FUNCTION OF MATRIX ATTACHMENT REGIONS IN THE IG LOCI

Scripps Research Institute, La Jolla CA

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Abstract

Matrix attachment regions (MAR) are AT-rich regions of DNA which mediate attachment of the chromosome to the nuclear matrix. MARs have been studied in a few model loci, two of which are the IgH and Ig- kappa loci. MARs flank both sides of the heavy chain E-mu intronic enhancer, are adjacent to some of the constant regions, and a MAR is located 5' of the kappa chain intronic enhancer. In addition, a MAR upstream of one VH gene has been described. Several studies have been performed on the Ig-kappa and IgH intronic enhancer-associated MARs as transgenes, stable integrants in cell lines, and recently gene targeting, and these studies suggest a role for the MARS in transcriptional enhancement, chromatin remodeling, demethylation, gene rearrangement and somatic hypermutation. We have found that many IgH and Ig-kappa V genes have AT-rich MAR sequences upstream of their promoters. These MARs bind nuclear matrix preparations and also bind the regulatory proteins SATB1, Cux/CDP and Bright in EMSAs. Here, we will investigate the role of these V region promoter MARs. First, we will characterize in detail the VH and V-kappa flanking MARs. We will analyze extended regions of flanking DNA for several human and murine VH genes for nuclear matrix binding, and binding to Cux/CDP and Bright. We will determine if there is a correlation between functional vs. non-functional V genes in the relative proximity of the MARs to the promoters. We will delete the MAR region (including the binding sites for Cux/CDP and Bright) from a VH gene promoter by gene targeting, and will analyze the resulting mice for (l) ability to undergo V(D)J rearrangement, (2) any evidence for aberrant regulation of V(D)J recombination, (3) production of germline transcripts from that V gene, (4) Ig transcription at the resting B cell stage, (5) antibody response to T dependent and independent antigenic stimulation, (6) somatic hypermutation, and other aspects of B cell development. We will determine if the effects are due to elimination of matrix binding, or to elimination of binding sites for these important regulatory proteins. These studies will hopefully elucidate the role(s) of these VH flanking MARs, and will determine which stage(s) and which function(s) in B cell differentiation are affected by them.