Bcl-2 Promoter Research Articles

Bcl6 is the nexus transcription factor of T follicular helper cell (TFH) differentiation via a set of repressor-of-repressor gene circuits

Abstract T follicular helper (TFH) cells are a distinct type of CD4 T cells that are essential for most antibody and B lymphocyte responses. TFH regulation and dysregulation is involved in a range of diseases. Bcl6 is the lineage defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl6 controls TFH biology has largely remained unclear, at least in part due to intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches. We ruled out the model that TFH is a default pathway of CD4 T cell by utilizing Bcl6 and Blimp1 double deficient mice in acute virus infection and protein immunization. We showed that Bcl6 has a strong negative autoregulatory loop in TFH cells, by generating mice containing a deletion mutation of an 8-nucleotide Bcl6 binding site in the Bcl6 promoter. To determine putative Bcl6 target TFs, we developed an integrated analytic approach using RNA-seq, BCL6 ChIP-seq, and ATAC-seq data and Bcl6 and Blimp1 double KO mice. Using this approach, Runx2, Runx3, and Klf2 were identified as Bcl6-targeted repressors (Bcl6-r TFs) that regulate important TFH genes. Taken together, Bcl6 controls pleiotropic attributes of TFH differentiation and function (migration, costimulation, inhibitory receptors, and cytokines) via multiple repressor-of-repressor gene circuits.

Critical role of histone H3 lysine 27 demethylase Kdm6b in the homeostasis and function of medullary thymic epithelial cells.

Medullary thymic epithelial cells (mTECs) play a central role in the establishment of T cell central immunological tolerance by promiscuously expressing tissue-restricted antigens (TRAs) and presenting them to developing T cells, leading to deletion of T cells responding to self-antigens. However, molecular mechanisms especially epigenetic regulation of mTEC homeostasis and TRA expression remain elusive. Here we show that the H3K27 demethylase Kdm6b is essential to maintain the postnatal thymic medulla by promoting mTEC survival and regulating the expression of TRA genes. Moreover, mice lacking Kdm6b developed pathological autoimmune disorders. Mechanically, Kdm6b exerted its function by reducing repressive H3K27 trimethylation (H3K27me3) at the promoters of anti-apoptotic gene Bcl2 and a set of Aire-dependent TRA genes. Thus, our findings reveal a dual role of Kdm6b in the regulation of mTEC-mediated T cell central tolerance.

The role of glucocorticoid receptors in the induction and prevention of hippocampal abnormalities in an animal model of posttraumatic stress disorder.

Since the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Numerous clinical studies have led to the hypothesis that elevated glucocorticoid levels in response to extreme stress might trigger a pathophysiological cascade which consequently leads to functional and morphological changes in the hippocampus. To elucidate the pathophysiology of PTSD, we examined the alteration of hippocampal gene expression through the glucocorticoid receptor (GR) in the single prolonged stress (SPS) paradigm, a rat model of PTSD. We measured nuclear GRs by western blot, and the binding of GR to the promoter of Bcl-2 and Bax genes by chromatin immunoprecipitation-qPCR as well as the expression of these 2 genes by RT-PCR in the hippocampus of SPS rats. In addition, we examined the preventive effects of a GR antagonist on SPS-induced molecular, morphological, and behavioral alterations (hippocampal gene expression of Bcl-2 and Bax, hippocampal apoptosis using TUNEL staining, impaired fear memory extinction (FME) using the contextual fear conditioning paradigm). Exposure to SPS increased nuclear GR expression and GR binding to Bcl-2 gene, and decreased Bcl-2 mRNA expression. Administration of GR antagonist immediately after SPS prevented activation of the glucocorticoid cascade, hippocampal apoptosis, and impairment FME in SPS rats. The activation of GRs in response to severe stress may trigger the pathophysiological cascade leading to impaired FME and hippocampal apoptosis. In contrast, administration of GR antagonist could be useful for preventing the development of PTSD.

