Globin Gene Region Research Articles

Naturally occurring methylation inhibitor: DNA hypomethylation and hemoglobin synthesis in human K562 cells.

A naturally occurring methylation inhibitor isolated from rabbit liver and named methinin inhibits a number of methyltransferases. Methinin is a low-molecular-weight compound (1,400) that has an active amine group. This compound inhibits the DNA methyltransferase of human erythroleukemia cells (K562) in vitro. When the K562 cells were grown in medium containing methinin, fetal hemoglobin was produced. Small but detectable amounts of adult hemoglobin were also produced. Methinin was not toxic to these cells. The overall rate of genomic DNA methylation was reduced by 60% in cells grown in medium containing methinin. Southern blots of genomic DNA from methinin-treated cells and untreated cells hybridized to a 32P-labeled globin gene probe showed that one site in the globin gene region was hypomethylated. Methinin is a naturally occurring compound which inhibits DNA methylation both in vitro and in vivo.

Rates of nuclear DNA evolution in pheasant-like birds: evidence from restriction maps.

To examine the tempo of genomic evolution in birds, we mapped 161 restriction sites in the nuclear DNA of seven species of birds belonging to the pheasant superfamily Phasianoidea. The three regions mapped lie on different chromosomes and bear eight genes, coding for lysozyme c, three "alpha-like" globins, and four "beta-like" globins. Together, the three regions span about 56 kilobases, most of which is presumably noncoding. The maps differed from one another at a minimum of 77 sites and by 9 length mutations. The extent of sequence divergence due to base substitutions was inferred to be similar for all three regions, even though the three coding regions differ by 5-fold from one another in mean rate of evolution at the amino acid level. A tree relating the maps differs in branching order from that implied by the traditional classification of phasianoid birds and is supported by published protein comparisons. Five of the nodes in the tree were associated with fossil evidence and historical biogeographic information, allowing us to estimate the mean rate of DNA divergence to be 0.34-0.40% per million years. This rate is similar to that estimated for the globin gene regions of higher primates, which validates the concept of an evolutionary clock at the DNA level. Our fossil-based calibration of DNA evolution differs by a factor of almost 2 from that proposed by others on the basis of biogeography. In consequence, published estimates of divergence times for birds and primates that are based on a biogeographically calibrated DNA clock may be too long.

A silent deletion in the β-globin gene cluster

A survey of the gamma-globin gene region of over 1000 normal individuals revealed a novel 2.5 kb deletion which removes the 5' end of the A gamma-globin gene. Unusually, this deletion in the beta-globin gene cluster is not associated with increased fetal haemoglobin production. Sequence analysis of the deletion endpoints revealed no significant homology at the breakpoint and failed to support a role for a proposed recombination hotspot in IVS-2 in the generation of this illegitimate recombination event. The existence of small "silent" deletions in the beta-globin gene cluster emphasizes the importance of deletion size in altering expression of the fetal globin genes.

Sequential alterations in globin gene chromatin structure during erythroleukemia cell differentiation.

During differentiation of murine erythroleukemia cells, adult beta-globin gene chromatin acquires site-specific, DNase I hypersensitivity and an increased sensitivity in the globin gene region toward micrococcal nuclease (MNase) digestion. The relationship of these changes in chromatin structure to globin gene activation and to cellular commitment events has been studied. Imidazole, which blocks globin gene transcription during induction does not affect the terminal differentiation of the cells nor does it prevent the acquisition of DNAse I hypersensitivity. The formation of the inducible DNase I-hypersensitive site near the globin gene accompanies the developmental events which lead to cellular differentiation independent of the transcription process. The increased MNase sensitivity of the adult beta-globin gene region, normally preceded by the acquisition of 5' DNase I hypersensitivity, was blocked by the addition of imidazole prior to but not after globin gene activation. The enhanced MNase sensitivity was not abolished by the addition of actinomycin D and, thus, reflects a part of chromatin alterations that define potential for transcription. Therefore, there is a sequential series of chromatin alterations in the globin gene region associated with murine erythroleukemia cell differentiation. The appearance of the inducible 5' DNase I-hypersensitive site precedes the onset of globin gene transcription and is strongly correlated with commitment events. The enhanced MNase sensitivity is closely related to globin gene transcription, but it is not a consequence of the transcription process. In addition, the commitment of cells to terminal differentiation is dissociable from the stimulation of globin gene transcription.

Association of heterocellular HPFH, beta(+)-thalassaemia, and delta beta(0)-thalassaemia: haematological and molecular aspects.

