baboon (P. anubis) is directly applicable to human. This investigation addressed the role of chromatin structure in developmental regulation of globin gene expression. DNA methylation of the ε- and γ-gene promoters and covalent histone modifications in chromatin associated with the ε- γ- and β-globin gene promoters have been investigated in 40d fetal primitive nucleated yolk sac-derived RBCs, and definitive erythroid precursor cells from fetal liver (40d to 56d), fetal BM (154d to 160d), and BM from phlebotomized adults. The methylation status of 3 CpG sites in the ε-globin promoter and 5 CpG sites in the γ-globin promoter was analyzed by sequencing 10 cloned PCR products of each sample following bisulfite modification. The ε-globin promoter was unmethylated in 40d primitive yolk-sac derived RBCs. Moderate methylation of the ε-globin promoter was observed in 40d fetal liver (33%: 50%) and was increased in fetal liver samples obtained 2 weeks later in gestation (54d: 76.6%, 56d: 79.1%) to levels observed in late term fetal BM ( 154d: 80%, 156d: 96.6%, 160 d: 93.1%) and adult BM (84.1%; n=2). Methylation of the γ-globin promoter was lowest in 40d primitive RBC (0%) and early fetal liver (40d: 3.1%, 54d: 0%, 56d: 7.1%) and was moderately increased in fetal BM (154d: 38.6%, 156d: 20%, 160d: 30%) compared to adult BM ( 67.3%; n=3). Levels of ac-H3, ac-H4, dimethyl H3 lys4 (H3-dimeK4), dimethyl H3 lys79 (H3-meK79), dimethyl H3 lys36 (H3-meK36), and RNA pol II bound to the ε-, γ-, and β-globin promoters were determined by immunoprecipitation of formaldehyde-fixed, sheared chromatin (ChIP) followed by real time PCR. The amount of RNA pol II, ac-H3, and ac-H4 associated with each globin promoter correlated with developmental-specific gene expression and differed from the pattern of H3-meK79 and H3-meK4 associated with these promoters during development. The amount of H3-meK79 and H3-dimeK4 bound to the the ε- and γ-globin promoters in 40d primitive RBC and fetal liver erythroid precursors (54 and 56d) was 5 times greater than to the β-globin promoter, while similar levels of each (< 2 fold difference) were associated with all three promoters in fetal and adult bone marrow cells. In contrast, the highest level of H3-meK36 was associated with developmentally silenced genes. The amount of H3-meK36 bound to the ε promoter was 2–3 fold higher than to the γ and β promoters in fetal liver (54 and 56d). Similar levels (<2 fold difference) of H3-meK36 were associated with the γ and ε promoters in late term fetal and adult BM and were 2–6 fold greater than bound to the β promoter. We conclude that the chromatin cofiguration of the β-globin locus undergoes distinctive changes associated with both gene activation and silencing during development. Changes in the levels of H3-dimeK4 and H3-meK79 may reflect generalized domain opening, while high levels of ac-H3 and ac-H4 are bound to the promoters of activated genes. In contrast, gene silencing is correlated with increased DNA methylation and enrichment of H3-meK36 bound to the promoters. Thus the baboon model offers unique opportunities to study developmental regulation of globin gene expression.

Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3627-3627
Author(s):  
Elliot M. Epner ◽  
Jin Wang ◽  
Jing Huang
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Globin Genes ◽  
Phenotypic Analysis ◽  
Pol Ii ◽  
Globin Gene Expression

Abstract The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.

Sign in / Sign up

Export Citation Format

Share Document