Human Β-globin Locus Research Articles

Genomic imprinting recapitulated in the human β-globin locus

A subset of genes in mammals are subject to genomic imprinting. The mouse H19 gene, for example, is active only when maternally inherited and the neighboring Igf2 gene is paternally expressed. This imprinted expression pattern is regulated by the imprinting control region (ICR) upstream of the H19 gene. A maternally inherited H19 ICR inhibits Igf2 gene activation by the downstream enhancer due to its insulator function while it suppresses H19 gene transcription by promoter DNA methylation when paternally inherited. These parent-of-origin specific functions depend on the allele-specific methylation of the ICR DNA, which is established during gametogenesis. Therefore, the ICR may also function as a landmark for epigenetic modifications. To examine whether the ICR confers these activities autonomously, we introduced a 2.9-kbp ICR-containing DNA fragment into a human beta-globin yeast artificial chromosome at the 3' end of the locus control region and established transgenic mouse lines. Expression of all of the beta-like globin genes was higher when the transgene was paternally inherited. In accord with this result, transgenic ICR DNA from nucleated erythrocytes was more heavily methylated when paternally transmitted. Chromatin immunoprecipitation assays confirmed that CCCTC binding factor is preferentially recruited to the maternal transgenic ICR in vivo. Surprisingly however, the parent-of-origin specific methylation pattern was not observed in germ cell DNA in testis, demonstrating that methylation was established after fertilization. Thus, the ICR autonomously recapitulated imprinting within the normally nonimprinted transgenic beta-globin gene locus, but the temporal establishment of imprinting methylation differs from that at the endogenous Igf2/H19 locus.

The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of β-like globin genes

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.

The Human β-Globin Locus Control Region Can Silence as Well as Activate Gene Expression

Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.

A humanized mouse model for a common β 0-thalassemia mutation

Accurate animal models that recapitulate the phenotype and genotype of patients with β-thalassemia would enable the development of a range of possible therapeutic approaches. Here we report the generation of a mouse model carrying the codons 41–42 (−TTCT) β-thalassemia mutation in the intact human β-globin locus. This mutation accounts for approximately 40% of β-thalassemia mutations in southern China and Thailand. We demonstrate a low level of production of γ-globins from the mutant locus in day 18 embryos, as well as production of mutant human β-globin mRNA. However, in contrast to transgenic mice carrying the normal human β-globin locus, 4-bp deletion mice fail to show any phenotypic complementation of the knockout mutation of both murine β-globin genes. Our studies suggest that this is a valuable model for gene correction in hemopoietic stem cells and for studying the effects of HbF inducers in vivo in a “humanized” thalassemic environment.

Onset and inheritance of abnormal epigenetic regulation in hematopoietic cells

Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.

LCR 5′HS3 Displays Specificity for ε-Globin Gene Activation during Primitive Erythropoiesis and γ-Globin Gene Activation during Fetal Definitive Erythropoiesis.

A 2.9 Kb deletion of 5′HS3 (Δ5′HS3) or a 234 bp deletion of the 5′HS3 core (Δ5′HS3c) in a 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) abrogate ε-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that HS sequences of the LCR are involved directly in ε-globin gene activation. The reduction of ε-globin gene transcription in Δ5′HS3 or Δ5′HS3c β-YAC transgenics can be explained by two hypotheses. The first is site-specificity. The interaction between the LCR and the ε-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the ε-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and ε-globin gene transcription is diminished. The second hypothesis is change in conformation of the LCR. Normally, in the embryonic stage, the LCR achieves a three-dimensional conformation that favors interaction with the first gene in the complex, i.e., the ε-globin gene. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, the LCR interacts with the next gene in order, the γ-globin gene. In Δ5′HS3c β-YAC mice, γ-globin gene expression is normal during primitive erythropoiesis, but is extinguished in the fetal stage of definitive erythropoiesis. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not. Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core. To distinguish between these possibilities, β-YAC lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to either an intact LCR, a 2.9 Kb 5′HS3 deletion or a 234 bp 5′HS3 core deletion. Δ5′HS3c Δε::βm β-YAC mice expressed βm-globin throughout development beginning at day 10 in the yolk sac. γ-globin was expressed in the embryonic yolk sac, but not in the fetal liver. Some wild-type β-globin was expressed in addition to βm-globin in adult mice. The γ-globin phenotype is consistent with published data on Δ5′HS3c β-YAC mice. Although ε-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δε::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for ε-globin expression during embryonic erythropoiesis, but not for βm-globin expression. These data support a site specificity model of LCR HS-globin gene interaction. In addition, nuclear ligation experiments provided evidence for a specific physical interaction between 5′HS3 and the γ-globin promoter during fetal definitive erythropoiesis, further supporting a site specificity model.

