Transcription Factor NF-κB Regulates Igλ Light Chain Gene Rearrangement

Bendall, Heather; Sikes, Michael L.; Oltz, Eugene M.

doi:10.4049/jimmunol.167.1.264

Cited by 17 publications

(17 citation statements)

References 27 publications

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“…By mapping in vivo targets of p65 through ChIP, we found that p65 binds proximal to 5Ј ends, as expected, but also with a high frequency at many other sites, including introns and sites distal to 5Ј ends. Finding NF-B-binding sites in introns is not unexpected because it was originally identified by its binding an enhancer element in the first intron of the light chain gene; since then, several examples of NF-B to binding intronic sequences have emerged (22,23,28,29). Our data also indicate that p65 not only binds genes whose expression is regulated by the proinflammatory cytokine TNF-␣ but also binds a large number of genomic loci whose expression is not regulated.…”

Section: Discussionsupporting

confidence: 50%

Distribution of NF-κB-binding sites across human chromosome 22

Martone

Euskirchen

Bertone

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

296

258

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We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-B family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity, and inflammation. The binding sites of the NF-B family member p65 were determined by using chromatin immunoprecipitation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5 end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-B binding was also found at genes whose expression was regulated by tumor necrosis factor ␣, a known inducer of NF-B-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor ␣. Many of these latter genes are either known to be activated by NF-B under other conditions or are consistent with NF-B's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-B binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.U nderstanding the targets regulated by transcription factors and where they bind relative to these targets in an unbiased fashion in mammalian cells is highly desirable. We and others have developed a procedure for mapping in vivo targets of transcription factors by chromatin immunoprecipitation (ChIP) with antibodies to a transcription factor of interest to isolate protein-bound DNA, followed by probing a microarray containing genomic DNA sequences with the immunoprecipitated DNA (ChIP chip) (1-3). This approach was first used successfully in yeast and has more recently been used in a limited fashion to identify transcription factor binding sites in mammalian cells (4-6). However, a large-scale, unbiased global analysis of the distribution of mammalian transcription factor binding sites along large genomic regions has not been previously explored.In this study we employ a microarray containing the entire nonrepetitive sequence of chromosome 22 to determine the chromosome-wide binding profile for the transcription factor NF-B. The NF-B͞Rel family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including immune and inflammatory responses (7,8), and the activation pathway has been studied extensively over the last two decades (9, 10). Numerous NF-B target genes have also been identified; however, it remains unclear how many of these are direct targets of the transcription factor (11).There are five mammalian NF-B family members (p50, p52, RelA͞p65, RelB, and c-rel), all of which function as homo-or heterodimers. The different dimers exhibit varying binding aff...

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Section: Discussionsupporting

confidence: 50%

Distribution of NF-κB-binding sites across human chromosome 22

Martone

Euskirchen

Bertone

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

296

258

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“…The cycling profile for J2͞3 SEs has been described (22). D␤ SE and RE cycling profiles were 94°C, 30 s; 62°C, 30 s; and 72°C, 30 s. A 2-l sample of PCR1 was used as template in 27-cycle (SE assays) or 30-cycle (RE assays) PCRs with nested primers.…”

Section: Methodsmentioning

confidence: 99%

“…Primers used in assays for 5ЈJ2͞3 SEs (22), VJ coding joins, J␤C transcripts, C, and ␤-actin have been described (12). Additional primers and probes are as follows.…”

Section: Methodsmentioning

confidence: 99%

Regulation of V(D)J recombination: A dominant role for promoter positioning in gene segment accessibility