R-loop-forming Sequences Analysis in Thousands of Viral Genomes Identify A New Common Element in Herpesviruses

R-loops are RNA-DNA hybrid sequences that are emerging players in various biological processes, occurring in both prokaryotic and eukaryotic cells. In viruses, R-loop investigation is limited and functional importance is poorly understood. Here, we performed a computational approach to investigate prevalence, distribution, and location of R-loop forming sequences (RLFS) across more than 6000 viral genomes. A total of 14637 RLFS loci were identified in 1586 viral genomes. Over 70% of RLFS-positive genomes are dsDNA viruses. In the order Herpesvirales, RLFS were presented in all members whereas no RLFS was predicted in the order Ligamenvirales. Analysis of RLFS density in all RLFS-positive genomes revealed unusually high RLFS densities in herpesvirus genomes, with RLFS densities particularly enriched within repeat regions such as the terminal repeats (TRs). RLFS in TRs are positionally conserved between herpesviruses. Validating the computationally-identified RLFS, R-loop formation was experimentally confirmed in the TR and viral Bcl-2 promoter of Kaposi sarcoma-associated herpesvirus (KSHV). These predictions and validations support future analysis of RLFS in regulating the replication, transcription, and genome maintenance of herpesviruses.

Characterization of interaction between Bcl-2 oncogene promoter I-Motif DNA and flavonoids using electrospray ionization mass spectrometry and pressure-assisted capillary electrophoresis frontal analysis

B-cell lymphoma 2 (Bcl-2) is an antiapoptotic protein which is believed to be a triggering factor in developing human tumors. The Bcl-2 C-rich promoter element has been shown to form the i-motif (IM) via cytosine−cytosine (C–C+) base pair building blocks, which can be targeted through the binding of ligands associated with Bcl-2 expression modulation. In this work, we monitored IM development and thermodynamic stability within the Bcl-2 promoter via circular dichroism (CD) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). The results demonstrated that at an acidic pH, as well as in a crowded molecular environment, the Bcl-2 promoter element predominantly exists in a stable intramolecular IM folded state. We further explored the potential of targeting of the Bcl-2 IM to increase chemotherapeutic efficacy. We first used a rapid ESI-MS screening assay to identify possible ligands, finding that three natural flavonoids (P1, P5 and P6) exhibited a clear affinity for IM binding at 1:1 stoichiometry. Relative to P6, P1 and P5 were expected to form the more stable complexes with the Bcl-2 IM in gas phase according to MS/MS data. We further used ESI-MS and pressure-assisted capillary electrophoresis frontal analysis (PACE-FA) to assess the binding constants for these flavonoids in gas and liquid phases, respectively, with the latter considering both specific and non-specific binding. We found P5 and P6 to specifically bind the Bcl-2 IM with binding constants of ~104 M−1. P1 binding was confirmed to be due to both specific and nonspecific interactions, and the specific binding constant (8.67 × 103 M−1) was found much less significant than the binding constant in gas phase. Taken all these observations into consideration, the specific binding of selected flavonoids to the Bcl-2 IM may prove to be a potential ligand for modulating Bcl-2 gene expression.

AID assists DNMT1 to attenuate BCL6 expression through DNA methylation in diffuse large B-cell lymphoma cell lines