An Italian family in which heterocellular hereditary persistence of fetal haemoglobin (HPFH) interacts with both beta(+)- and delta beta-thalassaemia is described. The index case was an 8 year old girl who was presumed to inherit both heterocellular HPFH and beta (+)-thalassaemia from her mother and delta beta-thalassaemia from her father. She was healthy and never needed blood transfusions. The possible contribution of heterocellular HPFH to the less severe expression of the compound delta beta/beta(+)-thalassaemia heterozygosity is discussed. By DNA analysis the specific delta beta-thalassaemia defect on the gamma delta beta globin gene region has been established. In addition, a previously unreported association of a polymorphic restriction site haplotype with a beta (+)-thalassaemia mutation has been observed.

Two novel arrangements of the human fetal globin genes: G gamma-G gamma and A gamma-A gamma.

We describe two novel arrangements of the human fetal globin gene region: one chromosome with two linked A gamma genes (A gamma-A gamma) and two chromosomes with two linked G gamma genes (G gamma-G gamma). The gamma genes of these three chromosomes were cloned and the unusual 5' A gamma gene and one of the unusual 3' G gamma genes were partially sequenced. Both of these unusual genes differ from the genes normally found at their respective locations by a nucleotide substitution at the site of the single coding region difference between normal G gamma and A gamma genes. In both cases, the substitution is identical to the nucleotide found at that position in the normal neighboring gene. The unusual 3' G gamma gene also differs from normal A gamma genes at two other nucleotide positions, but both differences appear to be "private" or exclusive to this particular gene. These unusual fetal globin gene arrangements could have arisen from point mutations or from gene conversions of limited extent, the boundaries of which have been determined for all three chromosomes.

The delta beta crossover region in Lepore boston hemoglobinopathy is restricted to a 59 base pairs region around the 5' splice junction of the large globin gene intervening sequence

Genomic DNA from a hemoglobin (Hb) Lepore Boston (delta 87 Gln beta 116 His) homozygote of Southern Italian origin has been studied in order to map the fusion point between the delta and beta genes. An Ava II restriction endonuclease recognition sequence, located 12 base pairs (bp) downstream from the 5' end of the beta gene large intervening sequence, has been taken as marker of the beta-like portion of the fusion gene. This site was present even in the delta beta gene, allowing the localization of the crossover area to a 59-bp region extending from the first nucleotide of the Leu codon in position 88 to the 11th nucleotide of the large intervening sequence. The analysis of the DNA restriction polymorphisms in the gamma delta beta globin gene region provides evidence that a single mutational event originated the Lepore delta beta genes, at least in the Italian population.

Restriction endonuclease mapping of globin genomic regions of HEL (human erythroleukemia) line.

The restriction endonuclease map of the alpha and beta globin genomic region of the new human erythroleukemia line, HEL, was compared with that of normal human DNA. The HEL line, which produces mainly fetal (G gamma and A gamma) but no adult (delta and beta) globin chains, was shown to have the same pattern of DNA fragments as that of normal human DNA. This suggests that the selective expression of the gamma globin genes observed in HEL cells is not due to a major deletion or rearrangement in the epsilon-G gamma-A gamma-delta-beta gene complex. Thus, the HEL line provides a model for studying the control of globin developmental switching in cells with structurally intact globin gene regions.

The deletion in a type of delta 0-beta 0-thalassaemia begins in an inverted AluI repeat.

In hereditary persistence of fetal haemoglobin (HPFH) and delta 0-beta 0-thalassaemia, increased levels of fetal haemoglobin (HbF) are found in adult individuals. HbF production is particularly noticeable in the former condition in which HbF levels of 20-40% are found in heterozygous patients, as opposed to 5-20% in delta 0-beta 0-thalassaemia. In a minority of these cases, no obvious abnormalities have been found in the globin gene region by DNA mapping, indicating that small deletions or perhaps even point mutations in critical DNA sequences in the globin gene cluster may be responsible for these conditions. However, most cases of HPFH and delta 0-beta 0-thalassaemia are associated with extensive deletions in the globin gene cluster. Genetic data and gene mapping investigations provided some evidence for the location of a regulatory area, whose deletion results in continuing activity of gamma-globin genes in adults, in a DNA region between the A gamma- and delta-globin genes, possibly 3-4 kilobases (kb) 5' to the delta gene. The precise nature of these sequences is of great interest, because it might help elucidate the molecular mechanisms regulating globin gene expression during development. Recently, Jagadeeswaran et al. cloned the DNA encompassing the region of a gene deletion in a type of HPFH and showed that the 5' end point of the deletion lies in the middle of an AluI repetitive DNA sequence. We have now cloned the corresponding region from the DNA of a delta 0 -beta 0-thalassaemia patient and we report here that the deletion ends in a different AluI sequence, congruent to 700 nucleotides 3' to the AluI repeat involved in the HPFH deletion, and in the opposite orientation.

