Deep sequencing of the murine IgH repertoire reveals complex regulation of nonrandom V gene rearrangement frequencies.

A diverse Ab repertoire is formed through the rearrangement of V, D, and J segments at the IgH (Igh) loci. The C57BL/6 murine Igh locus has >100 functional VH gene segments that can recombine to a rearranged DJH. Although the nonrandom 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. Chromatin immunoprecipitation-sequencing results for several histone modifications and RNA polymerase II binding, RNA-sequencing for sense and antisense noncoding germline transcripts, and proximity to CCCTC-binding factor (CTCF) and Rad21 sites were compared with 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 noncoding RNA in general, but having a CTCF site near their recombination signal sequence 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 noncoding RNA, which may compensate for their poorer recombination signal sequences 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.

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

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.

Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2 expression by the indole-3-carbinol metabolite 3,3'-diindolylmethane in breast cancer 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.

Cutting edge: developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development.

Contraction of the large Igh and Igkappa loci brings all V genes, spanning >2.5 Mb in each locus, in proximity to DJ(H) or J(kappa) genes. CCCTC-binding factor (CTCF) is a transcription factor that regulates gene expression by long-range chromosomal looping. We therefore hypothesized that CTCF may be crucial for the contraction of the Ig loci, but no CTCF sites have been described in any V loci. Using ChIP-chip, we demonstrated many CTCF sites in the V(H) and V(kappa) regions. However, CTCF enrichment in the Igh locus, but not the Igkappa locus, was largely unchanged throughout differentiation, suggesting that CTCF binding alone cannot be responsible for stage-specific looping. Because cohesin can colocalize with CTCF, we performed chromatin immunoprecipitation for the cohesin subunit Rad21 and found lineage and stage-specific Rad21 recruitment to CTCF in all Ig loci. The differential binding of cohesin to CTCF sites may promote multiple loop formation and thus effective V(D)J recombination.

Rosmarinic acid antagonizes activator protein-1-dependent activation of cyclooxygenase-2 expression in human cancer and nonmalignant cell lines.

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 micromol/L) reduced the 12-O-tetradecanoylphorbol-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 micromol/L, P < 0.05; 10 and 20 micromol/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 micromol/L; P < 0.01) and c-Fos (10 micromol/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 micromol/L, P < 0.05; 10 and 20 micromol/L, P < 0.01), the recruitment of c-Jun and c-Fos (10 micromol/L; P < 0.01) to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2) (10 micromol/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 micromol/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.

Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene.

INTRODUCTION: Increased estrogen level has been regarded to be a risk factor for breast cancer. However, estrogen has also been shown to induce the expression of the tumor suppressor gene, BRCA1. Upregulation of BRCA1 is thought to be a feedback mechanism for controlling DNA repair in proliferating cells. Estrogens enhance transcription of target genes by stimulating the association of the estrogen receptor (ER) and related coactivators to estrogen response elements or to transcription complexes formed at activator protein (AP)-1 or specificity protein (Sp)-binding sites. Interestingly, the BRCA1 gene lacks a consensus estrogen response element. We previously reported that estrogen stimulated BRCA1 transcription through the recruitment of a p300/ER-alpha complex to an AP-1 site harbored in the proximal BRCA1 promoter. The purpose of the study was to analyze the contribution of cis-acting sites flanking the AP-1 element to basal and estrogen-dependent regulation of BRCA1 transcription. METHODS: Using transfection studies with wild-type and mutated BRCA1 promoter constructs, electromobility binding and shift assays, and DNA-protein interaction and chromatin immunoprecipitation assays, we investigated the role of Sp-binding sites and cAMP response element (CRE)-binding sites harbored in the proximal BRCA1 promoter. RESULTS: We report that in the BRCA1 promoter the AP-1 site is flanked upstream by an element (5'-GGGGCGGAA-3') that recruits Sp1, Sp3, and Sp4 factors, and downstream by a half CRE-binding motif (5'-CGTAA-3') that binds CRE-binding protein. In ER-alpha-positive MCF-7 cells and ER-alpha-negative Hela cells expressing exogenous ER-alpha, mutation of the Sp-binding site interfered with basal and estrogen-induced BRCA1 transcription. Conversely, mutation of the CRE-binding element reduced basal BRCA1 promoter activity but did not prevent estrogen activation. In combination with the AP-1/CRE sites, the Sp-binding domain enhanced the recruitment of nuclear proteins to the BRCA1 promoter. Finally, we report that the MEK1 (mitogen-activated protein kinase kinase-1) inhibitor PD98059 attenuated the recruitment of Sp1 and phosphorylated ER-alpha, respectively, to the Sp and AP-1 binding element. CONCLUSION: These cumulative findings suggest that the proximal BRCA1 promoter segment comprises cis-acting elements that are targeted by Sp-binding and CRE-binding proteins that contribute to regulation of BRCA1 transcription.

