Cutting Edge: Developmental Stage-Specific Recruitment of Cohesin to CTCF Sites throughout Immunoglobulin Loci during B Lymphocyte Development

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...

Section: Resultsmentioning

confidence: 99%

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

Giallourakis

Franklin²,

Guo³

et al. 2010

“…At Igh and Igk, CTCF sites are generally intergenic except for those in the proximal one-third of the V H array, which are downstream of V H RSSs; moreover, CTCF sites are not associated with the major Igh and Igk enhancer elements (25,26,(48)(49)(50). At these loci, CTCF promotes long-distance interactions between CTCFbinding elements (24)(25)(26), but these looping interactions appear primarily to restrict or insulate the activities of the major enhancer elements.…”

Section: Discussionmentioning

confidence: 99%

Tcragene recombination is supported by aTcraenhancer- and CTCF-dependent chromatin hub

Shih

Verma-Gaur

Torkamani

et al. 2012

Self Cite

153

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4 − CD8 − double-negative thymocytes and Tcra gene segments in CD4 + CD8 + double-positive thymocytes. Initial V α -to-J α recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E α ) interacts directly with V α and J α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E α promotes interactions between these V α and J α segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E α and to many locus promoters, biases E α to interact with these promoters, and is required for efficient V α -J α recombination. Our data indicate that E α and CTCF cooperate to create a developmentally regulated chromatin hub that supports V α -J α synapsis and recombination.T-cell development | T-cell receptor | thymus T and B cells produce diverse antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at the T-cell receptor (Tcra, Tcrb, Tcrg, and Tcrd) and Ig (Igh, Igκ, and Igλ) loci. This V(D)J recombination is initiated by the lymphoid-specific recombination-activating gene-1 (RAG-1) and RAG-2 proteins, which recognize the recombination signal sequences (RSSs) that flank all V, D, and J gene segments and then cleave the DNA between the RSSs and the adjacent coding gene segments (1). A critical feature of the reaction is the assembly of a synaptic complex composed of two RSSs before the generation of RAG-dependent DNA doublestrand breaks (DSBs). As such, lineage-and developmental stagespecific V(D)J recombination events can be regulated not only by changes in RAG protein expression and RSS accessibility to RAG proteins but also by the ability of those RSSs to undergo synapsis (2).Conformational changes of antigen receptor loci are believed to support V(D)J recombination events because they can bring distant RSSs into proximity and therefore increase the probability of RSS synapsis (2, 3). Studies using 3D-FISH have demonstrated that lineage-and development stage-specific locus contraction marks the recombination windows at antigen receptor loci (3). For example, the 3-Mb Igh locus contracts specifically in pro-B cells to support V H -to-D H J H recombination (4-7). This contracted conformation brings distal and proximal V H segments, which are separated by megabases in the linear DNA sequence, to the vicinity of the D H J H cluster, presumably allowing all V H segments a similar opportunity for recombination (8). In addition, the mapping of Igh locus DNA-DNA...

“…The association of CTCF with boundaries of active and repressive genomic regions was also found to be cell type specific (25). How the specificity of CTCF-dependent chromatin loops is established is not known but might involve cell-type-specific proteins that support more frequent or more strongly stabilized higher-order chromatin interactions or might relate to lineage-specific recruitment of cohesin (26).…”

Section: Numerous Sites Of Ctcf Enrichment Reside Among Silent Olfactorymentioning

confidence: 99%

Cell type specificity of chromatin organization mediated by CTCF and cohesin

Hou

Dale

Dean

2010

229

214

CTCF sites are abundant in the genomes of diverse species but their function is enigmatic. We used chromosome conformation capture to determine long-range interactions among CTCF/cohesin sites over 2 Mb on human chromosome 11 encompassing the β-globin locus and flanking olfactory receptor genes. Although CTCF occupies these sites in both erythroid K562 cells and fibroblast 293T cells, the long-range interaction frequencies among the sites are highly cell type specific, revealing a more densely clustered organization in the absence of globin gene activity. Both CTCF and cohesins are required for the cell-type-specific chromatin conformation. Furthermore, loss of the organizational loops in K562 cells through reduction of CTCF with shRNA results in acquisition of repressive histone marks in the globin locus and reduces globin gene expression whereas silent flanking olfactory receptor genes are unaffected. These results support a genome-wide role for CTCF/cohesin sites through loop formation that both influences transcription and contributes to cell-type-specific chromatin organization and function.chromatin loops | insulator | CTCF | globin | transcription E ukaryotic chromatin is dynamically organized to form distinct transcriptionally active or silent domains (1). Chromatin insulators are proposed to play a role in the establishment of such domains within which proper enhancer-gene interactions occur and improper ones are excluded (2). Insulators can function as boundary elements between active and silent chromatin domains and can also interfere with enhancer-gene interaction when placed between them. Evidence also suggests that insulators are involved in higher-order chromatin organization by clustering together with other insulators. Such "insulator bodies" can be visualized at the nuclear periphery of certain cells in Drosophila and are thought to coalesce through the interaction of proteins such as Su(Hw) and Drosophila CTCF (3, 4).In mammalian cells, insulators are bound by CTCF, a protein with 11 zinc fingers through which it can bind to a range of DNA sequences, to itself, and to nuclear structural proteins such as nucleophosmin and matrix attachment regions (MARs) (2). Although insulators would be expected to reside primarily in intergenic regions where they could provide boundary or enhancer blocking activity, genome scans of CTCF enrichment show that CTCF sites are overrepresented in genes and promoter regions, with only 41-56% at intergenic locations (5). In Drosophila, between about 49% and 64% of insulator sites are located in intergenic regions (4). Thus, although CTCF sites are proposed to be insulators, their in vivo functions may not be limited to insulation. Recently, cohesin has been reported to colocalize at most sites of CTCF enrichment and to play a role in transcriptional insulation, but the significance of this joint occupancy to higher-order chromatin structures is unknown (6).In the Igf2/H19 locus, CTCF binding to the imprinting control region on the maternal allele is required to loop ...