Adult chicken alpha-globin gene expression in transfected QT6 quail cells: evidence for a negative regulatory element in the alpha D gene region.

The chicken adult alpha-globin genes, alpha A and alpha D, are closely linked in chromosomal DNA and are coordinately expressed in vivo in an approximate 3:1 ratio, respectively. When subcloned DNAs containing one or the other gene are stably transfected into QT6 quail fibroblasts, the alpha A-globin gene is expressed at measurable RNA levels, but the alpha D gene is not. The alpha A gene expression can be considerably increased by the presence of a linked Rous sarcoma virus long terminal repeat enhancer, but that of the alpha D gene remains undetectable. Transfection with subclones containing both genes, either in cis or in trans, leads to considerably reduced alpha A RNA levels and still no observable alpha D gene expression. Transfection with deleted subclones suggests that maximal expression levels in this system require the alpha A-globin gene promoter, as opposed to that of the alpha D gene, but that such expression is greatly reduced by one or more DNA sequences which lie approximately 2,000 base pairs upstream of the alpha A gene, within the body of the alpha D gene.     

Identification and characterization of a chicken alpha-globin enhancer.

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.     

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.     

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.     

The pattern of expression of the Xenopus laevis tadpole alpha-globin genes and the amino acid sequence of the three major tadpole alpha-globin polypeptides

We have isolated cDNA clones derived from three tadpole alpha-globin mRNAs of Xenopus laevis. The entire nucleotide sequence of the three mRNAs has been determined from the cDNA clones and is presented together with the deduced amino acid sequence of the encoded polypeptides. Two of the three polypeptide sequences are 96% homologous whilst the third sequence is highly diverged, with only a 72% homology. The three tadpole alpha-globin genes are all similarly diverged from the two X. laevis adult alpha-globin genes with which they display approximately 50% homology. Analysis of several independent clones from each class of tadpole alpha-globin sequence reveals a very high degree of coding region polymorphism for each of the three corresponding genes. Using the cloned DNA sequences as hybridisation probes, we have analysed the expression of the corresponding genes during larval development. We show that all three genes are activated simultaneously early in development and that thereafter all three are expressed at an approximately equivalent level. A fourth tadpole alpha-globin mRNA sequence, for which we do not have a cDNA clone, accumulates co-ordinately with the three major mRNA sequences but to a much lower concentration. This pattern of gene expression differs significantly from that of the tadpole beta-globin genes of X. laevis, despite the two classes of genes being closely linked in the genome.     

Human alpha-globin gene expression. The dominant role of the alpha 2-locus in mRNA and protein synthesis

The two human alpha-globin genes, alpha 1 and alpha 2, are coexpressed in normal erythroid cells and encode identical alpha-globin protein products. Based upon genetic studies, it has been assumed that these two adjacent and highly homologous genes are equally expressed. In previous studies we have, however, demonstrated that the alpha 2 gene encodes a 2-3-fold higher steady state level of mRNA than the alpha 1 gene. In the present study, we monitor the relative levels of protein production from these two loci by quantitating the synthesis of specific alpha-globin structural mutants encoded by each alpha-globin gene. These values are then used to infer the relative contributions of the normal alpha 1 and alpha 2 loci to total alpha-globin production. The results of eight separate studies, each based upon a different alpha-globin structural mutant mapped to either the alpha 1 or the alpha 2 locus, are internally consistent. The data demonstrate that the alpha 2 gene encodes 2-3-fold more protein than the alpha 1 gene. These results suggest that the human alpha-globin gene cluster contains a major and a minor locus. The dominant expression of the alpha 2 gene predicts a greater impact of mutations at this locus, in comparison to mutations at the alpha 1 locus, in the generation of the alpha-thalassemia phenotype.     

Demonstration of an enhancer activity in a fragment of DNA located at the 3' of the adult alpha globin gene in the duck

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.     

