The evidence that the human P-globin locus control region is a novel regulatory region, with properties distinct from classical enhancers, is not conclusive, but new approaches should help elucidate its real function.

The human j3globin locus comprises Iive linked, func- tional @like globin genes spanning roughly 50 kb on chromosome 11. The j%globin genes are arranged in the order in which they are expressed during development: expression switches from embryonic (E) in the yolk sac, to fetal (y) during intra-uterine life, and to adult (6 and j3) after birth. The way in which j3globin gene switching is regulated during development is still not completely understood. A region of chromosome 11 upstream of the E-globin gene, which has come to be known as the j3globin lo- cus control region (LCR), has been suggested to play a key part in regulating the transcription, replication timing and chromatin structure of the entire j3globin domain (reviewed in [ 1,2]). The LCR is operationally defined by Iive adjacent sites that are hypersensitive to cleavage by DNase I. This region was originalIy implicated in the con- trol of expression of all the p-globin genes by deletions of the region that result in failure to activate the linked globin genes [l--3]. Thus Hispanic (y@)-thalassemia was shown to be due to a 35-kb deletion removing four of the 6ve LCR hypersensitive sites (see Fig. l), resulting in inactivation of the erythroid-specific, DNase I-sensitive chromatin domain, and a change in replication timing of the locus from early to late in S-phase [3]. high level expression conferred by the LCR appears to be independent of chromosomal position, a Iinding that has been interpreted to suggest that the LCR is resistant to the ‘position effects’ observed for several classical en- hancers. For example, Grosveld and colleagues [4] re- ported that the human @globin gene linked to LCR is expressed in transgenic mice at levels comparable to those of the endogenous mouse P-globin genes, and that the expression level is linearly proportional to the num- ber of copies of the LCR-linked human P-globin gene in the host genome. Although several laboratories have confirmed the ‘position-independent’, red cell-specific expression of LCR-linked genes, the question of whether the LCR confers copy-number-dependent expression remains unresolved. The experiments in which LCR function has been in- vestigated in transfected cells and transgenic mice are, however, subject to a number of limitations. First, in all of the experiments the spatial relationship between the LCR and the reporter gene to which it is linked tiers from that between the LCR and the j3-globin genes in the natural context of the human p-globin locus. It has thus not been definitively established that the LCR enhances transcription of the downstream globin genes from its natural position. It has been argued that the LCR is a novel regulatory Second, the experiments leading to suggestion region with properties distinct from classical enhancers, the LCR confers position-Independent expression may be which allow it to have a profound influence on the entire confounded by an effect of the LCR on way a con- j3-globii gene domain. The basis for this lies in experi- struct integrates into a genome, increasing the chance of ments with transfected cells and transgenic mice, show- integration Into already ‘active’ regions. To prove that this ing that the LCR confers high levels of expression in is not a problem requires detailed analysis of sites erythroid cells on genes to which it is linked. Although integration with and without an LCR Furthermore, even the LCR does contain a classic enhancer element (that if constructs preferentially integrate into active regions is, it increases expression of a linked gene in transient whether or not they include an LCR, it may be that the assays), several LCR elements confer high level expres- LCR simply acts to prevent the developmentally-regulated sion only after Integration into cellular chromatin. The inactivation of chromatin at the integration site in the ery-