Induction of an embryonic globin gene promoter by short-chain fatty acids

Short-chain fatty acids (SCFAs) and dimethyl sulfoxide (DMSO) induce adult erythroid differentiation in murine erythroleukemia (MEL) cells, but only SCFAs concurrently up-regulate expression from the endogenous embryonic globin gene epsilony. The epsilony promoter, linked to a reporter gene and stably transfected into MEL cells, was tested during adult erythroid differentiation. Both the epsilony-CACCC site at -114 bp and enhancer sequences (hypersensitive site 2 [HS2]) from the beta-globin locus control region (LCR) were essential to maximal SCFA-mediated induction of expression from these constructs in MEL cells. Gel-shift analyses of binding activity from SCFA-induced MEL cell nuclear extracts showed in vitro binding by specificity proteins 1 and 3 (SP1, SP3) and basic or erythroid Krüppel-like factors (BKLF, EKLF) at the epsilony-CACCC site. In a functional analysis, transient cotransfections in nonerythroid NIH/3T3 cells of SP1, SP3, BKLF, or EKLF and HS2 epsilony promoter-luciferase constructs, with or without coactivators (p300, CREB-binding protein [CBP], or p300/CBP-associated factor [PCAF]) and SCFAs, were performed. SP1, SP3, and EKLF further increased expression from HS2 epsilony promoter constructs following exposure to SCFAs. This effect was variably augmented by coactivators and was diminished in EKLF mutants that were unable to undergo histone/factor-acetyl transferase (H/FAT)-mediated acetylation. In addition, acetylation of SP1 was detectable in NIH/3T3 cells following exposure to SCFAs. In sum, LCR sequence and an embryonic globin gene promoter CACCC site were essential to that promoter's up-regulation during SCFA-mediated induction of adult erythroid differentiation in vitro. Of factors that interact at the CACCC site, SCFA-mediated acetylation is implicated in SP1 and EKLF, and may be a mechanism through which SCFAs induce embryonic/fetal globin gene promoters during adult erythroid differentiation.     

A novel delta beta fusion gene expresses hemoglobin A (HbA) not Hb Lepore: Senegalese delta(0)beta(+) thalassemia

This study identified and characterized a novel delta beta fusion gene in which the delta-globin gene promoter is linked to intact beta-globin coding sequences in a Senegalese family. It results from a 7.4-kb deletion that removes the delta-globin coding sequences, the delta beta intergenic region as well as the beta-globin gene promoter and causes delta(0)beta(+) thalassemia with hemoglobin A expressed at the 11% to 15% range. The phenotype of this naturally occurring delta beta hybrid gene not only clarifies, in an in vivo context, the respective strength of delta- and beta-globin gene promoters, but also emphasizes the importance of beta-globin intragenic sequences in the expression of beta-globin chains. (Blood. 2001;98:1261-1263)     

The β-globin promoter is important for recruitment of erythroid Krüppel-like factor to the locus control region in erythroid cells

Erythroid Krüppel-like factor (EKLF), which binds to the CACCC box in the beta-globin promoter, is required for the expression of the beta-globin gene in adult erythroid cells. It was recently demonstrated that EKLF is also required for the activity of the beta-globin locus control region (LCR) 5'HS3. Some evidence suggests that the LCR and the beta-globin promoter interact in adult erythroid cells, and the network of protein-protein interactions that exists between these two elements may regulate how EKLF is recruited to the LCR. In this report, we use the PIN*POINT assay to study the role of the promoter on the recruitment of EKLF to 5'HS2 and 5'HS3 of the LCR. We find that recruitment of EKLF to 5'HS2 requires the TATA box, but recruitment to 5'HS3 depends on the CACCC and TATA boxes of the beta-globin promoter. Furthermore, recruitment of EKLF to 5'HS3 only occurred in beta-globin-expressing murine erythroid leukemia cells, whereas recruitment of EKLF to 5'HS2 occurred in both gamma-globin-expressing K562 cells and murine erythroid leukemia cells. Unlike EKLF, Sp1, which also binds to CACCC boxes, is not recruited to 5'HS3. We have also examined how one 5'HS affects the recruitment of EKLF to another 5'HS. We have found that the recruitment of EKLF to 5'HS3 depends on the presence of 5'HS2 in cis, but the recruitment to 5'HS2 does not depend on 5'HS3. Based on these results, we present a model that illustrates how EKLF may be recruited to the beta-globin locus.     

A delta-globin gene derived from patients with homozygous delta zero- thalassemia functions normally on transient expression in heterologous cells

Three Japanese individuals with homozygous delta zero-thalassemia from different families were the subjects of molecular genetic analysis. They were homozygous for seven polymorphic sites in the beta-globin gene cluster. Nucleotide sequence analysis of the delta-globin gene cloned from each patient revealed a single nucleotide substitution (T- C) 77 base pairs 5' to the cap site, just upstream of the CCAAC box of the delta-globin gene. When introduced into COS cells, the gene was expressed at normal levels with proper processing of RNA. These results suggest that the complete suppression of delta-globin chain synthesis in these patients is not due to a defective promoter, a defective RNA processing or a chain terminator mutation, but rather to impaired regulation of gene expression specific to erythroid cells. The region around the CCAAC box may have a significant role in expression of the delta-globin gene in erythroid cells.     

