The beta‐globin locus control region (LCR) confers high levels of position‐independent, copy number‐dependent expression onto globin transgenes. Here > 40 independent transgenic mouse lines and founders that carried the LCR in cis with the beta‐globin gene promoter driving a lacZ reporter gene were studied. Expression of the lacZ transgene was assayed by measuring beta‐galactosidase enzyme activity in fetal liver extracts, the levels of which correlated with the quantity of lacZ mRNA determined using RNase protection assays. Unexpectedly, expression of the lacZ transgene was found to show strong position effects, varying as much as 700‐fold per transgene copy. These position effects occurred even if the whole beta‐globin gene was incorporated as part of the lacZ reporter gene. Moreover, DNase I‐hypersensitive sites appeared in the transgene LCR in high expressing but not in low expressing lines, suggesting that the LCR itself was position dependent. In contrast, MEL cell clones, in which transcriptionally active integration sites were selected for, gave < 13‐fold variation in expression per copy of an LCR‐lacZ construct. These results show that the lacZ reporter affects the ability of the LCR to activate chromatin in mice and that culture cells are not an adequate model for position‐independent gene expression studies.

To define DNA regions involved in the neuron-specific expression of the neurofilament light (NF-L) gene, we generated transgenic mice bearing different NF-L constructs. A 4.9-kilobase human NF-L fragment including ؊292 base pairs of 5-flanking sequences contained sufficient elements for nervous system expression in transgenic mice. Deletion of introns 1 and 2 from this 4.9-kilobase DNA fragment resulted in reduced levels of transgene expression in the cortex, while deletion of intron 3 had little effect. Both introns 1 and 2 could act independently as enhancers to confer neuronal expression of the basal heat shock promoter (hsp68) fused to lacZ in transgenic mice. The hNF-L basal promoter (؊292 base pairs) was found to contain elements for directing neuronal expression of either the lacZ reporter gene or an intronless hNF-L construct. Sequence comparison revealed that intron 1, intron 2, and the basal human NF-L promoter all contain an ETS-like motif, CAGGA, present in a variety of genes expressed in the nervous system.Neurofilaments are formed by the copolymerization of three neuron-specific proteins with an apparent molecular mass on SDS gel of 70 kDa (NF-L), 150 kDa (NF-M), and 200 kDa (NF-H) (1-3). The genes coding for the three NF 1 proteins have been cloned and sequenced (4 -10). Like other intermediate filaments (IF) genes, NF genes are expressed in a cell typespecific and developmental manner (11-13). IF genes, from class IV and VI, are differentially regulated during the development of neuronal progenitor cells (5, 9, 14 -16).Neurofilaments are expressed in most neurons of the nervous system, and their expression coincides with terminal neuronal differentiation. There is growing evidence that deregulation of neurofilament expression may play a central role in motor neuron disease. Transgenic mice that overexpress neurofilament proteins show motor neuron degeneration (17-21). Furthermore, the levels of NF-L mRNA are decreased in dogs with hereditary canine spinal muscular atrophy (22) as well as in motor neurons of patients with amyotrophic lateral sclerosis (23). Yet, little is known about the mechanisms that regulate expression of neurofilament genes in the nervous system. This is in part due to the lack of suitable in vitro systems to study their expression (15). For instance, high levels of NF-L expression occurred after transfection of a complete genomic NF-L gene in non-neuronal cells such as cultured fibroblasts (6, 24), even though the endogenous NF-L gene remained silent. In contrast, DNA fragments containing either the complete human or mouse NF-L genes were correctly expressed in transgenic mice (25,26).We have shown previously that a human NF-L fragment including Ϫ292 bp of 5Ј-flanking sequences and intron sequences contained sufficient elements to drive NF-L expression in the nervous tissues of adult transgenic mice (27). To further clarify the potential role of introns in modulating expression of the human NF-L gene, we used the transgenic mouse approach to test the transcriptional activity...

Acting in cis with the beta-globin locus control region, the CAAT box of the beta-globin gene promoter stimulates transcription 10-fold in murine erythroleukemia (MEL) cells but is without effect in K562 cells. Our previous studies suggested that of four proteins from MEL cells that bind to this CAAT box region (CP1, GATA-1, and two factors that were denoted DSFr and DSF1) DSFr is involved in the up-regulation of transcription. In the present report, the DSFr protein in MEL cells was identified as C/EBPgamma through expression cloning and antibody studies. C/EBPgamma DNA binding activity could not be detected in K562 cells. However, K562 cells, but not MEL cells, were found to express LIP, which is a truncated form of C/EBPbeta and is an inhibitor of transcription. Thus, the differential expression of C/EBP members could account for the ability of the beta-globin CAAT box to stimulate transcription in MEL cells, but not function in K562 cells. Juxtaposing a specific C/EBP binding sequence next to the beta-globin promoter, in constructs in which the CAAT box had been rendered inactive by mutation or deletion, restored full promoter activity in MEL cells only if CP1 still bound to the promoter. In conjunction with previous mutation analyses, these results suggest that C/EBPgamma may collaborate with CP1 to enhance transcription through the beta-globin CAAT box.

We studied transgenic mice carrying the lacZ reporter gene linked to the erythroid-specific beta-globin promoter and beta-globin locus control region (LCR). Previously, we had demonstrated that the total level of expression of beta-galactosidase enzyme, which is the product of the lacZ gene, varies widely between different transgenic mice due to position effects at the sites of transgene integration. Here, using the X-gal based in situ assay for beta-galactosidase activity, we found that the percent erythroid cells that expressed the transgene also varied widely between the mice. Moreover, a kinetic analysis showed that the average beta-galactosidase content per expressing cell varied both between samples of different transgenic descent and between erythroid cells within each sample, demonstrating that the variable expression of this lacZ transgene was being controlled in a graded manner. These results suggest that the beta-globin LCR enhancers function through a graded model, which is described, rather than the binary mechanism that has been proposed previously for other enhancers.