A 46 base pair enhancer sequence within the locus activating region is required for induced expression of the gamma-globin gene during erythroid differentiation.

The locus activating region (LAR), contained within 30 kb of chromatin flanking the human beta-globin gene cluster, has recently been shown to be essential for high level beta-globin gene expression. To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma-globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNaseI hypersensitive site II (HS II), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression. We also showed that a 46 bp enhancer element within HS II was necessary and sufficient for the increased gamma-globin gene expression observed with hemin induced erythroid maturation of K562 cells. These results localize a distant regulatory element important for activation of globin genes during human erythroid cell maturation.     

Transcriptional potential of the gamma-globin gene is dependent on the CACCC box in a developmental stage-specific manner

To test the role of CACCC box on gamma-globin gene activation, the CACCC box was deleted or mutated and gamma-gene expression was monitored in transgenic mice. Disruption of the CACCC box had no effect on gamma-gene expression in the cells of embryonic erythropoiesis but it strikingly reduced gamma-gene expression in fetal erythropoiesis, and abolished gamma-gene expression in adult erythroid cells. The CACCC mutation diminished HS formation, as well as TBP and polII recruitment at the gamma-gene promoter; however, it only resulted in slight or no effects on histone H3 and H4 acetylation in adult erythropoiesis. Our findings indicate that each basic cis element of the proximal gamma-gene promoter, i.e. CACCC, CCAAT or TATA box, can be disrupted without affecting the activation of gamma gene in embryonic erythroid cells. We propose that the trans factors recruited by the three boxes interact with each other to form a 'promoter complex'. In embryonic erythropoiesis the locus control region enhancer is able to interact with the complex even when components normally binding to one of the motifs are missing, but it can only activate an intact 'promoter complex' in adult erythroid cells.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues.     

Evolutionary conservation of KLF transcription factors and functional conservation of human gamma-globin gene regulation in chicken

The Krüppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins with homology to Drosophila Krüppel. KLFs can bind to CACCC elements, which are important in controlling developmental programs. The CACCC promoter element is critical for the developmental regulation of the human gamma-globin gene. In the present study, chicken homologues of the human KLF2, 3, 4, 5, 9, 11, 12, 13, and 15 genes were identified. Phylogenetic analysis confirms that these genes are more closely related to their human homologues than they are to other chicken KLFs. This work also represents the first systematic study of the expression patterns of KLFs during erythroid development. In addition, transient transfections of human globin constructs into 5-day (primitive) chicken red blood cells show that human gamma-globin expression is regulated via its CACCC promoter element. This indicates that a CACCC-binding factor(s) important for gamma-globin expression functions in 5-day chicken red cells.     

The 5'HS4 core element of the human beta-globin locus control region is required for high-level globin gene expression in definitive but not in primitive erythropoiesis.

To assess the contribution of DNase I-hypersensitive site 4 (HS4) of the beta-globin locus control region (LCR) to overall LCR function we deleted a 280 bp fragment encompassing the core element of 5'HS4 from a 248 kb beta-globin locus yeast artificial chromosome (beta-YAC) and analyzed globin gene expression during development in beta-YAC transgenic mice. Four transgenic lines were established; each contained at least one intact copy of the beta-globin locus. The deletion of the 5'HS4 core element had no effect on globin gene expression during embryonic erythropoiesis. In contrast, deletion of the 5'HS4 core resulted in a significant decrease of gamma and beta-globin gene expression during definitive erythropoiesis in the fetal liver and a decrease of beta-globin gene expression in adult blood. We conclude that the core element of 5'HS4 is required for globin gene expression only in definitive erythropoiesis. Absence of the core element of HS4 may limit the ability of the LCR to provide an open chromatin domain and/or enhance gamma and beta-globin gene expression in the adult erythroid cells.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

A negative cis-element regulates the level of enhancement by hypersensitive site 2 of the beta-globin locus control region

The core of DNase hypersensitive site (HS) 2 from the beta-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5' and 3' ends of the core. Analysis of constructs with and without the 5' negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5' end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.     

A minimal ankyrin promoter linked to a human gamma-globin gene demonstrates erythroid specific copy number dependent expression with minimal position or enhancer dependence in transgenic mice

In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.     

Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.     

人β—簇珠蛋白基因LCR调控元件中HS3与反式调控因子相互作用的研究

Upstream of the human epsilon-globin gene is the Locus Control Region (LCR) of the human beta-globin cluster, which consists of four DNase-I hypersensitive sites(HS1-HS4). It has been reported in transgenic experiments that HS3 preferentially regulates epsilon-globin gene expression. In order to elucidate the regulatory function of HS3 in the expression of globin gene, nuclear extracts from mouse hematopoietic tissues at several developmental stages were prepared and the binding of the nuclear factors to HS3 was analysed by using electrophoresis mobility shift assay(EMSA). Our results showed that the binding patterns of HS3 with nuclear extracts of mouse hematopoietic tissues at day 13 and day 18 of gestation were completely different; furthermore, by Southwestern Blot, the distinction between both stages was also demonstrated. It has been known that GATA and CACCC binding motifs are contained within HS3 core region. Using competitive gel-retardation assay, we found that no shift bands could be competed by using CACCC motif as a competitor. However one shift band at day 13 and day 18 of gestation could be competed respectively by using GATA motif as a competitor. We suggested that the shift bands, which could not be competed by both motifs, might be novel and stage-specific factors. In addition, by using Western Blot, we demonstrated that the two shift bands at day 13 and day 18 of gestation, competed by GATA motif, were GATA-2 and GATA-1 respectively: GATA-1 was expressed in mouse hematopoietic tissues at day 18 of gestation and not expressed at day 13 of gestation; however, GATA-2 was only expressed in mouse hematopoietic tissues at day 13 of gestation. According to these results, we speculated that HS3 might play an important role in regulation of stage-specific expression of globin genes through interaction between stage-specific nuclear factors and HS3.