DNA methylation in chicken alpha-globin gene expression.

We have investigated certain specific methylation sites of the chicken alpha-globin gene cluster in DNA from embryonic and adult erythroid cells as well as from brain and sperm cells. Eight contiguous DNA fragments of the alpha-globin gene cluster were subcloned from a recombinant lambda phage. The subclones were used as probes to map all the Msp I/Hpa II and Hha I sites in the unmethylated cloned DNA and specific sites of methylation in and around the alpha-globin gene cluster in chromosomal DNA. The data show that sperm DNA is totally methylated at these restriction sites in the globin gene region, as is brain DNA, with some exceptions. Interestingly, the methylation status of specific sites 5' to the coding sequences is correlated with expression of the embryonic or adult alpha-globin genes in different stages of erythroid development. Some sites showing partial methylation, however, do not conform to the model that transcribed genes are unmethylated or undermethylated. We also find a well-defined 3.5-kilobase region of DNA 5' to the alpha-globin gene cluster in which all C-C-G-G sites are resistant to Msp I digestion in all tissues. This "Msp block" is presumably caused by 5-MeCpC methylation.

Hemoglobin switching in chickens. Is the switch initiated post-transcriptionally?

We have investigated the transcriptional specificity of chick embryo erythroid nuclei as a function of developmental age during progression of the hemoglobin switch. Nuclei were allowed to transcribe in vitro in the presence of high specific activity [alpha-32P]CTP and the radioactive transcripts were hybridized to a collection of plasmid clones spanning the beta-like globin gene region of the chicken genome. The results reveal locus-specific waves of transcription appearing during the interval between 5 and 12 days of incubation. The last wave, which comes in at 12 days, is highly specialized in transcription of the adult beta-globin gene locus. The most interesting wave of transcription occurs at 6-7 days. The most actively transcribed gene in the early part of this wave is the embryonic rho gene. As the wave progresses, the rho gene activity gradually gives way to beta gene transcription. Definitive red blood cells, which would be synthesizing little rho globin protein at this stage, appear to be responsible for the rho gene transcription. These results and additional data which we present indicate that during the initial stages of the hemoglobin switch the embryonic globin genes are silenced post-transcriptionally.

The chicken beta globin gene region. Delineation of transcription units and developmental regulation of interspersed DNA repeats.

The chicken beta globin gene region. Delineation of transcription units and developmental regulation of interspersed DNA repeats.

The gamma-delta-beta-globin gene region in G gamma-beta +-hereditary persistence of fetal hemoglobin

We report restriction endonuclease analysis of the gamma-delta-beta- globin gene region in a mother and child heterozygous for G gamma-beta +-hereditary persistence of fetal hemoglobin (HPFH). The affected chromosome in these persons directs the production of G gamma-chains and beta-chains but not A gamma-chains. DNA was digested with several restriction enzymes and was examined for gamma, delta, beta sequences by blot hybridization. Only normal digestion fragments were present. By sensitive methods, we were unable to detect a deletion in the entire gamma-delta-beta-globin gene region of the affected chromosome, indicating that in this family, G gamma-beta +-HPFH is not due to a large deletion.

Greek (A gamma) variant of hereditary persistence of fetal haemoglobin: globin gene organization and studies of expression of fetal haemoglobins in clonal erythroid cultures.

Individuals heterozygous for the Greek (A gamma) variant of hereditary persistence of fetal haemoglobin (HPFH) synthesize Hb F whose gamma-globin chains are predominantly of the A gamma type. DNA obtained from Greek HPFH heterozygotes was used to test for abnormalities in the organization of non alpha-globin genes. In addition, gamma- and beta-globin expression was studied in BFUe cultures. Restriction endonuclease mapping showed that the G gamma, delta and beta genes in cis to the Greek HPFH determinant are intact. Overproduction of gamma-globin chains synthesis was observed in the BFUe cultures. A significant portion of the gamma chain synthesis was of the G gamma type, suggesting that the G gamma genes cis and trans to the HPFH chromosome are active in culture. DNA mapping data indicate that in contrast to G gamma A gamma HPFH and the G gamma (delta beta) thalassaemia, the Greek (A gamma) HPFH is not due to a large deletion in the non-alpha globin gene region. It is possible that the anomaly may result either from a small deletion or point mutation which influences non alpha-globin transcription. The in vitro synthesis data suggest that the low level of G gamma-globin chain synthesis in vivo is not the result of transcriptional inactivation of the G gamma gene, since this gene appears to be expressed in erythroid cell cultures. We speculate that the genetic lesion in Greek (A gamma) HPFH is in regulatory sequences which control the level of G gamma and A gamma expression during development.

