Compaction and looping of the ∼2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V H genes to be brought in proximity with D H -J H segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ∼60 sites throughout the V H region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a D H -J H -Eμ-C H domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D H region and parts of the V H locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.A ntigen receptors are created through the highly regulated lineage-specific process of V(D)J recombination, creating a diverse repertoire of Ig and T-cell receptors. The generation of the mouse Ig heavy chain in pro-B cells begins with D H -to-J H rearrangement on both alleles, followed by V H -to-D H J H rearrangement. In order for the >100 functional murine V H genes spread across ∼2.5 Mb to gain access to the single D-J rearrangement on that allele, the Igh locus undergoes contraction and looping during the pro-B-cell stage of B-cell differentiation (1-5). By measuring spatial distances between 11 small probes spread throughout the Igh locus, Jhunjhunwala et al. (2) demonstrated that distal and proximal V H genes were approximately equidistant from the D genes specifically at the pro-B-cell stage when the V H genes are rearranging. Computational as well as geometrical approaches have suggested that the locus is organized into rosette-like clusters of loops that compact during rearrangement. Several proteins have been reported to influence Igh locus compaction, including Pax5, YY1, and Ikaros (5-7). These proteins and others, such as Ezh2 (8), are also necessary for the rearrangement of distal V H genes but not proximal V H genes. This is most likely a consequence of the lack of locus compaction in the absence of these proteins. How all these proteins funct...

Contraction of the large Diversity in the Ig Ab repertoire is achieved through V(D)J recombination, a lineage-specific process that is highly regulated during B cell development. IgH rearrangement in pro-B cells begins with D H to J H rearrangement followed by rearrangement of a V H gene segment to D H J H . The IgH is assembled before the L chains, and Ig rearrangement precedes Ig rearrangement. Strict regulation of accessibility allows for the lineage-specific and developmentally ordered rearrangement of V(D)J gene segments. The mechanisms controlling V H to D H J H rearrangement are exceptionally complex because Ͼ100 functional murine V H genes span a 2.5 Mb region. Likewise, the 96 functional V genes cover 3.1 Mb. The question arises of how all the V genes acquire access to the small J cluster (Ͻ2 kb) in either the Igh or Ig loci. Three-dimensional fluorescence in situ hybridization studies have demonstrated that the Igh and Ig loci undergo significant contraction to position gene segments in proximity for rearrangement at the appropriate time for rearrangement (1-4). In pro-B cells, the V H genes are brought into close proximity to D H gene segments via multiple loop structures, thus facilitating rearrangement through these long-range chromosomal interactions (4). The contraction and looping of the loci raises the question of which nuclear factors could be controlling these interactions. One potential nuclear protein that participates in long-range chromosomal interactions is CCCTC-binding factor (CTCF). 3CTCF is a ubiquitously expressed 11-zinc finger nuclear protein that is associated with all known vertebrate insulators (5). Chromatin insulators or boundary elements create distinct chromosomal domains preventing outside influences on the insulated region (5). The enhancer-blocking activity of CTCF prevents the interactions between enhancers and promoters separated by the insulator. Global mapping of CTCF binding has shown that CTCF binds in regions that could separate different chromosomal domains, consistent with the idea that CTCF may insulate the spread of repressive chromatin modifications into neighboring active domains (6 -8). One of the ways that CTCF may function as an insulator and regulate gene expression is through the facilitation of long-range intrachromosomal and interchromosomal looping (9 -11).CTCF has been reported to associate with a number of factors including YY1 (12), the chromodomain helicase CHD8 (13), and cohesin subunits (14 -17). Cohesin proteins have an established role of facilitating cohesion of sister chromatids during cell division (18)

A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5′-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that being positioned near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have higher levels of histone marks and non-coding RNA, which may compensate for their poorer RSSs and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for each of the four domains that comprise this locus.

One mechanism through which bioactive food components may exert anticancer effects is by reducing the expression of the proinflammatory gene cyclooxygenase-2 (COX-2), which has been regarded as a risk factor in tumor development.Rosmarinic acid (RA) is a phenolic derivative of caffeic acid present in rosemary (Rosmarinus officinalis). Previous research documented that RA may exert antiinflammatory effects. However, the mechanisms of action of RA on COX-2 expression have not been investigated. Here, we report that in colon cancer HT-29 cells, RA (5, 10, and 20 mmol/L) reduced the 12-Otetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity (P , 0.05) and protein levels (P , 0.05). In addition, the cotreatment with RA reduced (5 mmol/L, P , 0.05; 10 and 20 mmol/L, P , 0.01) TPA-induced transcription from a control activator protein-1 (AP-1) promoter-luciferase construct and repressed binding of the AP-1 factors c-Jun (10 mmol/L; P , 0.01) and c-Fos (10 mmol/L; P , 0.05) to COX-2 promoter oligonucleotides harboring a cAMP-response element (CRE). The anti-AP1 effects of RA were also examined in a nonmalignant breast epithelial cell line (MCF10A) in which RA antagonized the stimulatory effects of TPA on COX-2 protein expression (5 mmol/L, P , 0.05; 10 and 20 mmol/L, P , 0.01), the recruitment of c-Jun and c-Fos (10 mmol/L; P , 0.01) to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2) (10 mmol/L; P , 0.01), a member of the mitogen-activated protein kinase pathway. Additionally, RA antagonized ERK1/2 activation in colon HT-29 and breast MCF-7 cancer cells (10 mmol/L; P , 0.01). Thus, we propose that RA may be an effective preventative agent against COX-2 activation by AP-1-inducing agents in both cancer and nonmalignant mammary epithelial cells.

Ligands of the aryl hydrocarbon receptor (AhR) include the environmental xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl hydrocarbons, and the dietary compounds 3, 3'-diindolylmethane (DIM), a condensation product of indol-3-carbinol found in Brassica vegetables, and the phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE). Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may contribute to inflammation and tumorigenesis, we investigated the epigenetic regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7 breast cancer cells. Results of DNA binding and chromatin immunoprecipitation (ChIP) studies documented that the treatment with TCDD induced the association of the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter oligonucleotides. The TCDD-induced binding of the AhR was reduced by small-interfering RNA for the AhR or the cotreatment with synthetic (3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2 promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 micromol/L) abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter and the induction of COX-2 mRNA and protein levels. Taken together, these data suggest that naturally occurring modulators of the AhR such as DIM may be effective agents for dietary strategies against epigenetic activation of COX-2 expression by AhR agonists.