Fine mapping of an immunoglobulin gene activator.

Abstract

It has recently been shown that the promoter of a K immunoglobulin gene is activated for transcription by a downstream sequence element. Here we mapped the activating element to a resolution of about 20 base pairs by constructing a series of deletions in the cloned K gene. After transfection of each deleted gene into myeloma cells, a transient expression assay was used to measure the level of transcription from the K promoter. We found that the activating element extends through about 200 base pairs and encompasses a region of sequence that is conserved between mouse and human genes. As successively deeper deletions were made into the conserved region from either the 5' or 3' side, the activating ability was lost gradually rather than abruptly. Although several short segments in this region are homologous to sequences in viral enhancers, they did not seem to play a dominant role in the activating effect. We also found that the activating element remained functional when reversed in orientation or when moved upstream of the K gene.As a stem cell develops into a B-lymphocyte, rearrangements of the cellular DNA take place that form complete immunoglobulin genes from the separately encoded variable (V) and constant (C) regions (for a review, see reference 31). In B-lymphocytes that synthesize K light chains, one of many VK regions is joined to a JK segment located upstream of the unique CK region (Fig. 1A). After this rearrangement occurs, RNA transcripts of the complete K gene can be detected. The transcripts initiate from just before the rearranged VK region and proceed through the VK and JK regions, the JK-CK intron, and the CK region.As a developing B-lymphocyte enters the antibody-secreting plasma cell stage, the rate of transcription of the rearranged K gene increases, so that K transcripts may represent 5 to 10% of the total mRNA population (25). However, no transcription of the remaining unrearranged VK regions can be detected (12,19). The question therefore arises of how the cell activates the rearranged VK region for high-level transcription, while leaving the unrearranged VK regions completely inactive. Any explanation must be consistent with the fact that rearrangement does not alter the DNA sequences 5' to the VK region, where the signals for transcription initiation would normally be expected to occur (4,19).A guide to answering the question was provided by the existence of enhancer elements in certain viral genomes. Enhancers are DNA sequences that strongly stimulate transcription from many eucaryotic promoters; they can act from either an upstream or a downstream position on promoters that are thousands of base pairs (bp) away (2,7,15 in about 200 bp (1, 8), the K-activating element was only localized to the last third of the JK-CK intron (22). A logical candidate for its precise position is nrovided by a region of the intron that is strikingly well conserved in sequence among mouse, rabbit, and human genes (6). By constructing a series of K gene deletions and analyzing them in a transientexpression ...

Deregulation of the proto-oncogene c-myc through t(8;22) translocation in Burkitt's lymphoma

“…This element forms a DNaseI hypersensitive site in B-cells and is indispensible for cmyc deregulation in BL, but again, does not show enhancer activity in transient assays (HoÈ rtnagel et al, 1995). Whether or not the element residing within HSS 0 serves a similar function remains to be determined, but its properties are consistent with ®ndings reported by other groups (Aronow et al, 1995;Bergman et al, 1984;Forrester et al, 1994;Hug et al, 1992;Queen et al, 1984;Stief et al, 1989).…”

Multiple sequence motifs are involved in SV40 enhancer function.

“…However, when 10 393 Figure 2A and B, the sequence 5'-ATGCAAA-3' homologous to the immunoglobulin heavy chain enhancer (position 212 -206) is underlined, and the polyoma virus enhancer homology (P-motif, position 196-186) is boxed. References for the various sequence elements are as follows (see also text): LPV enhancer (Pawlita et al, 1985;Mosthaf et al, 1985); BK virus (BKV) enhancer (Rosenthal et al, 1983); Igx light chain enhancer (Picard and Schaffner, 1984;Queen and Stafford, 1984); consensus sequences of the upstream promoter elements of the immunoglobulin heavy (VH) and light (VL, opposite strand) chain genes (Falkner and Zachau, 1984;Parslow et al, 1984); Ig heavy chain enhancer (opposite strand, Ephrussi et al, 1985 and references therein; the star indicates the C complementary to the G protected against dimethylsulfate modification, see text); Xenopus Ul/U2 RNA genes (Mattaj et al, 1985;Ciiberto et al, 1985;Krol et al, 1985); polyoma virus enhancer (opposite strand, Veldman et al, 1985;Ruley and Fried, 1983). For comparison the ElA-like motif of the polyoma virus enhancer (Hearing and Shenk, 1983;Herbomel et al 1984) is shown.…”

“…Sequence elements closely related to the Sph-motifs are found as a single motif in the BK virus (Rosenthal et al, 1983) and as a repeat in the lymphotropic papovavirus (LPV) (Furuno et al, 1984;Pawlita et al, 1985;Mosthaf et al, 1985) (see Figure 6), where their function has not yet been studied. The Sph-motif is also present in the immunoglobulin x-light chain enhancer (Picard and Schaffner, 1984;Queen and Baltimore, 1983), where its deletion is detrimental to enhancer activity (Queen and Stafford, 1984). In addition, the juxtaposition of the two Sph-motifs in the SV40 enhancer generates a sequence element which is present in the immunoglobulin heavy chain enhancer (Banerji et al, 1983;Gilles et al, 1983;Neuberger, 1983) and in the upstream elements of the promoters of the VH and VL immunoglobulin genes (Falkner and Zachau, 1984;Parslow et al, 1984) (Figure 6).…”

“…[bovine papillomavirus (Lusky et al, 1983;Weiher and Botchan, 1984), adenovirus ElA (Hen et al, 1983), immunoglobulin xlight chain (Queen and Stafford, 1984) Figure 3B and pA213 in Figure 2A) does not abolish domain B activity. Furthermore, deleting GT-motif II can be compensated for by juxtaposing domain C to GT-motif I (compare pA104 with pA54 in Figure 2A).…”

“…These motifs belong to two major domains which have very little stimulatory activity by themselves, but generate a very potent enhancer when associated, suggesting the involvement of some type of cooperativity, the mechanism of which is unknown at present. All known viral and cellular enhancers contain short sequence stretches which exhibit some homology to one or several of the SV40 motifs, and when investigated, their importance for enhancer activity has indeed beeen demonstrated Hen et al, 1983;Weiher and Botchan, 1984;Queen and Stafford, 1984). It appears, therefore, that enhancers are made up of several sequence motifs which have been combined in various ways during evolution.…”