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 beta-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.

Participation of the human beta-globin locus control region in initiation of DNA replication.

The human beta-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 beta-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.

Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis.

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.

nti glucocorticoid receptor transcripts lack sequences encoding the amino-terminal transcriptional modulatory domain.

Glucocorticoid induction of cell death (apoptosis) in mouse lymphoma S49 cells has long been studied as a molecular genetic model of steroid hormone action. To better understand the transcriptional control of glucocorticoid-induced S49 cell death, we isolated and characterized glucocorticoid receptor (GR) cDNA from two steroid-resistant nti S49 mutant cell lines (S49.55R and S49.143R) and the wild-type parental line (S49.A2). Our data reveal that nti GR transcripts encode intact steroid- and DNA-binding domains but lack 404 amino-terminal residues as a result of aberrant RNA splicing between exons 1 and 3. Results from transient cotransfection experiments into CV1 cells using nti receptor expression plasmids and a glucocorticoid-responsive reporter gene demonstrated that the truncated nti receptor exhibits a reduced transcriptional regulatory activity. Gene fusions containing portions of both the wild-type and the nti GR-coding sequences were constructed and used to functionally map the nti receptor mutation. We found that the loss of the modulatory domain alone is sufficient to cause the observed defect in nti transcriptional transactivation. These results support the proposal that glucocorticoid-induced S49 cell death requires GR sequences which have previously been shown to be required for transcriptional regulation, suggesting that steroid-regulated apoptosis is controlled at the level of gene expression.

Elevated glutathione S-transferase gene expression is an early event during steroid-induced lymphocyte apoptosis.

Based on the finding that glutathione S-transferase Yb1 (GST) gene expression is elevated in the regressing prostate of androgen-ablated rats, we analyzed GST transcript levels during steroid-induced lymphocyte cell death. It was found that GST gene expression was induced in steroid-sensitive cells within 4 hr of dexamethasone treatment, required functional glucocorticoid receptor, and was dose-dependent with regard to hormone. GST expression was not induced in an apoptosis-defective variant that contained normal levels of functional receptor, indicating that GST up-regulation was the result of secondary events that occur during steroid-mediated apoptosis. Using the calcium ionophore A23817 to induce lymphocyte cell death, GST RNA levels were increased in both steroid-sensitive and steroid-resistant cell lines, supporting the conclusion that elevated GST expression was the result of cellular processes associated with apoptosis, rather than a direct consequence of steroid-mediated transcriptional control. The cells were also treated with dibutyryl cAMP to cause cell death; however, this mode of killing did not result in GST up-regulation. Taken together, these results suggest that GST induction in dexamethasone-treated T-lymphocytes occurs early in the steroid-regulated apoptotic pathway and that this may be a marker of calcium-stimulated cell death. Based on the known function of GST as an antioxidant defense enzyme and its transcriptional regulation by reactive oxygen intermediates, we propose that the gene product of a primary GR target gene(s) directly or indirectly effects the redox state of the cell. Thus activation of GST gene expression in apoptotic lymphocytes is likely a indicator of oxidative stress, rather than a required step in the pathway.