Both locus control region and proximal regulatory elements direct the developmental regulation of beta-globin gene cluster

Using ligation-mediated PCR and in vivo footprinting methods to study the status of DNA-protein interaction at hypersensitive site 2 of locus control region and beta(maj) promoter of erythroid cells of fetal liver and adult bone marrow, we found that during different developmental periods, the status of DNA-protein interaction at both hypersensitive site 2 and beta(maj) promoter changed significantly, and indicated that locus control region might function through a looping mechanism to regulate the expression of downstream genes, and that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of beta-globin gene cluster participate in the regulation of developmental specificity.     

Primary structure of the goat beta-globin locus control region

The goat beta-globin cluster is composed of a triplicated four-gene set. A locus control region (LCR) containing elements homologous to 5'DNase I hypersensitive sites (HS) 1, 2, and 3 of the human beta-globin LCR has been identified at the 5' end of this locus. We determined 10.2 kb of nucleotide sequence from the goat beta-globin locus control region. Self-comparison of this sequence by dot matrix analysis revealed the presence of six complete and three incomplete artiodactyl repeats. A novel repeated element, termed D repeat, was also identified. Southern blotting analysis demonstrated that these elements exist in the goat genome as a low to medium frequency interspersed repeat family. The absence of any other large region of self-homology (direct or inverted) in the goat LCR suggests that 5'HSs 1, 2, and 3 did not arise through duplication, but rather evolved independently. By comparing goat 5'HS 1 to those of human, rabbit, and mouse, we show a greater than 80% conservation in sequence between the four species. This level of evolutionary conservation suggests that 5'HS 1 plays an important role in the regulation of beta-globin loci.     

Structural polymorphism exhibited by a quasipalindrome present in the locus control region (LCR) of the human beta-globin gene cluster

Structural polymorphism of DNA is a widely accepted property. A simple addition to this perception has been our recent finding, where a single nucleotide polymorphism (SNP) site present in a quasipalindromic sequence of β-globin LCR exhibited a hairpin-duplex equilibrium. Our current studies explore that secondary structures adopted by individual complementary strands compete with formation of a perfect duplex. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, circular dichroism (CD) techniques, we have demonstrated the structural transitions within a perfect duplex containing 11 bp quasipalindromic stretch (TGGGG(G/C)CCCCA), to hairpins and bulge duplex forms. The extended version of the 11 bp duplex, flanked by 5 bp on both sides also demonstrated conformational equilibrium between duplex and hairpin species. Gel-electrophoresis confirms that the duplex coexists with hairpin and bulge duplex/cruciform species. Further, in CD spectra of duplexes, presence of two overlapping positive peaks at 265 and 285 nm suggest the features of A- as well as B-type DNA conformation and show oligomer concentration dependence, manifested in A → B transition. This indicates the possibility of an architectural switching at quasipalindromic region between linear duplex to a cruciform structure. Such DNA structural variations are likely to be found in the mechanics of molecular recognition and manipulation by proteins.     

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.     

A silent deletion in the beta-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.     

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.