Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures, the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay" to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp7O promoter:lacZ fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements are capable of blocking the action of the yp-l enhancer when placed between it and the hsp7O promoter. In contrast, a MAR/SAR DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.In eukaryotes the cis-acting elements controlling gene expression include not only the sequences that surround the transcription start site and define the promoter but also additional auxiliary elements, enhancers and silencers, which modulate the activity of the promoter in response to specific developmental cues. Unlike the cis-acting sequences contained within the promoter, enhancers and silencers can be located both 5' and 3' relative to the transcription start site and in either orientation (1, 29). In the yeast Saccharomyces cerevisiae, the auxiliary regulatory elements, the upstream UAS, are usually located rather close to the promoter to effectively modulate gene activity (12). In contrast, the regulatory elements of higher eukaryotes are often able to exert their effects over considerable distances. For example, in Drosophila melanogaster the regulatory elements of complex developmental loci like bithorax, antennapedia, or decapentaplegic can control promoter activity from distances as great as 25 to 50 kb (4,14,16,19,22,26).In addition to modulating gene activity from distant sites, these auxiliary regulatory elements are also capable of interacting with more than one promoter. For example, Garabedian and coworkers (9, 10) have shown that the sex-, stage-, and tissue-specific expression of the divergently transcribed yolk protein genes of D. melanogaster, yp-1 and yp-2, are controlled by enhancer elements which act on both genes. One enhancer directs expression of yp-l and yp-2 in the adult female fat body, while the other directs expression in the ovary (18). The yp-l enhancer can activate not only the yp-J and yp-2 promoters but also a heterologous promoter. T...