Cyclooxygenase-2 promoter activation by the aromatic hydrocarbon receptor in breast cancer mcf-7 cells: repressive effects of conjugated linoleic acid.

The role of the aromatic hydrocarbon receptor (AhR) in transcriptional regulation of the human cyclooxygenase-2 (COX-2) gene remains elusive. We report that the AhR-ligands benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced transcription activity of COX-2 in breast cancer MCF-7 cells. The TCDD-dependent activation of the COX-2 promoter was abrogated by mutation of 2 xenobiotic response elements (XREs) = CGTG). We found that TCDD stimulated the binding of the AhR to COX-2 and cytochrome P4501A1 (CYP1A1) oligonucleotides containing consensus XREs. Conversely, the cotreatment with TCDD plus a mixture of conjugated linoleic acid (CLA) or selected CLA isomers prevented (CLAmix = t10,c12-CLA > c9,t11-CLA) the induction of transcription from the COX-2 promoter. The TCDD-induced binding of the AhR to COX-2 and CYP1A1 oligonucleotides was repressed by cotreatment with CLA (t10,c12-CLA > c9,t11-CLA), and the AhR antagonists, 3-methoxy-4-naphthoflavone, and resveratrol. We conclude that the AhR may be a suitable target for prophylactic strategies that target COX-2 expression.

Trans-10, cis-12 conjugated linoleic acid inhibits prolactin-induced cytosolic NADP+ -dependent isocitrate dehydrogenase expression in bovine mammary epithelial cells.

Conjugated linoleic acid (CLA) has been found to exert beneficial effects on lipid profile and repress de novo fatty acid synthesis in mammary gland during lactation. However, the underlying mechanisms responsible for the antilipogenic effects of CLA have not been established. The cytosolic NADP+ -dependent isocitrate dehydrogenase (IDH1) plays a critical role in cholesterol and fatty acid biosynthesis by providing reducing equivalents as NADPH. In previous studies, we documented that the expression of IDH1 in bovine mammary epithelium was modulated by regulators of mammary differentiation and metabolic effectors. In this study, we investigated the short-term effects of prolactin (PRL) and CLA on IDH1 expression in BME-UV bovine mammary epithelial cells. In time-course experiments, we found that the treatment with PRL for 60 and 90 min elicited a significant increase in IDH1 transcript levels. Conversely, the cotreatment of BME-UV cells with PRL plus a CLA mixture for 90 min prevented the accumulation of IDH1 mRNA induced by PRL. In addition, we found that the trans-10, cis-12 CLA, but not the cis-9, trans-11 CLA isomer, inhibited basal- and PRL-induced IDH1 mRNA expression. The inhibitory effects of the trans-10, cis-12 CLA isomer on PRL-induced IDH1 expression accumulation were confirmed by quantitative real time PCR and western-blotting analysis. We propose that the inhibitory effects of CLA on milk fat synthesis in mammary epithelial cells may derive, at least in part, from repression of IDH1 expression.

Conjugated linoleic acid attenuates cyclooxygenase-2 transcriptional activity via an anti-AP-1 mechanism in MCF-7 breast cancer cells.

Overexpression of cyclooxygenase-2 (COX-2) is regarded as a causative factor in the onset of tumorigenesis of the breast. In this study, we investigated the effects of conjugated linoleic acid (CLA) on COX-2 transcription in MCF-7 breast cancer cells. Results of transient transfection studies revealed that treatment with a CLA mix or selected isomers (c9, t11-CLA; t10, c12-CLA) at concentrations ranging from 20 to 80 micromol/L, attenuated COX-2 transcription induced by the proinflammatory agent 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, the CLA mix inhibited TPA-induced activity of the collagenase-1 promoter. Using electrophoretic mobility shift assays, we found that the CLA mix reduced TPA-induced recruitment of nuclear proteins to a cAMP response element (CRE) in the COX-2 promoter and a consensus TPA-responsive element (TRE) in the collagenase-1 promoter. Both CRE and TRE are binding sites for activator protein-1 (AP-1). Binding studies revealed that the t10, c12-CLA isomer was more effective than the CLA mix or c9, t11-CLA in reducing binding of cJun to either the COX-2 CRE or collagenase-1 TRE, whereas linoleic acid increased binding to both elements. Overexpression of the AP-1 member, c-Jun, reversed the inhibitory effects of the CLA mix on COX-2 transcription, and restored binding of nuclear proteins to the CRE and TRE. Collectively, these results suggest that CLA represses AP-1-mediated activation of COX-2 transcription.