The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements.

We have located presumptive chromosomal loop anchorage elements within the mouse heavy chain immunoglobulin locus. Analysis of 31 kilobases spanning diversity, joining, enhancer, switch, and the mu and delta constant regions reveals that only a single 1-kilobase segment exhibits specific binding to nuclear matrices. It is of particular significance that the transcriptional enhancer element resides within this matrix association region (MAR). Fine structure mapping indicates that binding is mediated by A+T-rich approximately 350-base pair segments that reside on either side of the enhancer. The MAR sequences residing 5' of the enhancer contain topoisomerase II consensus sequences like the MAR located upstream of the kappa light chain gene enhancer. The heavy chain gene MARs, however, exhibit a lower affinity for matrix association compared to the kappa gene MAR. Significantly, the juxtaposition of enhancer elements with MARs appears to be evolutionarily conserved within the immunoglobulin genes, suggesting that MARs may act as positive and/or negative regulators of enhancer function.

conserved within the immunoglobulin genes, suggesting that MARs may act as positive and/or negative regulators of enhancer function.
DNA within interphase nuclei and mitotic chromosomes is organized into topologically constrained looped domains of about 10-100 kilobases in length (1-4). Given the chemical complexity of the mammalian genome, one can estimate that roughly lo5 chromosomal loop anchorage sites would exist in a single diploid nucleus. With these considerations in mind, a number of interesting questions can be entertained. Do anchorage sites punctuate gene clusters into functionally distinct chromatin domains? Do such sites target genes in the nucleus to specific compartments? Are common anchorage elements shared by functionally diverse genes or only by related gene families? Finally, are different regions of a given gene anchored depending on transcription, replication, development, or tissue type?
To search for presumptive chromosomal loop anchorage elements, in an earlier study we developed an in vitro DNA binding assay that localizes matrix association regions (MARs)' within cloned genes (5). This approach can be complemented by the nuclear halo mapping procedure of Laemmli * This research was supported by Grants GM22201, GM29935, and GM31689 from the National Institutes of Health and by Grant 1-823 from the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed.
The abbreviations used are: MAR or M, matrix association region; regions of immunoglobulin genes: D, diversity; V, variable; J, joining; S, switch; C, constant; H, heavy chain; E, enhancer; Top0 11, topoisomerase II; LIS, lithium diiodosalicylate; SDS, sodium dodecyl sulfate; CTAB, cetyltrimethylammonium bromide; kb or bp, kilobase or base pairs; SAR, scaffold attached region. and co-workers (6), which employs nuclear fractionation of endogeneous sequences to identify scaffold attached regions (SARs) (7). A limited comparison between these two procedures reveals that SARs appear to be strictly analogous to MARs ((5, 7 ) vide infra). Interestingly, SARs are located nonrandomly and at specific sites adjacent to a series of functionally distinct Drosophila class I1 genes (6-9). Available evidence suggests that the positions of these contact points appear not to vary with gene expression, but technical limitations preclude a definitive conclusion that anchorage is constitutive (7). Furthermore, studies on the K light chain immunoglobulin gene in mouse cells reveal a MAR adjacent to the transcriptional enhancer element that is in the same position both before and after recombination and gene expression (5). In addition, certain SARs of Drosophila genes also appear to be located near enhancer-like elements (7). Significantly, Drosophila gene SARs share a number of features in common with the mouse K gene MAR, including overall A+T richness, the presence of topoisomerase 11 consensus sequences, short clusters of certain characteristic A+T-rich sequences, and the ability to compete for abundant binding sites in mouse nuclear matrix preparations (5-9). The presence of topoisomerase I1 sites is interesting, considering that this protein is the major component of the mitotic chromosome scaffold (10-12) and is recovered in high yield in certain nuclear matrix preparations (13). Whether topoisomerase I1 is responsible for anchoring these sequences at the base of chromosomal loops in the interphase nucleus in uiuo, however, remains to be demonstrated.
The juxtaposition of the K gene enhancer with a MAR that contains topoisomerase I1 sites suggests that a functional relationship may exist between transcriptional enhancement and DNA swiveling at the base of chromosomal loops ( 5 ) . Consistent with this view is the apparent in uivo localization of topoisomerase I1 adjacent to the SV40 enhancer (14) and the in vitro activation of dynamic supercoiling of the Xenopus 5 S gene by trans-acting factors (15). In the present study we address these issues further by an analysis of the mouse heavy chain immunoglobulin locus. Previous studies have identified a powerful tissue-specific enhancer (EH) in the intron between the joining (JH) and switch recombination sequences (S,) just upstream of ~1 constant region exons (C,) (16, 17) (for review of immunoglobulin genes, see Ref. 18). We show here that this enhancer is flanked on both sides by presumptive chromosomal loop anchorage elements. The 5' MAR appears similar to the K gene MAR as it contains topoisomerase I1 sites. Therefore, the heavy and light chain mouse immunoglobulin genes have a strikingly similar DNA sequence organization with respect to MARs and enhancers.

RESULTS AND DISCUSSION^
Portions of this paper (including "Experimental Procedures,"

Regions within the Heavy Chain Locus
, .