Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.     

Functional organization of the hepatitis B virus enhancer.

We have studied the functional constituents of the hepatitis B virus enhancer in a number of cell lines. The sequence of this enhancer, being embedded within an open reading frame of the virus, is in part evolutionarily frozen and therefore serves as a good model to investigate the fundamental enhancer elements. The hepatitis B virus enhancer contains three functionally important DNA sequence elements, EP, E, and NF-1a, each of which is bound by a distinct protein(s). The synergistic action of these elements accounts for all of the enhancer activity in a nonliver cell line and for most, but not all, of the activity in liver-derived cell lines. Multimers of the E but not of the EP element act as an autonomous enhancer. Conversely, a single element of either the E or the NF-1a element can act only when linked to the EP element. These results suggest that EP is a crucial enhancer element that acts only in interaction with a second enhancer element with intrinsic enhancer activity. Interestingly, a highly similar enhancer structure is found in a number of distinct viruses.     

Exploring Functional Redundancy in the Immunoglobulin μ Heavy-Chain Gene Enhancer

Immunoglobulin (Ig) μ heavy-chain gene enhancer activity is mediated by multiple DNA binding proteins. Mutations of several protein binding sites in the enhancer do not affect enhancer activity significantly. This feature, termed redundancy, is thought to be due to functional compensation of the mutated sites by other elements within the enhancer. In this study, we identified the elements that make the basic helix-loop-helix (bHLH) protein binding sites, μE2 and μE3, redundant. The major compensatory element is a binding site for interferon regulatory factors (IRFs) and not one of several other bHLH protein binding sites. These studies also provide the first evidence for a role of IRF proteins in Ig heavy-chain gene expression. In addition, we reconstituted the activity of a monomeric μ enhancer in nonlymphoid cells and defined the domains of the ETS gene required for function.     

Discrete elements within the SV40 enhancer region display different cell-specific enhancer activities.

The SV40 enhancer contains three genetically defined elements, called A, B and C, that can functionally compensate for one another. By using short, synthetic DNA oligonucleotides, we show that each of these elements can act autonomously as an enhancer when present as multiple tandem copies. Analysis of a progressive series of B element oligomers shows a single element is ineffective as an enhancer and that the activity of two or more elements increases with copy number. Assay in five different cell lines of two separate enhancers containing six tandem copies of either the B or C element shows that these elements possess different cell-specific activities. Parallel oligomer enhancer constructs containing closely spaced double point mutations display no enhancer activity in any of the cell lines tested, indicating that these elements represent single units of enhancer function. These elements contain either a 'core' or 'octamer' consensus sequence but these consensus sequences alone are not sufficient for enhancer activity. The different cell-specific activities of the B and C elements are consistent with functional interactions with different trans-acting factors. We discuss how tandem duplication of such dissimilar elements, as in the wild-type SV40 72-bp repeats, can serve to expand the conditions under which an enhancer can function.     

Murine hoxd4 expression in the CNS requires multiple elements including a retinoic acid response element

We have identified a retinoic acid response element (RARE) within a neural enhancer located 3' to the Hoxd4 gene. This RARE is required for the initiation and maintenance of Hoxd4 transgene expression in neurectoderm, and for full anteriorized expression upon retinoic acid (RA) treatment. Mutations within the sequence TTTTCTG, located 2 bp downstream of the RARE, posteriorized transgene activity. However, the onset of transgene expression and its response to RA were indistinguishable from wild type. While the TTTTCTG motif resembles a CDX binding site, human CDX1 protein did not interact with this element in vitro. Three additional regions were also shown to control transgene expression in neurectoderm, establishing that multiple elements constitute the Hoxd4 neural enhancer.