The BCL6 proto-oncogene encodes a transcriptional repressor, which is required for germinal centers (GCs) formation and lymphomagenesis. Previous studies have been reported that the constitutive expression of BCL6 leads to diffuse large B cell lymphoma (DLBCL) through activation-induced cytidine deaminase (AID) mediated chromosomal translocations and mutations. However, other DLBCLs (45%) without structural variants were characterized by abnormally high level of BCL6 expression through an unknown mechanism. Herein, we report that deficiency in AID or methyltransferase 1 (DNMT1) triggers high level of BCL6 expression. AID-DNMT1 complex binds to −0.4 kb −0 kb region of BCL6 promoter and contributes to generate BCL6 methylation which results in inhibition of BCL6 expression. The proteasome pathway inhibitor MG132 induces accumulation of AID and DNMT1, causes decreased BCL6 expression, and leads to cell apoptosis and tumor growth inhibition in DLBCL cell xenograft mice. These findings propose mechanistic insight into an alternative cofactor role of AID in assisting DNMT1 to maintain BCL6 methylation, thus suppress BCL6 transcription in DLBCL. This novel mechanism will provide a new drug selection in the therapeutic approach to DLBCL in the future.

Crlz-1 Controls Germinal Center Reaction by Relaying a Wnt Signal to the Bcl-6 Expression in Centroblasts during Humoral Immune Responses.

Crlz-1 was expressed along with Wnt3a in the rapidly proliferating centroblasts within the dark zone of germinal center (GC) during humoral immune responses. Significantly, Crlz-1 relayed a Wnt/β-catenin signal to the expression of Bcl-6, the master regulator of centroblasts, by mobilizing the cytoplasmic CBFβ into the nucleus to allow Runx/CBFβ heterodimerization and its subsequent binding to the Bcl-6 promoter. The knockdown of Crlz-1 or β-catenin, as well as inhibition of Wnt signaling in the centroblasts, led to the decreased expression of Bcl-6 and, thereby, the altered expression of its various target genes, resulting in their diminished proliferation. Consistently, the administration of Wnt inhibitors into the immunized mice impaired or abolished GC reaction, with concomitant decreases of Crlz-1 and Bcl-6 expression and, thus, centroblastic proliferation. Our observation that Wnt/β-catenin signaling via Crlz-1 regulates GC reaction would suggest developmental strategies for vaccine adjuvants and cancer therapeutics because both immune efficacy and accidental lymphoma depend on GC reaction. Our studies of Crlz-1 were performed using human cell lines, mice, and their primary cells.

An OCT2 / OCA-B / MEF2B Ternary Complex Controls the Activity and Architecture of an Essential Locus Control Region for Normal and Malignant Germinal Center B-Cells

Melnick: Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding.

Rationale for Targeting BCL6 in MLL-Rearranged B-ALL

Background and significance: MLL-gene rearrangements occur in ~10% of B-cell acute lymphoblastic leukemia (B-ALL) and 70% of infant B-ALL and define a group of patients with dismal outcomes. MLL belongs to the family of histone methyltransferases and plays a critical role in maintaining hematopoietic stem cells (Jude et al. 2007). Analyzing gene expression data from 207 children with high-risk ALL, we found that BCL6 is a predictor of poor clinical outcome in B-ALL, particularly in cases with MLL rearrangements. Thus, while the mechanisms of drug-resistance in MLL-rearranged B-ALL are largely unknown, we studied here a potential role of BCL6 in promoting the aggressive and refractory phenotype in this subtype of B-ALL. Results: Immunohistochemical staining of bone marrow biopsies from 10 of 11 MLL-rearranged B-ALL samples studied revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug-resistance in B-ALL (Duy et al. Nature 2011). Our genetic and ChIP-seq analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and upregulated BCL6 expression. While oncogenic MLL-fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to upregulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL-fusions strongly induced transcriptional activation of the pro-apoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small-molecule (FX1) BCL6-inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL-fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL. Discussion: Pharmacological inhibition of BCL6 using a retro-inverso peptide inhibitor (RI-BPI) compromised MLL-rearranged B-ALL leukemia-initiation and subverted vincristine-resistance. A central mechanistic aspect of BCL6-function in MLL-rearranged B-ALL involves transcriptional repression of the pro-apoptotic BH3-only molecule Bim (BCL2L11). Profiling for BH3-only proteins in various B-ALL subtypes revealed that MLL-rearranged B-ALL is associated with increased expression of the pro-apoptotic protein BIM (Benito et al. 2015). ABT-199 (venetoclax) is a BH3-mimetic that disrupts the interaction between BCL2 and BIM, leading to BIM release and induction of apoptosis. Previous studies have found that MLL-rearranged leukemia cells are sensitive to the BCL2 inhibitor ABT-199. Besides BCL2, we here identified BCL6 as a central antagonist of pro-apoptotic BIM function in MLL-rearranged B-ALL cells. In genetic experiments, we showed that oncogenic MLL-fusions strongly activated BCL2L11 transcription reinforcing the notion that constitutively high Bim expression levels represents an important and selective vulnerability in MLL-rearranged B-ALL. Both BCL2 and BCL6 represent crucial antagonists of BIM in MLL-rearranged B-ALL, BCL2 mediating BIM-sequestration and BCL6 being required for transcriptional repression of BIM. In support of this scenario, peptide (RI-BPI) and small molecule (FX1) inhibitors of BCL6 strongly synergized with blockade of BCL2-mediated BIM sequestration (ABT-199) in killing MLL-rearranged B-ALL cells. Figure 1 Disclosures Armstrong: AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; Janssen: Research Funding; Novartis: Research Funding; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership. Melnick:Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Milne:OxStem Ltd.: Equity Ownership.

