Elliot M. Epner

Participation of the Human β-Globin Locus Control Region in Initiation of DNA Replication

The human β-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human β-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3′ to 5′ direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.

The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.

The Locus Control Region Is Necessary for Gene Expression in the Human β-Globin Locus but Not the Maintenance of an Open Chromatin Structure in Erythroid Cells

ABSTRACT Studies in many systems have led to the model that the human β-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the β-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous β-globin locus in an erythroid background, transcription of all β-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the β-globin locus are not affected by the deletion. In contrast, deletion of 5′HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on β-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an “open” conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the β-like globin genes.

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.