Hemoglobins from bacteria to man: evolution of different patterns of gene expression.

1998 ◽  
Vol 201 (8) ◽  
pp. 1099-1117 ◽  
Author(s):  
R Hardison
Keyword(s):  
Dna Sequences ◽  
Cpg Island ◽  
Globin Gene ◽  
Gene Clusters ◽  
Regulatory Elements ◽  
Chromatin Domain ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Ancestral Gene ◽  
Beta Globin Gene

The discovery of hemoglobins in virtually all kingdoms of organisms has shown (1) that the ancestral gene for hemoglobin is ancient, and (2) that hemoglobins can serve additional functions besides transport of oxygen between tissues, ranging from intracellular oxygen transport to catalysis of redox reactions. These different functions of the hemoglobins illustrate the acquisition of new roles by a pre-existing structural gene, which requires changes not only in the coding regions but also in the regulatory elements of the genes. The evolution of different regulated functions within an ancient gene family allows an examination of the types of biosequence data that are informative for various types of issues. Alignment of amino acid sequences is informative for the phylogenetic relationships among the hemoglobins in bacteria, fungi, protists, plants and animals. Although many of these diverse hemoglobins are induced by low oxygen concentrations, to date none of the molecular mechanisms for their hypoxic induction shows common regulatory proteins; hence, a search for matches in non-coding DNA sequences would not be expected to be fruitful. Indeed, alignments of non-coding DNA sequences do not reveal significant matches even between mammalian alpha- and beta-globin gene clusters, which diverged approximately 450 million years ago and are still expressed in a coordinated and balanced manner. They are in very different genomic contexts that show pronounced differences in regulatory mechanisms. The alpha-globin gene is in constitutively active chromatin and is encompassed by a CpG island, which is a dominant determinant of its regulation, whereas the beta-globin gene is in A+T-rich genomic DNA. Non-coding sequence matches are not seen between avian and mammalian beta-globin gene clusters, which diverged approximately 250 million years ago, despite the fact that regulation of both gene clusters requires tissue-specific activation of a chromatin domain regulated by a locus control region. The cis-regulatory sequences needed for domain opening and enhancement do show common binding sites for transcription factors. In contrast, alignments of non-coding sequences from species representing multiple eutherian mammalian orders, some of which diverged as long as 135 million years ago, are reliable predictors of novel cis-regulatory elements, both proximal and distal to the genes. Examples include a potential target for the hematopoietic transcription factor TAL1.

Screening regulatory sequences from bacterial artificial chromosome DNA of alpha- and beta-globin gene clusters

2002 ◽  
Vol 80 (4) ◽  
pp. 415-420 ◽  
Author(s):  
Shen Zhang ◽  
Hua-Bing Zhang ◽  
De-Pei Liu ◽  
Xing-Guo Li ◽  
De-Long Hao ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Globin Gene ◽  
K562 Cells ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Nuclear Proteins ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Regulatory Dna

In the forthcoming postgenomic era, identification of regulatory DNA sequences is becoming increasingly important for characterizing DNA-binding proteins and for elucidating the regulatory mechanisms of gene expression. Presently, there lack efficient methods to broadly screen and identify DNA regulatory elements on a large scale. We established herein an efficient strategy to screen regulatory sequences from bacterial artificial chromosome (BAC) DNAs containing human alpha- and beta-globin gene clusters based on polymerase chain reaction and electrophoretic mobility shift assay (EMSA) techniques without purified transcription factors. Twenty-three subclones derived from alpha-BAC DNA by bulk EMSA selection retained the ability to bind nuclear proteins of K562 cells when retested by EMSA. In 19 clones sequenced, 14 are identical to those registered in GenBank and five have one base difference. All of the 24 randomly picked beta-BAC clones showed specific binding with nuclear proteins of K562 cells. In 11 clones sequenced, eight are identical to those registered in GenBank and three have one base difference. This approach could be particularly powerful if combined with other systematic methods for identifying cis-regulatory DNA elements.Key words: gene expression regulation, bacterial artificial chromosome, alpha- and beta-globin gene clusters.

