Enhancer-dependent transcriptional oscillations in mouse erythroleukemia cells.

It has long been known that expression of transfected or translocated genes is not always stable and is subject to position effects. In metazoans, the two best-studied such phenomena are heterochromatin-induced position effect variegation (PEV) (24), in which transgenes are clonally inactivated in a fraction of a cell population, and extinction of transgenes in cell culture (50) and transgenic animals (15, 35, 41, 47). We have recently developed a novel method called recombinase-mediated cassette exchange (RMCE) that allows integration of single copies of different expression cassettes at the same predetermined chromosomal locations (1). This method is ideally suited to study position effects and gene regulation because interesting chromosomal locations can be identified and then revisited by insertion of different expression cassettes. To study position effects and means to prevent them, we have used as a model components of the human β-globin gene cluster. This cluster is located on chromosome 11 and contains five genes that are sequentially expressed during development and are regulated by the locus control region (LCR) (14, 34, 44). The LCR is located 6 to 20 kb upstream of the ɛ-globin gene and consists of five DNase I-hypersensitive sites (HSs) first reported by Tuan et al. (48). The importance of the LCR for regulation of the globin genes was inferred from studies of naturally occurring deletions of the LCR that are associated with transcriptional silencing of the locus and with a switch from early to late replication and a change of chromatin conformation (13). More recent analyses of experimentally induced deletions of HS1 to HS5 on mouse chromosomes (6) and HS2 to HS5 on human chromosomes (40) revealed, however, that the locus was still sensitive to DNase I in the absence of the LCR and that in the case of the murine locus, expression was decreased but not abolished by the deletion. Taken together, these data suggest that the LCR, as currently defined, acts as an enhancer but is not required to render the chromatin sensitive to DNase I at its native location. Transfection and transgenic experiments show that the LCR confers high-level expression on the globin genes. When multiple copies of LCR-driven cassettes are integrated at ectopic loci, expression is often found to be copy number dependent and site of integration independent (19). On the basis of these experiments, it has been proposed that the LCR differs from other enhancers in having a dominant chromatin-opening activity (19). No single HS of the LCR is indispensable for transcriptional activation of the globin genes, since deletions of individual HSs introduced at the endogenous mouse locus (8, 26), in yeast artificial chromosomes (3, 38), or in cosmids containing large fragments encompassing the entire human locus (35) are associated with relatively mild phenotypes. These results have led many authors to the conclusion that the LCR acts as a holocomplex composed of all of the HSs. Nevertheless, HS2, HS3, HS4, and, to a lesser extent, HS1 have enhancer activity when tested individually in cell culture and in transgenic mice (reviewed in reference 18). In a previous study (1), RMCE was used to investigate the function of some of the HSs constituting the LCR by integrating five expression cassettes in the genome of mouse erythroleukemia (MEL) cells at the same randomly chosen chromosomal location. This chromosomal location was termed RL1, for random locus 1. The cassettes studied included the β-globZ gene (the human β-globin promoter fused to lacZ coding sequences) alone or driven by HS2, HS3, or HS234 (a large LCR fragment containing HS2, HS3, and HS4) of the LCR (Fig. ​(Fig.1a).1a). This revealed that expression of these five cassettes occurred in mosaic patterns reminiscent of PEV (1). FIG. 1 Mosaic expression patterns at RL1 in the presence of various cassettes. (a) Cassette integrated at RL1. The HYG cassette contains the PGK-HYG gene and the Lox sites that are required for RMCE. All other cassettes were exchanged with the PGK-HYG gene by ... These mosaic expression patterns appeared particularly interesting because the proportion of expressing cells was dependent on the HS and on the number of genes present at the locus. In the present study this phenomenon has been further investigated and found to be clearly different from PEV and to be caused by dynamic activation and inactivation of β-globZ resulting in oscillations of expression. The oscillations occur once every few cell cycles at a rate specific to the enhancer present at the locus. We have also found that treatment of the cells with trichostatin A (TSA) (51) eliminates the oscillations in a cell cycle-dependent manner. Oscillations at a second locus were also observed.