Expression of the (Recombinant) Endogenous Immunoglobulin Heavy-Chain Locus Requires the Intronic Matrix Attachment Regions

Abstract

The elements which regulate gene expression have traditionally been identified by their effects on reporter genes which have been transfected into cell lines or animals. It is generally assumed that these elements have a comparable role in expression of the corresponding endogenous locus. Nevertheless, several studies of immunoglobulin heavy-chain (IgH) gene expression have reported that the requirements for expressing IgH-derived transgenes differ from the requirements for expression of the endogenous IgH locus. Thus, although expression of transgenes requires multiple elements from the J H -C intron-the E core enhancer, the matrix attachment regions (MARs) which flank E, and several switch-associated elements-B-cell lines in which expression of the endogenous heavy-chain gene is maintained at the normal level in the absence of these intronic elements have occasionally been reported. Gene targeting offers an alternative method for assessing regulatory elements, one in which the role of defined segments of endogenous genes can be evaluated in situ. We have applied this approach to the IgH locus of a hybridoma cell line, generating recombinants which bear predetermined modifications in the functional, endogenous heavy-chain gene. Our analysis indicates the following. Chromatin fibers appear to exist as loops which are anchored to the nuclear matrix, or scaffold (reviewed in reference 25). The loops are thought to correspond to functional domains in which the transcriptional state of one domain is largely independent of its neighbors. It is generally considered that the sites of anchorage in the DNA have a particular affinity for the matrix and can therefore be identified as matrix attachment regions (MARs) or scaffold attachment regions.As illustrated in Fig.

Complex regulation of somatic hypermutation by cis-acting sequences in the endogenous IgH gene in hybridoma cells

“…1 A) (ref. 20 and M.J.S., unpublished observation). Loss of E or of both E and the MARs resulted in cells in which expression was variegated, such that two types of stable subclones could be isolated: those that continued to make WT levels of the heavy chain and those that had lost the ability to express the heavy chain.…”

“…The MARs recombinant, in which the MARs are the only intronic regulatory elements in the J-C intron, was used to generate the MARs-ϩ and MARs-Ϫ subclones, which are active and silent for IgH expression, respectively (18,19). ⌬, a recombinant with a complete deletion of the intronic elements, was used to generate the subclones ⌬-ϩ and ⌬-Ϫ , in which IgH is active or silent, respectively (14,20).…”

“…The WT recombinant was generated by inserting the guanyl phophorybosyl transferase (gpt) between the C region and C␦ region ( Fig. 1 A) (17), resulting in insulation from the influence of the 3Ј locus control region on the expression of the heavy chain (20). In WT, all of the cells still expressed WT levels of IgM (14).…”

A Nuclear Matrix Attachment Region Upstream of the T Cell Receptor β Gene Enhancer Binds Cux/CDP and SATB1 and Modulates Enhancer-dependent Reporter Gene Expression but Not Endogenous Gene Expression

“…In this regard, it is worthwhile to consider the putative functions of MARs at the IgH and Ig loci. By reporter gene assays in cell lines or transgenic mice, MARs flanking the enhancer E were shown to modulate IgH gene chromatin accessibility (31) and transcription in both positive and negative manners (27)(28)(29)(30). Similarly, MAR associated with the Ig intronic enhancer is thought to be involved in Ig gene transcription, demethylation, recombination, and somatic hypermutation (18,(33)(34)(35)(36).…”

“…In the immunoglobulin heavy chain (IgH) locus, MARs flank the intronic enhancer E and are in close proximity to V H promoters (22)(23)(24)(25)(26). Reporter gene assays in cell lines and transgenic mice have suggested that these MARs exert both positive and negative effects on IgH gene transcription and promote long range chromatin accessibility (27)(28)(29)(30)(31). A highly conserved MAR is also found 200 base pairs (bp) upstream of the intronic immunoglobulin (Ig) enhancer in mouse, human, and rabbit (22,32).…”