A system for precise analysis of transcription-regulating elements of immunoglobulin genes

BACKGROUND: Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state. RESULTS: As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin μ heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci

A Weakened Transcriptional Enhancer Yields Variegated Gene Expression

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ∼1.4 kb 3′ of the promoter, but inactive ∼6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ∼1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators

Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID

AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and single-stranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors

The indicated cell lines were stained for intracellular IgM using μ-specific, FITC-labeled antibody and analyzed by flow cytometry

<p><b>Copyright information:</b></p><p>Taken from "A system for precise analysis of transcription-regulating elements of immunoglobulin genes"</p><p>BMC Biotechnology 2005;5():27-27.</p><p>Published online 4 Oct 2005</p><p>PMCID:PMC1266055.</p><p>Copyright © 2005 Cheng et al; licensee BioMed Central Ltd.</p> Intensity of staining is represented on the horizontal axis, cell number on the vertical. The geometric mean (M) for the staining is noted in each panel

Flow cytometry of μ expression from reporters with mutant enhancers.

<div><p>a) Two independent transfectants expressing the #654 reporter were analyzed by flow cytometry.</p> <p>b) As in (a) two independent transfectants bearing reporter #651 were analyzed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000033#pone-0000033-g003" target="_blank">Figure 3</a>. </p> <p>The transfectant 651/b was biphasic and yielded subclones with different levels of expression, 651/b3 and 651/b5.</p> <p>To assess the stability of this difference, these subclones were re-subcloned, and the mean fluorescence, M, and normalized fluorescence, N, of eight re-subclones were measured.</p> <p>N_m, the mean value for N for the re-subclones and the associated standard deviation, were calculated.</p> <p>The values of N_m for each set of subclones were significantly different and close to the value of the N for their respective parents.</p> <p>The statistical parameters, M, N, σ, and C are defined in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000033#pone-0000033-g003" target="_blank">Figure 3</a>.</p></div