Functional interplay between p53 and Δ133p53 in adaptive stress response.

Apart from its well-known prodeath activity, p53 is also implicated in promoting cell survival. How p53 can mediate such seemingly opposing effects is largely unclear. We report here a novel mechanism in which p53-mediated proapoptosis is switched to antiapoptosis via its interaction with a p53 isoform, Δ133p53. We show that the expression of Δ133p53 is induced by mild or a moderate level of stress via an HIF1-dependent mechanism. Increased Δ133p53 levels contribute to the adaptive response by shifting the p53 binding at the Bcl2 promoter from suppressive responsive elements (RE) to activating REs, resulting in induction of Bcl2. In accordance with this mode of action, pretreatment of mice with mild stress induces Δ133p53 and Bcl2, which is associated with protection of animals from toxicity caused by high doses of DNA damage agents. Collectively, our work uncovers a novel functional interplay between p53 and Δ133p53 determining cell fate; survival or death in response to stress.

Estrogen suppresses bcl2 expression through dnmt1 in Japanese eel ovarian follicle.

Japanese eels are commercially valuable species in Asian aquaculture. This study evaluated whether salmon pituitary extract (SPE) or 17β-estradiol (E2) treatment can induce cytotoxic activity in eel ovarian follicles. Follicular cells died after exposure SPE for 24-h culture in an in vitro culture. Moreover, the E2 treatment also significantly reduced follicular cell counts. These results reveal that the inhibition of follicular cell numbers by SPE may occur through SPE-induced steroidogenesis. Results of a quantitative PCR analysis indicated that adding E2 to the culture decreased bcl2 and increased dnmt1 mRNA expression in Japanese eel follicular cells after 24 h. The results of a promoter assay revealed that E2 significantly increase dnmt1 promoter activity through estrogen receptor-binding site. An in silico analysis predicted several putative transcription factors targeting the bcl2 gene promoter region. Methylation of the bcl2 promoter accounted for the downregulation of bcl2 by E2-mediated dnmt1. The DNA methylation level of the bcl2 gene was significantly higher in E2-treated follicular cells than that in the control group. Finally, the E2-induced hypermethylation pattern of the bcl2 promoter and the reduction in follicular cell numbers were suppressed by adding an MTase inhibitor. Our findings demonstrate that estrogen has a negative effect on the reproductive system of female eels by regulating an epigenetic mechanism during artificial maturation.

DHX33 Interacts with AP-2β To Regulate Bcl-2 Gene Expression and Promote Cancer Cell Survival.