Effect of LCR 5′HS4 and 5′HS1 Deletions on β-Like Globin Gene Expression in β-Yac Transgenic Mice.

A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently.To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.

Multiple interactions between regulatory regions are required to stabilize an active chromatin hub.

The human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the beta-globin gene in the maintenance of the ACH. When the human beta-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the beta-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the beta-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the beta-globin gene are required to maintain the appropriate spatial configuration in vivo.

The human beta-globin replication initiation region consists of two modular independent replicators.

Previous studies have shown that mammalian cells contain replicator sequences, which can determine where DNA replication initiates. However, the specific sequences that confer replicator activity were not identified. Here we report a detailed analysis of replicator sequences that dictate initiation of DNA replication from the human beta-globin locus. This analysis suggests that the beta-globin replication initiation region contains two adjacent, redundant replicators. Each replicator was capable of initiating DNA replication independently at ectopic sites. Within each of these two replicators, we identified short, discrete, nonredundant sequences, which cooperatively determine replicator activity. Experiments with somatic cell hybrids further demonstrated that the requirements for initiation at ectopic sites were similar to the requirements for initiation within native human chromosomes. The replicator clustering and redundancy exemplified in the human beta-globin locus may account for the extreme difficulty in identifying replicator sequences in mammalian cells and suggest that mammalian replication initiation sites may be determined by cooperative sequence modules.

An embryonic-specific repressor element located 3′ to the Aγ-globin gene influences transcription of the human β-globin locus in transgenic mice

Objective Persistent expression of the human fetal γ-globin genes in the adult stage is often associated with naturally occurring deletions in the human β-globin locus. The mapping of the 5′ breakpoints of these deletions within the Aγ- to δ-globin intergenic region has suggested that regulatory elements involved in the silencing of the γ-globin genes in the adult may be present. We previously identified two elements in this region, termed Enh and F, located 3′ to the Aγ-globin gene acting as silencers in transient transfection assays. Here, we tested directly the in vivo significance of these elements in the developmental regulation of the human β-like globin genes. Materials and methods We selectively deleted both Enh and F elements in the context of a 185-kb human β-globin locus PAC (P1 phage artificial chromosome) and tested the effects of this deletion on the expression of the human β-like globin genes in transgenic mice. Results The Enh/F deletion resulted in an increase in ϵ- and γ-globin mRNA levels in the embryonic yolk sac stage of erythropoiesis, which appears to be due to an increase in the rate of transcription rather than to an increase in the number of cells transcribing the human globin locus. However, the human developmental switching from fetal γ-globin to adult β-globin gene expression in transgenic mice was not affected by this deletion. Conclusion These results identify Enh and F as locus-wide regulatory elements capable of down-regulating transcription of the human β-globin locus in an embryonic-specific manner.

Developmental stage–specific epigenetic control of human β-globin gene expression is potentiated in hematopoietic progenitor cells prior to their transcriptional activation

AbstractTo study epigenetic regulation of the human β-globin locus during hematopoiesis, we investigated patterns of histone modification and chromatin accessibility along this locus in hematopoietic progenitor cells (HPCs) derived from both humans and transgenic mice. We demonstrate that the developmentally related activation of human β-like globin genes in humans and transgenic mice HPCs is preceded by a wave of gene-specific histone H3 hyperacetylation and K4 dimethylation. In erythroid cells, expression of β-like globin genes is associated with histone hyperacetylation along these genes and, surprisingly, with local deacetylation at active promoters. We also show that endogenous mouse β major and human β-like genes are subject to different epigenetic control mechanisms in HPCs. This difference is likely due to intrinsic properties of the human β-globin locus since, in transgenic mice, this locus is epigenetically regulated in the same manner as in human HPCs. Our results suggest that a defined pattern of histone H3 acetylation/dimethylation is important for specific activation of human globin promoters during development in human and transgenic HPCs. We propose that this transient acetylation/dimethylation is involved in gene-specific potentiation in HPCs (ie, before extensive chromatin remodeling and transcription take place in erythroid cells).

