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Locus Control Regions Coming of Age at a Decade Plus

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Locus Control Regions Coming of Age at a Decade Plus The b-globin locus control region Reviews Locus control regions coming of age at a decade plus The b-globin locus control region (LCR) is the founding member of a novel class of cis-acting regulatory elements that confer high level, tissue-specific, site-of-integration-independent, copy number-dependent expression on linked transgenes located in ectopic chromatin sites. Knowledge from b-globin and other LCR studies has shed light on our understanding of the long-range interaction between enhancers and promoters, the relationship between chromatin conformation and transcriptional regulation, and the developmental regulation of multiple gene loci. After over a decade of investigation and discovery, we take a retrospective look at the b-globin LCR and other LCRs, summarize their properties and review models of LCR function. peculation regarding the presence of a cis-acting regu- They confer tissue-specific and physiological levels of Slatory element upstream of the b-globin genes, and the expression on linked genes, activate the transcription of subsequent discovery of the locus control region (LCR), transgenes in a position-independent, copy number- was a result of inferential data obtained from several dependent manner, and determine DNA replication timing sources. First, early studies of transgenic mice carrying and the origin utilized (Box 1). LCRs are novel elements, only the human b- or g-globin gene demonstrated that distinct from promoters, classical enhancers, chromoso- gene expression was significantly lower than endogenous mal insulators and matrix-attachment regions or scaffold- b-globin gene expression1–3. Moreover, transgene expres- attachment regions (MARs or SARs). They are more sion was observed only in a small proportion of transgenic complex than enhancers and insulators; in fact, they animals, indicating that they were susceptible to position incorporate the functions of both these elements. effects. These studies implied that a regulatory element Enhancers activate transcription (Box 1), but gene expres- must be present in the native locus that was missing in sion might be absent or variable at non-physiological these constructs. Second, in a number of human b- levels. This variation has been termed position effect (PE) thalassemias, the region upstream of the locus is deleted, and is thought to result from the positive or negative but the globin genes are intact. These genes function nor- effects of the chromatin surrounding the transgene. Unlike mally in vitro, but are silent and reside in inactive enhancers, LCRs buffer against PE. Insulators function as chromatin in vivo, suggesting that a regulatory element is boundary elements of chromatin structure, preventing PEs missing in the mutant loci4,5. Third, the existence of a regu- associated with transgenes without enhancing transcrip- latory element was suggested by the presence of develop- tion. In contrast to insulators, LCRs activate gene expres- mentally stable, erythroid-specific DNAse I-hypersensitive sion and can also contain MARs as part of their collection Qiliang Li* 9 e 6 sites (HSs) located 5 of the -globin gene . The functional of elements. li111640@u. involvement of these HSs in globin gene expression was washington.edu implied from studies of somatic cell hybrids7. When the Transcription-enhancer activity + chromosome carrying the b-globin locus in non-erythroid In the absence of the LCR, the transcription of the gene Susanna Harju [email protected] cells, where the HSs are normally absent, was introduced encoding human b-globin is usually less than 1% of the into an erythroid cell environment, the HSs were formed. murine b-globin mRNA in transgenic mice, if it is Kenneth R. Peterson+o Finally, studies in transgenic mice provided positive evi- expressed at all1–3. Inclusion of the LCR increases the [email protected] dence that this region conferred erythroid-specific, high- expression of the human b-globin gene to a level compara- *Division of Medical level expression upon a linked b-globin transgene in a ble with endogenous b-globin gene expression in all trans- Genetics, Mail Box position-independent, copy number-dependent manner8. genic animals, indicating that the LCR has strong 357720, Department of The b-globin LCR is located upstream of the b-globin enhancer activity8. LCR enhancer activity is also signifi- Medicine, University of gene and consists of five DNAse I-hypersensitive sites cant in situ, as demonstrated by LCR-deletion experi- Washington, Seattle, (59HS1–5) (Fig. 1). 59HS 1–4 are erythroid-specific, but ments9,10. These deletions in the native chromosome of WA 98195, USA. 59HS5 is not. mouse or human cell lines severely reduce the expression + of globin genes. Department of Properties of LCRs The enhancer activity of the LCR resides in 59HS2, 3 Biochemistry and 9 Molecular Biology and Technically, DNA fragments that are capable of enhanc- and 4, but not in 5 HS1 or 5 (Ref. 11 and references o 9 Department of Anatomy ing the expression of a linked gene that is integrated in therein). 