Sikes

Meade

Tripathi

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Antigen receptor gene assembly is regulated by transcriptional promoters and enhancers, which control the accessibility of gene segments to a lymphocyte-specific V(D)J recombinase. However, it remained unclear whether accessibility depends on the process of transcription itself or chromatin modifications that accompany transcription. By using T cell receptor ␤ substrates that integrate stably into nuclear chromatin, we show that promoter location, rather than germ-line transcription or histone acetylation, is a primary determinant of recombination efficiency. These spatial constraints on promoter positioning may reflect an RNA polymerase-independent mechanism to target adjacent gene segments for chromatin remodeling events that facilitate rearrangement.A ntigen receptor genes are assembled in developing lymphocytes by a series of recombination events that fuse variable (V), diversity (D), and joining (J) gene segments. Two lymphocyte-specific proteins, termed RAG-1 and RAG-2, are essential components of the V(D)J recombinase complex (1, 2). These proteins introduce double-strand DNA breaks at selected recombination signal sequences (RSSs), which flank all Ig and T cell receptor (TCR) gene segments. The RSSs contain conserved heptamer and nonamer sequences separated by a nonconserved spacer (12 or 23 bp). Under physiological conditions, RAGmediated cleavage requires the synapsis of a 12-bp and a 23-bp RSS (3, 4). Ubiquitous DNA repair enzymes then fuse participating gene segments and RSSs to generate a chromosomal coding join and a signal join, respectively (5). Although most RSSs are interchangeable, V(D)J recombinase is targeted to specific Ig and TCR loci during lymphocyte development. For example, assembly of TCR genes is restricted to thymocytes, initiating with rearrangement of D␤ and J␤ segments, followed by V␤ to D␤J␤ and ultimately V␣ to J␣ recombination (6).Prevailing models for the developmental control of V(D)J recombination invoke changes in the accessibility of specific gene segments to the RAG-1͞2 complex (6-8). Extensive correlations exist between transcription of unrearranged gene segments and their recombination potential (9, 10), suggesting that germ-line transcription and RAG accessibility share key regulatory components. Indeed, deletion of transcriptional enhancers from most Ig and TCR loci dramatically impairs the assembly of linked gene segments (3, 6). We and others have shown that the TCR␤ enhancer (E␤) mediates efficient D␤1J␤ rearrangement via activation of a promoter located directly upstream of the D␤1 gene segment (PD␤; refs. 11-13). Because prior studies have shown that chromatin impairs the access of RAG proteins to RSSs (14), promoter activation likely regulates recombination by initiating a cascade of chromatin modifications that culminate in gene transcription. In this regard, the acetylation of histones H3 and H4 correlates tightly with the transcription and recombination of antigen receptor loci (7,15,16). Despite these correlations, it remains unknown whether promoter-direct...

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“…Furthermore, NF-B-binding sites are present in the human and mouse IgH, Ig, and Ig enhancer modules (15)(16)(17)(18)(19)(20). Different studies using mice or murine B cell lines have shown that impairing NF-B function-either by knocking down/repressing its component subunits or by increasing IB␣ expression-has dramatic effects on the level of Ig gene rearrangement (21)(22)(23). In vitro studies have demonstrated that the RAG proteins are critical in the joining phase of recombination and interact with the postcleavage coding ends (24).…”

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confidence: 99%

The NF-κB Canonical Pathway Is Involved in the Control of the Exonucleolytic Processing of Coding Ends during V(D)J Recombination

Souto-Carneiro

Fritsch

Sepúlveda

et al. 2008

The Journal of Immunology

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V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-κB-binding sites are present in the human and mouse IgH, Igκ, and Igλ enhancer modules and RAG expression is controlled by NF-κB, it is not known whether NF-κB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-κB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-κB essential modulator (NEMO/Ikkγ). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkγ-mediated canonical NF-κB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3H from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-κB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-κB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.

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Transcription Factor NF-κB Regulates Igλ Light Chain Gene Rearrangement

Cited by 17 publications

References 27 publications

Distribution of NF-κB-binding sites across human chromosome 22

Distribution of NF-κB-binding sites across human chromosome 22

Regulation of V(D)J recombination: A dominant role for promoter positioning in gene segment accessibility

The NF-κB Canonical Pathway Is Involved in the Control of the Exonucleolytic Processing of Coding Ends during V(D)J Recombination

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