Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression.

We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.     

Gm typing by immunoglobulin heavy-chain gene RFLP analysis.

This study was undertaken to investigate a means of assigning Gm allotypes to Caucasians by RFLP analysis. A single immunoglobulin heavy-chain gamma-4 cDNA probe (HU gamma 4) was hybridized with genomic DNA digested separately with two restriction enzymes, TaqI and PvuII. Results showed excellent correlation (P less than .001) between serologically defined Gm allotypes G1m(1), G1m(2), G2m(23), and G1m;G3m (3;5,10) and RFLPs identified with the (HU gamma 4) probe. We conclude that it is now possible to define common Gm haplotypes in Caucasians by RFLP analysis. This method provides a useful adjunct to serological allotyping and indeed has several important advantages over traditional serology: it allows confident Gm assignment and the definition of homozygous and heterozygous Gm arrangements, is highly reproducible, and is readily executed in any molecular genetic laboratory.     

Study of long-distance functional interactions between Su(Hw) insulators that can regulate enhancer-promoter communication in Drosophila melanogaster

The Su(Hw) insulator found in the gypsy retrotransposon is the most potent enhancer blocker in Drosophila melanogaster. However, two such insulators in tandem do not prevent enhancer-promoter communication, apparently because of their pairing interaction that results in mutual neutralization. Furthering our studies of the role of insulators in the control of gene expression, here we present a functional analysis of a large set of transgenic constructs with various arrangements of regulatory elements, including two or three insulators. We demonstrate that their interplay can have quite different outcomes depending on the order of and distance between elements. Thus, insulators can interact with each other over considerable distances, across interposed enhancers or promoters and coding sequences, whereby enhancer blocking may be attenuated, cancelled, or restored. Some inferences concerning the possible modes of insulator action are made from collating the new data and the relevant literature, with tentative schemes illustrating the regulatory situations in particular model constructs.     

Evolution of the rat immunoglobulin gamma heavy-chain gene family

The sequences of the four immunoglobulin gamma heavy chains of the rat (gamma 1, gamma 2a, gamma 2b, gamma 2c) have been determined. These sequences reveal that the rat genes have evolved differently from the closely related mouse gamma genes (gamma 1, gamma 2a, gamma 2b, gamma 3): in rat two of the four genes (gamma 2a and gamma 1) are 94% homologous to each other and best resemble the single mouse gamma 1 gene. Rat gamma 2b is equivalent to the mouse gamma 2a/gamma 2b pair as regards both nucleotide sequence and antibody effector functions whilst rat gamma 2c resembles mouse gamma 3. In evolutionary terms this suggests the existence of a set of three common C gamma genes before separation of rat and mouse as individual species. In addition, two independent duplication events must have occurred after species separation affecting different constant regions; this yielded rat gamma 2a and gamma 1 as a recently evolved pair and mouse gamma 2a and gamma 2b as a different pair. Furthermore, the sequence comparisons reveal several other features of interest; rat IgG2b lacks two amino acids in CH1 which are conserved in all other sequenced gamma chains. Residues believed to be essential for monocyte interaction (FcRI) are retained only in rat gamma 2b and not in the other rat gamma genes whilst a particular motif involved in C1q interaction shows a variation in both rat IgG1 and rat IgG2a which has not been observed previously.     

Polymorphisms of the immunoglobulin heavy-chain delta gene and association with other constant-region genes.

Polymorphisms have previously been reported for the C mu, C alpha, C epsilon, and C gamma genes of the immunoglobulin heavy-chain (IGH) gene cluster. Here we report polymorphisms of the IGH C delta gene region, observed using the enzymes ApaI, AvaII, TaqI, and XbaI. The TaqI and XbaI polymorphisms were used in an investigation of linkage disequilibrium throughout the cluster of constant-region genes. The TaqI polymorphism, located 5' to the C delta gene, is in linkage disequilibrium with a polymorphism of the C mu switch region. The XbaI polymorphism, which is in the vicinity of the C delta 2 exon, is not strongly associated with any other polymorphisms, including the TaqI polymorphism and the Gm polymorphism of C gamma 3. Although there is a high degree of association between most genes of the IGH region, there is a lack of association between C delta and C gamma 3, which may indicate a hot spot for recombination.     

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.     

Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.     

Enhancer-promoter communication at the yellow gene of Drosophila melanogaster: diverse promoters participate in and regulate trans interactions

The many reports of trans interactions between homologous as well as nonhomologous loci in a wide variety of organisms argue that such interactions play an important role in gene regulation. The yellow locus of Drosophila is especially useful for investigating the mechanisms of trans interactions due to its ability to support transvection and the relative ease with which it can be altered by targeted gene replacement. In this study, we exploit these aspects of yellow to further our understanding of cis as well as trans forms of enhancer-promoter communication. Through the analysis of yellow alleles whose promoters have been replaced with wild-type or altered promoters from other genes, we show that mutation of single core promoter elements of two of the three heterologous promoters tested can influence whether yellow enhancers act in cis or in trans. This finding parallels observations of the yellow promoter, suggesting that the manner in which trans interactions are controlled by core promoter elements describes a general mechanism. We further demonstrate that heterologous promoters themselves can be activated in trans as well as participate in pairing-mediated insulator bypass. These results highlight the potential of diverse promoters to partake in many forms of trans interactions.