Chicken CD69 and CD94/NKG2-like genes in a chromosomal region syntenic to mammalian natural killer gene complex

In mammals, natural killer (NK) cell C-type lectin receptors were encoded in a gene cluster called natural killer gene complex (NKC). The NKC is not reported in chicken yet. Instead, NK receptor genes were found in the major histocompatibility complex. In this study, two novel chicken C-type lectin-like receptor genes were identified in a region on chromosome 1 that is syntenic to mammalian NKC region. The chromosomal locations were validated with fluorescent in situ hybridization. Based on 3D structure modeling, sequence homology, chromosomal location, and phlylogenetic analysis, one receptor is the orthologue of mammalian cluster of differentiation 69 (CD69), and the other is highly homologous to CD94 and NKG2. Like CD94/NKG2 gene found in teleostean fishes, chicken CD94/NKG2 has the features of both human CD94 and NKG2A. Unlike mammalian NKC, these two chicken C-type lectin receptors are not closely linked but separated by 42 million base pairs according to the chicken draft genome sequence. The arrangement of several other genes that are located outside the mammalian NKC is conserved among chicken, human, and mouse. The chicken NK C-type lectin-like receptors in the NKC syntenic region indicate that this chromosomal region existed before the divergence between mammals and aves. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences have been submitted to the GenBank nucleotide sequence database under the accession number chicken CD69 (DQ156495), CD94/NKG2 (DQ156496), and CD94/NKG2 variant (DQ241793).     

Sequence and comparative analysis of the rabbit alpha-like globin gene cluster reveals a rapid mode of evolution in a G + C-rich region of mammalian genomes

A sequence of 10,621 base-pairs from the alpha-like globin gene cluster of rabbit has been determined. It includes the sequence of gene zeta 1 (a pseudogene for the rabbit embryonic zeta-globin), the functional rabbit alpha-globin gene, and the theta 1 pseudogene, along with the sequences of eight C repeats (short interspersed repeats in rabbit) and a J sequence implicated in recombination. The region is quite G + C-rich (62%) and contains two CpG islands. As expected for a very G + C-rich region, it has an abundance of open reading frames, but few of the long open reading frames are associated with the coding regions of genes. Alignments between the sequences of the rabbit and human alpha-like globin gene clusters reveal matches primarily in the immediate vicinity of genes and CpG islands, while the intergenic regions of these gene clusters have many fewer matches than are seen between the beta-like globin gene clusters of these two species. Furthermore, the non-coding sequences in this portion of the rabbit alpha-like globin gene cluster are shorter than in human, indicating a strong tendency either for sequence contraction in the rabbit gene cluster or for expansion in the human gene cluster. Thus, the intergenic regions of the alpha-like globin gene clusters have evolved in a relatively fast mode since the mammalian radiation, but not exclusively by nucleotide substitution. Despite this rapid mode of evolution, some strong matches are found 5' to the start sites of the human and rabbit alpha genes, perhaps indicating conservation of a regulatory element. The rabbit J sequence is over 1000 base-pairs long; it contains a C repeat at its 5' end and an internal region of homology to the 3'-untranslated region of the alpha-globin gene. Part of the rabbit J sequence matches with sequences within the X homology block in human. Both of these regions have been implicated as hot-spots for recombination, hence the matching sequences are good candidates for such a function. All the interspersed repeats within both gene clusters are retroposon SINEs that appear to have inserted independently in the rabbit and human lineages.     

Superhelicity induces hypersensitivity of a human polypyrimidine . polypurine DNA sequence in the human alpha 2-alpha 1 globin intergenic region to S1 nuclease digestion--high resolution mapping of the clustered cleavage sites.

Supercoiled recombinant DNAs containing the human adult alpha-globin gene region have been probed with nuclease S1 in vitro. While agarose gel electrophoresis showed only one predominant, double-stranded cleavage generated by S1 within 6 kb of human DNA and 4 kb of pBR322 sequence, a high resolution gel analysis reveals that the unique S1-hypersensitive locus in the human adult alpha-globin gene region actually contains more than 15 authentic S1 cleavage sites closely spaced together. The mapping approach used here locates the specific S1 cleavage sites on both DNA strands at the nucleotide sequence level. Interestingly, most of these sites are mapped within a 90 bp stretch of GC-rich (66%) polypyrimidine . polypurine DNA that is located 1060 to 1150 bp upstream from alpha 1-globin gene. These results provide the first high resolution map of double-stranded S1-cleavage sites induced within a specific DNA sequence under supercoil strain. The distribution and relative cutting frequencies of these sites mapped are consistent with a slippage mechanism in which the simple repeating sequences are organized into base-mismatched duplex on supercoiled DNA.     

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.