Variegated Expression of the Endogenous Immunoglobulin Heavy-Chain Gene in the Absence of the Intronic Locus Control Region

Abstract

The expression of chromosomally integrated transgenes usually varies greatly among independent trans-fectants. This variability in transgene expression has led to the definition of locus control regions (LCRs) as elements which render expression consistent. Analyses of expression in single cells revealed that the expression of transgenes which lack an LCR is often variegated, i.e., on in some cells and off in others. In many cases, transgenes which show variegated expression were found to have inserted near the centromere. These observations have suggested that the LCR prevents variegation by blocking the inhibitory effect of heterochromatin and other repetitive-DNA-containing structures at the insertion site and have raised the question of whether the LCR plays a similar role in endogenous genes. To address this question, we have examined the effects of deleting the LCR from the immunoglobulin heavy-chain locus of a mouse hybridoma cell line in which expression of the immunoglobulin heavy-chain gene is normally highly stable. Our analysis of expression in single cells shows that deletion of this LCR resulted in variegated expression of the gene. That is, in the absence of the LCR, expression of the gene in the recombinant locus could be found in either of two epigenetically maintained, metastable states, in which transcription occurred either at the normal rate or not at all. In the absence of the LCR, the on state had a half-life of 100 cell divisions, while the half-life of the off state was 40,000 cell divisions. For recombinants with an intact LCR, the half-life of the on state exceeded 50,000 cell divisions. Our results thus indicate that the LCR increased the stability of the on state by at least 500-fold. Most genes in complex, differentiated organisms, such as metazoa, are expressed in a tissue-specific fashion, on in one subset of cells and off in others. Tissue-specific gene expression is initially established as cells in different environments are subjected to different signals. The signals are each presumed to induce the production of a distinct complement of transcription factors, which are then directed by cis-acting elements to activate or silence adjoining genes. The ontogenetic state of cells is thus considered to reflect their past and present production of transacting factors. Much of our knowledge about gene expression in complex organisms comes from studying the expression of transgenes, DNA segments which have been introduced into animals or cell lines. Early work revealed that the expression of chromo-somally integrated transgenes varies greatly, and traditionally this variability has been ascribed to the effects of the neighboring genome. The variability in transgene expression has led to the definition of locus control regions (LCRs) as elements which render expression consistent, at least among cells which are in the same ontogenetic state. Analyses of expression in single cells revealed that the expression of transgenes which lack the LCR is often variegated, i.e., cells which are presumed to be in the same ontogenetic state differ in their expression of the transgene (for a review, see reference 25). The variability in transgene expression thus reflects variations in the fraction of expressing cells. In many cases, transgenes which showed var-iegated expression were found to have inserted near the cen-tromere (11, 26). The variegated expression of transgenes is thus similar to position effect variegation in Drosophila mela-nogaster, whereby events such as chromosomal translocations move an endogenous gene into a heterochromatic region. Repeated DNA segments can themselves induce variegation (10, 14), and the variegated expression of transgenes can sometimes result from their insertion as a tandem array. Such observations have suggested that the LCR prevents variegation by blocking the inhibitory effect of heterochromatin and other repetitive-DNA-containing structures (25, 26). Most genes are located in euchromatin, raising the question of whether the LCR also serves to prevent the repression of endogenous genes. The immunoglobulin heavy chain (IgH) locus of the mouse contains an LCR which includes three distinct elements in the major (VDJ-C) intron: the E core enhancer, the matrix attachment regions (MARs) which flank E, and the switch region (S) (13, 16, 37). A system in which expression of the endogenous heavy-chain gene of a mouse hybridoma cell line depends on the integrity of this LCR has been previously described (29, 43). The data in the present analysis indicate that deletion of the LCR resulted in varie-gated expression of the gene. That is, in the absence of the LCR, expression of the gene in the recombinant locus could exist in either of two epigenetically maintained, metastable states, in which transcription occurred either at the normal rate or not at all. We have measured the rates at which cells switched between the on and off states. In the absence of the LCR, the on state had a half-life of 100 cell divisions, while the half-life of the off state was 40,000 cell divisions. Our analysis of recombinants bearing an intact LCR indicated that the LCR increased the stability of the on state by at least 500-fold.