Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy.

genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.

SummaryV(D)J recombination is the process by which antibody and T-cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non-homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence-specific nuclease and its pathological role as a structure-specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence-specific and structurespecific nuclease. It also deals with the off-target cleavage of RAGs and its implications in genomic instability.

The RAG (recombination-activating gene) complex is responsible for the generation of antigen receptor diversity by acting as a sequence-specific nuclease. Recent studies have shown that it also acts as a structure-specific nuclease. However, little is known about the factors regulating this activity at the genomic level. We show in the present study that the proximity of a V(D)J nonamer to heteroduplex DNA significantly increases RAG cleavage and binding efficiencies at physiological concentrations of MgCl(2). The position of the nonamer with respect to heteroduplex DNA was important, but not orientation. A spacer length of 18 bp between the nonamer and mismatch was optimal for RAG-mediated DNA cleavage. Mutations to the sequence of the nonamer and deletion of the nonamer-binding domain of RAG1 reinforced the role of the nonamer in the enhancement in RAG cleavage. Interestingly, partial mutation of the nonamer did not significantly reduce RAG cleavage on heteroduplex DNA, suggesting that even cryptic nonamers were sufficient to enhance RAG cleavage. More importantly, we show that the fragile region involved in chromosomal translocations associated with BCL2 (B-cell lymphoma 2) can be cleaved by RAGs following a nonamer-dependent mechanism. Hence our results from the present study suggest that a non-B DNA can replace the heptamer of RSS (recombination signal sequence) when present adjacent to nonamers, explaining the generation of certain chromosomal translocations in lymphoid malignancies.

Background Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. Results We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation. Conclusion This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

BackgroundUbiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL is normally only present in testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription.ResultsWe demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites, to regulate transcription, chromatin looping and insulation.ConclusionThis study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

Integrase inhibitors are a class of antiretroviral drugs used for the treatment of AIDS that target HIV integrase, an enzyme responsible for integration of viral cDNA into host genome. RAG1, a critical enzyme involved in V(D)J recombination exhibits structural similarity to HIV integrase. We find that two integrase inhibitors, Raltegravir and Elvitegravir, interfered with the physiological functions of RAGs such as binding, cleavage and hairpin formation at the recombination signal sequence (RSS), though the effect of Raltegravir was limited. Circular dichroism studies demonstrated a distinct change in the secondary structure of RAG1 central domain (RAG1 shares DDE motif amino acids with integrases), and when incubated with Elvitegravir, an equilibrium dissociation constant (Kd) of 32.53±2.9 μM was determined by Biolayer interferometry, leading to inhibition of its binding to DNA. Besides, using extrachromosomal assays, we show that Elvitegravir inhibited both coding and signal joint formation in pre-B cells. Importantly, treatment with Elvitegravir resulted in significant reduction of mature B lymphocytes in 70% of mice studied. Thus, our study suggests a potential risk associated with the use of Elvitegravir as an antiretroviral drug, considering the evolutionary and structural similarities between HIV integrase and RAGs.

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms of how CTCF dynamically contributes to genome organization is not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs; promoting its oligomerization. Here we found that inhibition of transcription or interfering with CTCF ability to bind RNA through mutations of two of its 11 zinc fingers that are not involved with CTCF binding to its cognate site in vitro, zinc finger-1 (ZF1) or -10 (ZF10), disrupt CTCF association to chromatin. These mutations alter gene expression profiles as CTCF mutants lose their ability to promote local insulation. Our results highlight the importance of RNA as a structural component of the genome, in part by affecting the association of CTCF with chromatin and likely its interaction with other factors. Bullet Points (4 of max 75 words):• Transcriptional inhibition disrupts CTCF binding to chromatin • RNA-binding regions (RBR) in CTCF are found within ZF1 and ZF10 • Local insulation is markedly decreased in ZF1∆ and ZF10∆ mutant rescues • Gene expression and chromatin organization are disrupted by RBR mutants