The RNA helicase DHX33 has been found to be overexpressed in human cancers, where it promotes cancer development. Previous reports have shown that DHX33 deficiency caused cancer cell apoptosis, but the underlying mechanism remains unknown. In this study, we discovered that DHX33 regulates Bcl-2 family protein expression. In multiple human cancer cell lines, DHX33 was found to stimulate the transcription of Bcl-2 Mechanistically, we found that DHX33 interacts with the AP-2β transcription factor and acts as a coactivator to stimulate Bcl-2 gene transcription. DHX33 deficiency abolished the loading of AP-2β onto the promoter of Bcl-2 and thereby reduced the recruitment of active RNA polymerase II during transcription initiation. Acute knockdown of DHX33 in multiple human cancer cells caused decreased Bcl-2 protein level, which ultimately triggered mitochondrion-mediated cellular apoptosis. In addition, we found that normal human lung and mammary epithelial cells were less sensitive to acute DHX33 knockdown, implying that cancer cells are uniquely responsive to DHX33 reduction. These data support the notion that disruption of DHX33 function could be an important application for cancer therapy.

Rationale for targeting BCL6 in MLL-rearranged acute lymphoblastic leukemia.

Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene occur in ∼10% of B-cell acute lymphoblastic leukemia (B-ALL) and define a group of patients with dismal outcomes. Immunohistochemical staining of bone marrow biopsies from most of these patients revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug resistance in B-ALL. Our genetic and ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and up-regulated BCL6 expression. While oncogenic MLL fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to up-regulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL fusions strongly induced transcriptional activation of the proapoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small molecule (FX1) BCL6 inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL.

HOXA10 induces BCL2 expression, inhibits apoptosis, and promotes cell proliferation in gastric cancer.

Homeobox A10 (HOXA10) has been implicated critical for the promotion of carcinogenesis, but the underlying mechanism between HOXA10 and malignant gastric cancer (GC) phenotype remains elusive. In the present study, we analyzed and validated that HOXA10 and BCL2 expressions were elevated both at the mRNA and protein levels in GC tissues. Upregulated HOXA10 promoted GC cell proliferation with reduced apoptosis in vitro and accelerated GC tumor growth in vivo. Bioinformatics analysis and quantitative real‐time polymerase chain reaction (qRT‐PCR) experiment inferred that HOXA10 might upregulate the expression of BCL2. By performing western blot, chromatin immunoprecipitation and quantitative PCR (ChIP‐qPCR), and rescue experiment, we found that HOXA10 might bind to BCL2 promoter region, induce its expression, and thus inhibit intrinsic apoptosis pathway. Moreover, higher expression of HOXA10 and BCL2 predicted poor overall survival (OS) in GC patients. In summary, our study indicated that HOXA10 was upregulated in GC, and that HOXA10 might promote cell proliferation by elevating BCL2 expression and inhibiting apoptosis.

Bach2 Controls T Follicular Helper Cells by Direct Repression of Bcl-6.

T follicular helper (Tfh) cells are a specialized T cell subset that regulates the long-lived production of highly specific Abs by B cells during the germinal center (GC) reaction. However, the transcriptional network sustaining the Tfh cell phenotype and function is still incompletely understood. In this study, we identify the transcription factor Bach2 as a central negative regulator of Tfh cells. Ectopic overexpression of Bach2 in murine Tfh cells resulted in a rapid loss of their phenotype and subsequent breakdown of the GC response. Low Bach2 expression levels are required to maintain high expression of the signature cytokine IL-21, the coinhibitory receptor TIGIT and the transcriptional repressor Bcl-6. In stark contrast to the regulatory network in GC B cells, Bach2 in Tfh cells is not coexpressed with Bcl-6 at high levels to inhibit the antagonizing factor Blimp-1, but suppresses Bcl-6 by direct binding to the promoter. These data reveal that by replacing an activating complex of Batf and Irf-4 at the Bcl-6 promoter, Bach2 regulates the transcriptional network of Tfh cells in a different way, as in GC B cells.