5 HS2 behaves as a classical enhancer, in that its and Cell Biology, ectopic chromatin sites in a copy number-dependent man- activity can be detected in transient transfection assays. University of Kansas ner in transgenic mice are grouped together as LCRs. Enhancer activity in 59HS3 or 4 can be detected only when Medical Center, 3901 LCRs are powerful genetic regulatory elements that are they are integrated into chromatin. A requirement for Rainbow Blvd, Kansas characterized phenotypically by three major properties. chromosomal integration suggests that alteration of City, KS 66160, USA. 0168-9525/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01780-1 TIG October 1999, volume 15, No. 10 403 Reviews The b-globin locus control region FIGURE 1. The human β-globin locus BOX 1. Cis-acting transcriptional regulatory elements (DCR) Locus control region (LCR) (LAR) Opens locus chromatin domains LCR Insulates against effects of surrounding positive or negative chromatin Has cell lineage-specific enhancer activity 5′HS 54 32 1 3′HS1 Influences timing of replication and choice of origin utilized ε Gγ Aγ ψβ δ β Enhancer Stimulates transcription in an orientation-independent manner (V)(IV)(III)(II)(I) (1) (2)(3)(4) Classical enhancers function independently of orientation and distance Hispanic deletion 10 kb Insulator Human LCR deletion Creates independent functional domain without enhancement or Mouse LCR deletion trends in Genetics activation function by blocking the effects of surrounding positive or negative chromatin The locus consists of five functional genes, indicated as white boxes, arrayed in their order of Interrupts communication between a promoter and another regulatory developmental expression, 59-e-Gg-Ag-d-b-39. There are two developmental switches in globin chain element when placed between them synthesis coincident with changes in site and type of erythropoiesis. During primitive erythropoiesis, the e-globin gene is expressed in the embryonic yolk sac. The first switch occurs at approximately Promoter eight weeks gestation; the e-globin gene is silenced and the Gg- and Ag-globin genes are expressed Region of DNA at which RNA polymerase binds and initiates transcription during definitive erythropoiesis in the fetal liver. The second switch occurs shortly after birth; the g- globin genes are silenced and the b-globin gene and, to a lesser extent, the d-globin gene, are Matrix attachment region (MAR) or scaffold attachment activated in the bone marrow. 59HS1–5 are located 26, 211, 215, 218 and 222 kb relative to the region (SAR) e-globin gene, respectively, and are indicated by arrows. Another HS is located 20 kb downstream of DNA segment that might bind the nuclear scaffold the b-globin gene (39HS1). 39HS1 is found only in erythroid cells; its function remains unclear. A DNA loop between two MARs might form an independent chromosomal Transcription start sites for the functional globin genes are at the left edge of the white boxes in the domain diagram. The LCR and its HSs were named differently in early literature. At the Seventh Conference on Has no enhancer activity Hemoglobin Switching held at Airlie House, Virginia in 1990, senior participants agreed upon a uniform nomenclature. The Locus Control Region (LCR) was the compromise name to be used in place of Locus Activation Region (LAR) or Dominant Control Region (DCR). A standard numbering system for Copy number-dependent expression and chromatin- the individual HSs was adapted. The obsolete names and numbering for HSs are shown in domain opening parenthesis. This should be helpful to those reading earlier LCR-related publications. Lines below the Another property of the LCR is its ability to confer diagram of the locus indicate deletions of the LCR discussed in the text. The Hispanic deletion, which position-independent, copy number-dependent expression causes (gdb)0 thalassemia in humans, extends an additional 20 kb 59 of the LCR 59HS5 (Ref. 20). on a linked gene8. Copy number-dependent expression is widely considered to be indicative of open chromatin chromatin structure is involved in propagating the structure; that is, DNA that is accessible to transcription enhancer activity of 59HS3 or 4. The functions of 59HS1 factors. Involvement of the LCR in creating open chro- and 5 remains to be identified. matin was suggested from the analysis of the naturally Like classical enhancers, individual HS activity is inde- occurring Hispanic b-thalassemia deletion. In this condi- pendent of orientation. However, unlike classical enhancers, tion, a region upstream of 59HS1 of approximately 35 kb LCR activity is orientation-dependent12 and distance- is deleted, but the remainder of the globin locus is intact. dependent13–15, suggesting that the LCR does not act as a However, none of the globin genes are expressed. The simple enhancer of gene transcription. A proximal gene is deletion produces a closed chromatin conformation that more likely to be transcribed than a distal one.
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