The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity

1988 ◽  
Vol 8 (11) ◽  
pp. 4958-4965
Author(s):  
V Dhar ◽  
D Mager ◽  
A Iqbal ◽  
C L Schildkraut
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Hypersensitive Sites ◽  
Flanking Sequences ◽  
Globin Gene Domain

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

1987 ◽  
Vol 7 (11) ◽  
pp. 4024-4029
Author(s):  
M Trudel ◽  
J Magram ◽  
L Bruckner ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Base Pairs ◽  
Beta Globin Gene ◽  
Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

scholarly journals Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human beta-globin gene.

1982 ◽  
Vol 2 (2) ◽  
pp. 190-198 ◽  
Author(s):  
E F Wagner ◽  
B Mintz
Keyword(s):  
Stem Cells ◽  
Thymidine Kinase ◽  
Dna Sequences ◽  
Human Gene ◽  
Globin Gene ◽  
Mouse Strains ◽  
Gene Probe ◽  
Beta Globin ◽  
Selection Scheme ◽  
Beta Globin Gene

Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, we investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH beta 1 containing the human genomic beta-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human beta-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2067-2067
Author(s):  
Andrew J. Woo ◽  
Jonghwan Kim ◽  
Jian Xu ◽  
Hui Huang ◽  
Alan Cantor
Keyword(s):  
Globin Gene ◽  
Regulatory Elements ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Histone 3 ◽  
Cd34 Cells ◽  
Alpha Globin ◽  
Globin Gene Regulation ◽  
Erythroid Development

Abstract Abstract 2067 The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

1994 ◽  
Vol 14 (10) ◽  
pp. 6936-6943 ◽  
Author(s):  
P J Detloff ◽  
J Lewis ◽  
S W John ◽  
W R Shehee ◽  
R Langenbach ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Second Step ◽  
Globin Genes ◽  
Beta Globin ◽  
Es Cell ◽  
Beta Globin Gene ◽  
Targeting Strategy

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

scholarly journals Eastern European (delta beta) zero-thalassemia: molecular characterization of a novel 9.1-kb deletion resulting in high levels of fetal hemoglobin in the adult [see comments]

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3738-3745 ◽  
Author(s):  
A Palena ◽  
A Blau ◽  
G Stamatoyannopoulos ◽  
NP Anagnou
Keyword(s):  
Globin Gene ◽  
Adult Life ◽  
Fetal Hemoglobin ◽  
Direct Repeat ◽  
Regulatory Elements ◽  
Negative Control ◽  
Beta Thalassemia ◽  
Eastern European ◽  
Beta Globin ◽  
Beta Globin Gene

A novel deletion in the human beta-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heterocellular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the beta-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the delta-globin gene, and terminates within the first intron of the beta-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (delta beta) zero-thalassemias. The 5′ breakpoint of the new deletion is close to that of the Yugoslavian delta beta-thalassemia deletion, whereas the 3′ breakpoint is very close to those of the Turkish and the Greek beta zero-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an “illegitimate” or “nonhomologous” recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, delta beta-thalassemia v HPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the gamma-globin gene expression.

Eastern European (delta beta) zero-thalassemia: molecular characterization of a novel 9.1-kb deletion resulting in high levels of fetal hemoglobin in the adult [see comments]

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3738-3745
Author(s):  
A Palena ◽  
A Blau ◽  
G Stamatoyannopoulos ◽  
NP Anagnou
Keyword(s):  
Globin Gene ◽  
Adult Life ◽  
Fetal Hemoglobin ◽  
Direct Repeat ◽  
Regulatory Elements ◽  
Negative Control ◽  
Beta Thalassemia ◽  
Eastern European ◽  
Beta Globin ◽  
Beta Globin Gene

Abstract A novel deletion in the human beta-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heterocellular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the beta-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the delta-globin gene, and terminates within the first intron of the beta-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (delta beta) zero-thalassemias. The 5′ breakpoint of the new deletion is close to that of the Yugoslavian delta beta-thalassemia deletion, whereas the 3′ breakpoint is very close to those of the Turkish and the Greek beta zero-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an “illegitimate” or “nonhomologous” recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, delta beta-thalassemia v HPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the gamma-globin gene expression.

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