High level expression in mice of transgenes derived from the immunoglobulin heavy chain (IgH) locus requires both the core enhancer (E) and the matrix attachment regions (MARs) that flank E. The need for both elements implies that they each perform a different function in transcription. While it is generally assumed that expression of the endogenous IgH locus has similar requirements, it has been difficult to assess the role of these elements in expression of the endogenous heavy chain gene,

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... ause B cell development and IgH expression are strongly interdependent and also because the locus contains other redundant activating elements. We have previously described a gene-targeting approach in hybridoma cells that overcomes the redundancy problem to yield a stable cell line in which expression of the IgH locus depends strongly on elements in the MAR-E-MAR segment. Using this system, we have found that expression of the endogenous gene persists at substantially (ϳ50%) normal levels in recombinants which retain either the MARs or E. That is, despite the dissimilar biochemical activities of these two elements, either one is sufficient to maintain high level expression of the endogenous locus. These findings suggest new models for how the enhancer and MARs might collaborate in the initiation or maintenance of transcription. Expression activating elements in the immunoglobulin heavy chain (IgH) 1 locus ( Fig. 1 ) have been identified and characterized in diverse ways. An enhancer lying in the VDJ-C intron was originally detected by its capacity to stimulate transcription from linked reporter genes that were transfected into myeloma cell lines (1, 2). Experiments using transgenic cell lines from which this enhancer could be deleted after transcription had been initiated indicated that the enhancer was required to maintain high level expression (3, 4). The enhancer-bearing segment can be divided into two subregions on the basis of sequence motifs and protein-binding characteristics. Thus, multiple transcription factors that bind to charac-teristic motifs define a central core (E) region; this region is flanked by matrix attachment regions (MARs) identified by their affinity for the nuclear matrix (reviewed in Refs. 5 and 6). Both elements, E and the MARs, are required for high level expression of IgH-derived transgenes in mice, albeit not in stably transfected cell lines (7) . The switch (S) regions were first recognized as repetitive DNA segments that lie 5Ј of the exons encoding heavy chain constant regions and which are the sites of most of the breakage and rejoining events underlying the heavy chain isotype switch. These switch regions also harbor elements that stimulate expression of IgH-derived reporter genes in transgenic mice, but again this activity has not been detected in assays of stably transfected cell lines (8, 9) . That all three types of elements are required for high level expression in transgenic mice indicates that each element performs a different and necessary function in transcription. Evidence of the E-MAR collaboration can also be seen in assays of chromatin accessibility (10) and DNA demethylation (11). It is generally assumed that the expression-activating elements defined in transgene expression assays function similarly in the transcription of genes in their endogenous chromosomal loci. Thus, while the intronic MARs and E core enhancer are not needed to maintain expression of the endogenous IgH locus in some B-cell lines (12-15), this difference is generally ascribed to the presence of other, functionally redundant, activating elements present in the IgH locus but absent from the transgenes, viz. the MARs that are located 5Ј of the C␥ exons (16) and enhancers that are located 3Ј of the C␣ exons (reviewed in Ref. 17). In fact, the capacity of the intronic elements to contribute to IgH expression can be observed under conditions that ablate this redundancy. Thus, targeted replacement of one of these 3Ј E␣ enhancers by the neo gene extinguishes IgH expression specifically in those myeloma cells that lack the intronic activating elements (18). We have previously described a related method of ablating redundancy in which introduction of the gpt cassette 3Ј of the endogenous heavy chain gene of hybridoma cells apparently insulates the transcription unit from other activating elements and renders expression dependent on the intronic activating elements (6). That is, in this configuration, deletion of the 5Ј-MAR-E-3Ј-MAR segment depresses expression to ϳ2% the normal level (6). Although the mechanism by which the gpt cassette renders the gene expression dependent on the intronic elements is unknown, this approach offers the possibility of examining how these elements function in a reproducible and nearly normal context. Thus, using this system we showed that recombinants that retain a substantial segment of the MARs maintain expression at a high (ϳ60% normal) level. In the context of models in which expression requires both MAR and E functions, this result implies that other enhancers in