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

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.     

Physical mapping of the globin gene deletion in hereditary persistence of foetal haemoglobin (HPFH).

We have mapped the globin gene region in the DNA of two HPFH patients. In a patient homozygous for the G gamma A gamma type of HPFH at least 24 kb of DNA in the globin gene region has been deleted to remove most of the gamma-delta intergenic region and the delta and beta globin genes. The 5' break point of the deletion is located about 9 kb upstream from the delta globin gene. The 3' break point has not been precisely located but is at least 7 kb past the beta globin gene. DNA from an individual heterozygous for the Greek (A gamma) type of HPFH, however, shows no detectable deletion in the entire gamma delta beta-globin gene region. HPFH, therefore, appears to occur in different molecular forms. These results are discussed in terms of a model for the regulation of globin gene expression in man.     

Localization and characterization of members of a family of repetitive sequences in the goat beta globin locus.

We have characterized a family of repetitive DNA elements in the beta-globin locus of the goat. These sequences are structurally analogous to the Alu families of repeats of other mammals. Repetitive elements are located both in the intervening sequences and in the intergenic regions of the goat beta-globin locus. Nucleotide sequence analysis of five repetitive elements located within the large intervening sequence of the beta-like globin genes, and four repeats located 5' to the major developmentally regulated beta-globin genes has resulted in the definition of a consensus sequence for this family of repeats.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

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.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Characterization of 5'-terminal methylation of human alpha- and beta-globin mRNAs

After incubating peripheral blood of various anemic individuals with [methyl-3H]methionine, 5'-terminal cap structures and the sequential methylation of human globin mRNAs were determined. Human alpha- and beta-globin mRNAs have the same methylated nucleotides at their 5' ends as the alpha- and beta-globin mRNAs of mouse and rabbit: m7G(5')ppp(5')[m6Am/Am]pCmp. The order of methyl group addition to human globin mRNA is also similar to that of mouse globin mRNA in that methylation of the 5'-terminal guanosine and the ribose of adenosine occurs earlier than that of the base of adenosine and the ribose of cytidine. Thus, both the cap structures and the order of methylation are conserved in the globin mRNAs of these species, suggesting that variation in the structure of the 5' terminus of globin mRNAs may alter the function of these molecules.     

Nonuniform recombination within the human beta-globin gene cluster.

Population genetic analysis of 15 restriction site polymorphisms demonstrates nonuniform recombination within the human beta-globin gene cluster. These DNA polymorphisms show two clusters of high nonrandom associations, one 5' and another 3' to the beta-globin structural gene, with no significant linkage disequilibrium between the two clusters. The 5'- and 3'-association clusters are 34.6 kilobases (kb) and 19.4 kb long, respectively, and are separated by 9.1 kb of DNA immediately 5' to the beta-globin gene. For each of these three DNA regions, we have observed a relationship between nonrandom associations and physical distance between the polymorphisms. However, this relationship differed for each of these regions. On the assumption that the effective population size (Ne) is 5,000-50,000, we estimate the total recombination rate to be 0.0017%-0.0002% in the 5' cluster, 0.0931%-0.0093% in the 3' cluster, and 0.2912%-0.0219% in the 9.1-kb region between them. The beta cluster thus shows nonuniformity in recombination. Moreover, the recombination rate in the 9.1-kb DNA segment is 3-30 times greater than expected and is thus a hot spot for meiotic recombination.     

Mapping of the goat beta-globin gene cluster to a region of chromosome 7 by in situ hybridization

We mapped the beta-globin gene cluster of the goat (Capra hircus) to a specific chromosomal region of the species, using in situ hybridization. The probe we used was a 1.2-kb Eco RI/Bam HI genomic DNA fragment that was 3H labeled to high specific activity by the random primer method. This DNA fragment contains part of the 5' flanking region and the first two exons of the epsilon IV gene of the goal beta-globin gene cluster. Our results show that this gene cluster is on chromosome 7 in the region of bands 33 to 35 based on BrdU G-banded chromosome preparations of this species.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.     

Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

We have confirmed the result that chicken beta-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on beta-globin DNA, we have studied chromatin assembly of cloned chicken beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into chromatin possessing a regular 180 +/- 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the beta A gene and promoter region, but lacking the downstream intergenic region between the beta A and epsilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta A gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta A-epsilon intergenic region.     

An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene.

A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene. This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant. The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene. Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence. This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron. This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron. The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.