Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination

Abstract

Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5′ to 3′, several hundred V H gene segments, 13 D segments, and 4 J H segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J H rearrangement occurring on both alleles before appendage of a V H segment. Also, IgH V H to DJ H rearrangement does not occur in T cells, even though DJ H rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V H and D clusters (generating ΔV H -D alleles). In both B and T cells, ΔV H -D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V H segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J H rearrangement and substantial V H to DJ H rearrangement of proximal IgH V H segments. Thus, the V H -D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination. The activity of the common V(D)J recombinase is regulated in several contexts (8, 9). First, V(D)J recombination is regulated in a lineage-specific manner. Thus, Ig variable-region exons are completely assembled in B cells and not in T cells, whereas TCR variable-region exons are assembled in T but not B cells (6). Second, within a given lineage, V(D)J recombination is highly ordered. The IgH locus contains a large cluster of V H segments, a cluster of 13 D segments, and a cluster of 4 J H segments in a several-megabase region (1). In progenitor (pro) B lymphocytes, IgH variable-region exons are assembled via a process in which D to J H rearrangements occur first and on both alleles, followed by appendage of a V H segment to the DJ H complex (10). Direct joining of a V H to a D is not observed, even though permitted by the 12/23 rule (10). Although not strictly ordered, the most Dproximal V H families and, in particular, the most D-proximal V H 81X gene segment, rearrange more frequently than the most distal V H gene families, such as the V H J558 family (11). After assembly of a productive IgH variable-region exon and expression of a μ heavy chain, pro-B cells advance to the precursor (pre) stage at which Ig light-chain (IgL) variable-region assembly occurs. Similarly ordered V(D)J recombination occurs during assembly of TCR variable-region exons in developing T lineage cells (12). F...

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

“…Surprisingly, we found an increase in antisense transcription from the Adam6 gene through the DSP genes, suggesting a broader regulatory role for CTCF/DFL. Indeed, a very recent study has shown that deletion of a 108-kb intergenic region extending from the proximal V H genes through DFL16.1, including the CTCF/DFL sites, resulted in increased D H antisense transcription throughout the D H locus in both B and T cells (25). Because we have shown that CTCF knockdown results in the same increase in D H antisense transcription, we propose it is the decrease in looping at CTCF/DFL-Eμ-CTCF/3′RR that mediates this enhancing effect on D H antisense transcription.…”

“…Antisense transcription through the D H locus precedes D H -to-J H rearrangement and has been proposed to make the D H region accessible for subsequent rearrangement (23,24). Some antisense transcription begins near Eμ and is dependent on the presence of Eμ, whereas other antisense transcription begins near DST4 (23,25). Recently, it was shown that antisense transcription decreases just upstream of the CTCF/DFL sites, suggesting that this CTCF region is a boundary that prevents antisense transcription from continuing toward the V H locus (22).…”

Determinación de la clonalidad en tejidos humanos

“…Este proceso se conoce como rearreglo o reordenamiento, términos que se utilizarán indistintamente en este texto. Cuando el reordenamiento de los genes del receptor de antígeno no es perfecto, bien sea por falta de alguna enzima o por otro defecto, se presentan graves inmunodeficiencias (13). Por otra parte, cuando una célula linfoide se transforma en cancerosa y se divide sin cesar, sus células hijas poseen el mismo reordenamiento de los genes de receptor de antígeno que la célula madre que las originó.…”

“…El segmento D solo está presente en los loci de las cadenas pesadas de Ig y los loci β y δ son exclusivos de las del TCR (figura 2). La recombinación alélica ocurre cuando se unen los fragmentos variables, de diversidad y unión, llamada recombinación V(D)J que se regula por medio de exclusión alélica; una vez que un alelo se ha reordenado, se envía una señal al otro para interrumpir el proceso (12,13). Los reordenamientos se efectúan por medio de pasos secuenciales e invariables:…”

“…Durante largo tiempo se utilizó con este fin el Southern Blot. La técnica es muy específica, pero sus exigencias metodológicas limitan la aplicación (13). Desde hace diez años existe un grupo de protocolos basados en PCR (reacción en cadena de la polimerasa, por la sigla en inglés de polymerase chain reaction) (7).…”