Persistent γ-globin expression in adult transgenic mice is mediated by HPFH-2, HPFH-3, and HPFH-6 breakpoint sequences

AbstractDeletions at the 3′ end of the human β-globin locus are associated with the hereditary persistence of fetal hemoglobin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal γ-globin genes. We have tested how sequences at the HPFH-2, HPFH-3, and HPFH-6 breakpoints, which act as enhancers in vitro, affect the silencing of a locus control region Aγ (LCRAγ) transgene in the adult stage of mice. We found persistent Aγ expression in the adult blood of most of the multicopy HPFH-2, HPFH-3, or HPFH-6 lines, in contrast to the control LCRAγ lines which were silenced. Cre-mediated generation of single copy lines showed persistent γ gene expression maintained in some of the HPFH-2 and HPFH-6 lines, but not in any of the HPFH-3 or LCRAγ lines. In the HPFH-2 and HPFH-6 lines, persistent γ gene expression correlated with euchromatic transgene integrations. Thus, our observations provide support for a model whereby HPFH conditions arise from the juxtaposition of enhancers as well as permissive chromatin subdomains in the vicinity of the γ-globin genes.

Dynamic Alterations of Replication Timing in Mammalian Cells

Background: The eukaryotic genome is divided into distinct replication timing domains, which are activated during S phase in a strictly conserved order. Cellular differentiation can alter replication timing in some loci, but recent experiments yielded conflicting data regarding the relationship between gene expression and replication timing. The genetic and epigenetic determinants of replication timing in mammalian cells have yet to be elucidated.Results: We developed a mammalian experimental system in which the timing of DNA replication can be altered in a controlled manner. This system utilizes sequences from the human β-globin locus that exhibit orientation-dependent transcriptional silencing when inserted into the murine genome. We found that before insertion, the murine target site replicated late during S phase. After insertion, replication timing depended on the orientation of the transgene. In a transcription-permissive orientation, the transgene and flanking sequences replicated early. In the reverse (silencing-prone) orientation, these sequences replicated late. Early replication correlated with histone modifications of the transgene chromatin but could be observed in the absence of the β-globin promoter. Importantly, the replication timing switch did not require a replication origin within the transgene.Conclusions: Transgene insertions into mammalian heterochromatin can alter the timing of DNA replication at the insertion site. This differentiation-independent replication timing switch did not necessitate insertion of an active promoter or a replication origin. These observations suggest that the timing of DNA replication can be manipulated by changes in DNA sequence, but that the determinants of replication timing are distinct from the sequences that specify replication initiation sites.

Targeted modification of a human β-globin locus BAC clone using GET Recombination and an I- Scei counterselection cassette

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human β-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I- SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I- SceI site in the counterselection cassette is cut by I- SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.

Insertion of common mutations into the human β-globin locus using GETRecombination and an EcoRI endonuclease counterselection cassette

A large number of mutations have been described in the human β-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human β-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan R counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG→AAG mutation and the codon 41–42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human β-globin locus. The counterselection cassette was first inserted into the target sequence in the β-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI q . Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.

Autocrine stimulation by erythropoietin in transgenic mice results in erythroid proliferation without neoplastic transformation

Erythropoietin (Epo) autocrine stimulation has been implicated in erythroleukemia. To develop a model of Epo autocrine stimulation, we made transgenic mice using a construct that linked the human Epo gene to an erythroid-specific regulatory element, designated 5′HS-2, from the human β-globin locus control region. We hypothesized that Epo gene expression would be targeted to erythroid cells in these mice, resulting in autocrine stimulation of erythroid progenitor cell growth in culture, and that chronic autocrine Epo stimulation would result in erythroleukemia. Transgenic mice containing intact copies of the 5′HS-2Epo construction had elevated hematocrits, reticulocyte counts and serum Epo levels and marked splenic enlargement. Analysis of RNA isolated from organs of transgenic mice revealed constitutive Epo mRNA expression primarily in spleen, blood and bone marrow. RNA samples from anemic transgenic mice revealed Epo gene induction only in the liver. Marrow derived from 5′HS-2Epo mice grew BFU-E in the absence of exogenous Epo. Despite observation of up to 2 years, no mouse developed erythroleukemia, demonstrating that Epo autocrine stimulation alone is insufficient for progression to malignancy. These studies show that 5′HS-2 can be used to target Epo gene expression to erythroid tissue. These mice could provide a model system for studying autocrine growth regulation.

Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human β-globin locus in erythropoietic cells

Reactivation of fetal hemoglobin genes has been proposed as a potential therapeutic procedure in patients with beta-thalassemia, sickle cell disease, or other beta-hemoglobinopathies. In vitro model systems based on small plasmid globin gene constructs have previously been used in human and mouse erythroleukemic cell lines to study the molecular mechanisms regulating the expression of the fetal human globin genes and their reactivation by a variety of pharmacologic agents. These studies have led to great insights in globin gene regulation and the identification of a number of potential inducers of fetal hemoglobin. In this study we describe the development of enhanced green fluorescence protein (EGFP) reporter systems based on bacterial artificial chromosomes (EBACs) to monitor the activity of the epsilon-, (G)gamma-, (A)gamma-, delta-, and beta-globin genes in the beta-globin locus. Additionally, we demonstrate that transfection of erythroleukemia cells with our EBACs is greatly enhanced by expression of EBNA1, which also facilitates episomal maintenance of our constructs in human cells. Our studies in human cells have shown physiologically relevant differences in the expression of each of the globin genes and also demonstrate that hemin is a potent inducer of EGFP expression from EGFP-modified epsilon-, (G)gamma-, and (A)gamma-globin constructs. In contrast, the EGFP-modified delta- and beta-globin constructs consistently produced much lower levels of EGFP expression on hemin induction, mirroring the in vivo ontogeny. The EGFP-modified beta-globin eukaryotic BAC (EBAC) vector system can thus be used in erythroleukemia cells to evaluate induction of the epsilon- and gamma-globin genes from the intact human beta-globin locus.

Chromatin structure and control of beta-like globin gene switching.

The human beta-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional beta-like globin genes, epsilon, (G)gamma, (A)gamma, delta, and beta, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNaseI-hypersensitive sites located 6-22 Kb upstream of the epsilon -globin gene. During ontogeny two switches occur in beta-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic epsilon - to fetal gamma-globin occurs at six weeks of gestation. The second switch from gamma- to adult delta- and beta-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.

Cloning MafF by Recognition Site Screening with the NFE2 Tandem Repeat of HS2: Analysis of Its Role in Globin and GCSl Genes Regulation

ABSTRACT The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human β-globin locus control region is required for high level globin gene expression. We used an oligonucleotide of the NF-E2 tandem repeat, within HS2, as recognition site probe to screen a K562 cDNA library for interacting transcription factors. A 2.3 kb full length cDNA encoding the b-zip transcription factor MafF was isolated. MafF can form both homodimers and high affinity heterodimers with Nrf1, Nrf2 and Nf-E2, three members of the CNC-bZip family. Despite obvious structural similarities with the other small Maf proteins, MafF differs in its tissue distribution and its inability to repress transcription when overexpressed as homodimer. In fact, in different cell lines and on different promoters (γ-globin, β-globin and glutamylcysteine synthetase genes) the MafF homodimers do not appreciably affect transcription of target promoters, whereas MafF/CNC member heterodimers act as weak transcriptional activators. Even though MafF was cloned using probes derived from the globin LCR, it is in the context of the GCSl promoter and in combination with Jun that MafF shows a rather distinct and specific regulatory role. These observations suggest that a complex network of small Maf and CNC—AP1 protein interactions might be involved in regulating transcription in diverse tissues or developmental stages.

Sickle cell gene therapy

The autosomal recessive hemoglobinopathy sickle cell disease (SCD) has been the subject of much pioneering genetic research developed to determine the molecular basis of disease. However, turning the results of this research into a new therapeutic approach for SCD has been a long time coming.A recent study by Pawliuk et al. [1xCorrection of sickle cell disease in transgenic mouse models by gene therapy. Pawliuk, R. et al. Science. 2001; 294: 2368–2371Crossref | PubMed | Scopus (362)See all References][1] represents a major breakthrough. In this study, an optimized lentiviral vector based on HIV-1 was used to transduce murine bone marrow cells with a modified human β-globin gene, βA-T87Q, that inhibits the effect of abnormal sickle cell hemoglobin (HbS). To ensure cell- specific expression of the βA-T87Q gene, elements from the human βglobin locus control region were used to promote transcription. In normal mice, βA-T87Q-positive cells were detected in 99% of red blood cells ten months after transplantation of transduced bone marrow. Analysis of secondary transplants from these mice showed evidence for transduction of hematopoietic stem cells suggesting that long-term correction of SCD could be possible.To assess the possible clinical benefit of expressing the βA-T87Q variant, bone marrow from two different transgenic models of SCD were transduced and transplanted into normal or SCD transgenic recipients. Marked changes were seen in several clinically relevant markers in those animals that received βA-T87Q-transduced bone marrow when compared with animals that received mock-transduced bone marrow. Improvements observed included a decreased percentage of sickle cells induced under increased oxygen pressure, a change in the kinetics of HbS polymerization towards values seen in HbS heterozygous individuals and a prevention of the splenomegaly associated with one of the transgenic models of SCD.Although these findings are exciting, several issues must first be addressed before clinical studies can begin. In particular, there needs to be an extensive assessment of the safety of the lentiviral vector system and a means of ensuring efficient transduction and engraftment of transduced cells so that the highest percentage of modified red blood cells possible is obtained.