Known and new delta globin gene mutations and their diagnostic significance

Mutations in the delta-globin gene (HBD, MIM# 142000) are not pathologically relevant. However, since high HbA2 levels are diagnostic for beta-thalassemia trait and a lowered level for an alpha- or delta-mutation, co-inheritance of delta- and beta-gene defects may lead to misinterpretation of diagnostic results. We examined 29 cases with low HbA2 level diagnosed in our laboratory, in the presence or absence of a second HbA2 fraction. We found a delta globin gene mutation in 20 cases. In total four different known mutations were found, three structural and one expressional. Moreover, two new defects were observed, one causing a structural abnormality and one a beta-thalassemia. The structural abnormality HBD c.431A->G (p.His144Arg)(dcd 143 CAC->CGC) was homologous to the beta-globin gene variant called Hb-Abruzzo and we have named this mutation HbA2 -Abruzzo. The new delta-thalassemia defect HBD c.-118C->T (d -68 C->T) has no homology on the beta-globin gene (HBB, MIM# 141900). All mutations caused a low HbA2 level and through this could lead to misdiagnosis when inherited together with a beta-thalassemia.     

Development of a condensed locus control region cassette and testing in retrovirus vectors for A gamma-globin

Retrovirus vectors for A gamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various A gamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, A gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the beta pr.A gamma cassettes.     

Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.     

Regulation of human fetal and adult globin genes in mouse erythroleukemia cells

We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta-globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5' end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.     

Beta-thalassemia intermedia with exceptionally high hemoglobin A2: relationship to mutations in the beta-gene promoter.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression.

The locus control region of the human beta-globin cluster consists of four major DNase I hypersensitive sites (HS). When linked to globin genes, the locus control region confers a high level of erythroid-specific expression of these genes in transgenic mice or transfected erythroid cell lines. We have examined the effect of one of these sites, HS2, on human beta-globin gene expression in a murine erythroleukemia cell line (MEL) after retrovirus-mediated gene transfer. We incorporated a 732- or 412-base-pair (bp) segment of HS2 in the retroviral construct carrying the human beta-globin gene. These fragments rendered the viruses unstable as the human beta-globin gene was rearranged or deleted in all the packaging cell lines examined. On the other hand, when a 36-bp fragment containing the NFE-2/AP-1 binding consensus in this region was inserted into the retroviral construct, we recovered 6 stable packaging cell lines of 12 examined, similar in percentage to the construct with the beta-globin gene alone. The virus titers of the packaging cell lines from these two constructs were similar. We infected MEL cells with viruses produced from three packaging cell lines of each of the two constructs and measured the ratio of human beta-globin to mouse alpha-globin mRNA after hexamethylenebisacetamide induction. The overall level of expression increased 2-fold from 6.0% to 12.7% with the addition of this 36-bp enhancer.     

Activation of the human delta-globin gene promoter in primary adult erythroid cells

Restoration of the CCAAT box or insertion of an erythroid Krüppel-like factor (EKLF) binding site in the delta promoter activates its expression in several erythroid cell lines. We extended these studies using a novel primary human adult erythroid cell ( h AEC) system to investigate these effects at the late erythroblast stage. Restoration of the CCAAT box at −70 bp, or insertion of an EKLF binding site at −85 bp or −95 bp in the promoter significantly increased delta globin gene expression in h AEC. Our results demonstrate that the altered CCAAT box (CCAAC) and the lack of an EKLF binding site in δ-globin contribute to its low level of expression in the h AEC model as well.     

Spontaneous delta- to beta-globin switching in K562 human leukemia cells.

Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta-globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.     

An erythroid-specific chromatin opening element reorganizes beta-globin promoter chromatin structure and augments gene expression.

In erythroid tissues the chromatin structure of the beta-globin gene locus is extensively remodeled. Changes include the formation of DNase I hypersensitive sites (HSs) over the promoters of actively expressed genes. To test the hypothesis that such "opening" of promoter chromatin structure is important for beta-globin gene expression, we placed a 101-bp erythroid-specific hypersensitive-site forming element (HSFE) from the core of LCR HS4 immediately upstream of a minimal beta-globin gene promoter. We then studied the effects of this element alone and in combination with other cis-acting elements on globin gene chromatin structure and gene expression in MEL cells and transgenic mice. Single or tandem HSFEs increased the size of the portion of the promoter accessible to DNase digestion, increased the proportion of promoters in an accessible conformation, and increased gene expression approximately 5-fold. These were equivalent to expression levels attained using a 2.8-kb microLCR construct. Inclusion of the LCR HS2 enhancer did not increase expression further. In transgenic mouse fetal liver cells the HSFE increased average expression 2.5-fold compared to the minimal promoter alone. These results indicate that a small cis-acting element is capable of remodeling local beta-globin promoter chromatin structure and producing expression similar to that seen with a microLCR construct.     

Delta +-thalassemia in Sardinia

We have defined a new type of delta-thalassemia in which beta-globin chain synthesis is incompletely suppressed. Homozygotes have unusually low HbA2 levels, and double heterozygosity for this delta-thalassemia gene and beta-thalassemia normalizes the HbA2 level. The delta- thalassemia occurs on a chromosome that is identifiable using polymorphic restriction endonuclease sites. We call this condition delta +-thalassemia, to distinguish it from the previously described delta 0-thalassemia syndromes in which no delta-globin chain synthesis occurs.