Base substitutions, length differences and DNA strand asymmetries in the human Gγ and Aγ fetal globin gene region

We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G γ and A γ fetal globin genes. The local occurrence of base substitutions in the duplicated 5 kb region correlates positively with the local AT base pair content. This correlation also occurs in two mouse β-globin genes and in two mouse immunoglobulin genes. The relationship is valid for transcribed or nontranscribed DNA and for DNA that contains only coding sequences. Length differences in the fetal globin duplicated regions correlate positively with the occurrence of short direct repeats of ≥5 base pairs. Path analysis of the interrelationships of base composition, base substitutions, repeats and length differences provides an integrated view of the relative effects on chromosomal changes of these variables and of selection. The distributions along the chromosome of simple sequences and of base compositions show highly significant local asymmetries between the transcribed and nontranscribed strands of the DNA, which permit us to divide the fetal globin gene region into chromosomal domains. Comparable domains are present in DNA from other sources, including the mammalian viruses SV40 and polyoma virus strain A-2 in which some of the domains appear related to discrete functions.

Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes.

The human gamma-, delta-, and beta-globin genes are located within a 30-kilobase (kb) region of DNA, of which only 20% represents the globin genes. We have attempted to define the nature of flanking and intergenic sequences by isolating recombinants containing the human epsilon, both gamma-, or the 3' end of the beta-globin gene from a bacteriophage library of cloned human DNA. Comparison of these recombinants and a recombinant containing the delta- and beta-globin genes (H beta G1) has provided the following results. The epsilon-globin gene is located 14 kb 5' to the G gamma gene. DNA sequence homology between the region containing the two G gamma genes and the delta nd beta gene region is limited to only a few hundred nucleotides which include the globin coding sequences. Repetitive DNA sequences have been found in the region 3' to the beta-globin gene. Sequences located adjacent to the beta-globin gene are repeated in the globin gene region. A repetitive DNA sequence more than 3.2 kb long is repeated frequently in the human genome but is not repeated in the globin gene region in the clones examined.

DNA methylation in the human γδβ-globin locus in erythroid and nonerythroid tissues

We have analyzed DNA modification in the human γδβ-globin gene region at 17 cleavage sites of restriction endonucleases which are unable to cleave DNA if 5-methylcytosine is present at certain positions in their respective cleavage sites. Using this criterion, all sites tested in the globin gene region are fully modified in the germ line (sperm) DNA. In somatic tissues, however, methyl groups are absent at specific sites in the globin gene region. In tissues not expressing the genes, these losses range from one of these cleavage sites in lymphocyte DNA to essentially all of these sites in the entire region in placental DNA. In the DNA of tissues expressing the globin genes, the region surrounding and including the genes expressed shows a low level of modification, whereas the neighboring DNA regions have a high level of modification. The data suggest that a low level of DNA methylation may be a necessary, but not a sufficient, condition for gene expression in higher eucaryotes.

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.

Physical mapping of the globin gene deletion in (δβ)° - thalassaemia

We have constructed a physical map of restriction endonuclease cleavage sites in the (δ+β)-globin gene region in the DNA of patients with (δβ)°-thalassaemia. This map shows that a 10 kb deletion has occurred in (δβ)°-thalassaemia to remove the entire β-globin gene and the 3′ portion of the δ-globin gene. The 5′ terminus of the deletion is in the large intron of the δ-globin gene and the 3′ terminus 1.8 kb to the 3′-side of the β-globin gene. A similar deletion of about 7 kb has been described previously in the DNA of patients with Hb Lepore; the 5′ terminus of the deletion is also in the δ-globin gene but the 3′ terminus is in the β-globin gene. Comparison of the foetal (γ) globin gene expression in adults with (δβ)°-thalassaemia and Hb Lepore suggests that the 3′ extragenic regions of the β-globin gene contain DNA sequences involved in the regulation of γ-globin gene expression.

Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin.

In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.