Steven Fiering

Regulation of β-globin gene expression: straightening out the locus

Abstract A casual examination of the globin literature would leave most readers with the impression that all aspects of β-globin gene regulation are controlled by the upstream locus control region (LCR). There is no clear evidence, however, that the LCR affects transcription in the β-globin locus other than by altering its topology to maintain it in a state permissive for expression of the globin genes. Developmental switching of the globin genes may be independent of the LCR, relying only on elements close to the genes and the arrangement of the genes with respect to each other.

Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids

Targeted modification of human chromosomal alleles by homologous recombination is a powerful approach to study gene function, but gene targeting in mammalian cells is an inefficient process. In contrast, gene targeting in a chicken pre-B cell line, DT40, is highly efficient. We have transferred human chromosome 11 into DT40 cells by microcell fusion, and find that the resulting hybrids are recombination-proficient. In these cells, targeting efficiencies into the chicken ovalbumin locus were >90% and into the human p-globin and Ha-ras loci were 10–15%. These modified human chromosomes can be transferred subsequently to mammalian cells for functional tests. This chromosome shuttle system allows for the efficient homologous modification of human chromosomal genes, and for subsequent phenotypic analyses of the modified alleles in different mammalian cell types

Reduced β‐Globin Gene Expression in Adult Mice Containing Deletions of Locus Control Region 5′ HS‐2 or 5′ HS‐3

Abstract: To gain insights into the functions of individual DNAse hypersensitive sites within the β globin locus control region (LCR), we deleted the endogenous 5′ HS‐2 and HS‐3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5′ HS‐2 or 5′ HS‐3 reduced the expression of the embryonic ɛy and βh1 globin genes minimally in yolk sac‐derived erythrocytes, but that both knockouts reduced the output of the adult β (β‐Major +β‐Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK‐Neo cassette was retained within either the HS‐2 or HS‐3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK‐Neo was shown to be expressed in an erythroid‐specific fashion when it was retained in the HS‐3 position. These results show that neither 5′ HS‐2 nor HS‐3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult β‐globin gene expression in adult red blood cells. Each site therefore contains some non‐redundant information that contributes to adult globin gene function.