Folding/unfolding kinetics of G-quadruplexes upstream of the P1 promoter of the human BCL-2 oncogene

The G-rich Pu39 region of the P1 promoter of the oncogene BCL-2, an apoptosis regulator, can fold into multiple G-quadruplex (G4) structures. Bcl2-2345 and Bcl2-1245 are two major G4 species forming with high thermal stability and distinct topologies in the Pu39 region, but their folding/unfolding kinetics have not yet been investigated. Here, we used magnetic tweezers to measure the mechanical stability and the folding/unfolding kinetics of the Bcl2-2345 and Bcl2-1245 G4 structures. We report that the hybrid-stranded Bcl2-2345 G4 had a lower mechanical stability than the parallel-stranded Bcl2-1245 G4. We observed that the Bcl2-2345 G4 is a kinetically favored structure, whereas the Bcl2-1245 G4, with a slow unfolding rate, may function as a kinetic barrier for transcription. We also determined that in addition to the Bcl2-2345 and Bcl2-1245 G4s, other stable DNA secondary structures, such as a hybrid-stranded Bcl2-1234 G4, can also form in the Pu39 sequence. The characterization of the folding/unfolding kinetics of specific G4s reported here sheds light on the participation of G4s during gene transcription and provides information for designing G4-targeting small molecules that could modulate BCL-2 gene expression.

Structure and Dynamics of the Bcl-2 Promoter G-Quadruplex using the Drude Polarizable Force Field

G-quadruplexes (GQ) are noncanonical nucleic acid structures that form in guanine-rich sequences and exist in a variety of topological forms. The guanine bases in GQ arrange in a square planar configuration and stack to form tetrads via Hoogsteen hydrogen bonding and coordination of ions like K+. These highly ordered secondary structures are implicated in the regulation of gene expression and genome maintenance, and alterations to their structures contribute to diseases such as cancer. The B-cell CLL/lymphoma 2 (bcl-2) oncogene is associated with a variety of cancers and its promoter features a GQ that arises due to two consecutive substitutions of guanine with thymine. Understanding the forces driving GQ folding and stabilization is required for developing novel therapeutics that target these structures. To develop a greater understanding of the atomistic details in the bcl-2 GQ, we performed molecular dynamics simulations using the CHARMM36 additive and Drude-2017 polarizable force fields. In the classical Drude oscillator model, electronic degrees of freedom are modeled as negatively charged particles attached harmonically to all heavy atoms. This approach overcomes limitations of nonpolarizable force fields, which approximate average polarization via assignment of partial charges. We performed simulations of the mutant bcl-2 promoter GQ and created the wild-type sequence by reverting the point mutations. The wild-type sequence was then thermally denatured and allowed to re-fold to determine the structural differences between the mutant bcl-2 GQ and the native, wild-type fold. Our analysis provides insight into the stability of the bcl-2 GQ structure, backbone conformational ensembles in dihedral space, base-ion interaction energetics, and the magnitude and fluctuation of dipole moments. These simulations illustrate that polarization is integral for tetrad stability and suggests that the Drude force field is essential for simulating GQ structures.

Genetic and epigenetic analysis of the BAX and BCL2 in the placenta of pregnant women complicated by preeclampsia.

The current study examined the effects of BAX and BCL2 polymorphisms and methylation as well as mRNA expression on susceptibility to PE. After delivery, the placentas were collected from 92 women with PE, as well as 106 normotensive pregnant women. The BAX rs4645878 and BCL2 rs2279115 polymorphisms were genotyped by the PCR-RFLP method. Methylation-specific PCR (MSP) was used for analysis of promoter methylation. mRNA expression was assayed by Quantitative RT-PCR. In addition, in silico analysis was performed by bioinformatics tools. There was no relationship between PE and placental BAX rs4645878 and BCL2 rs2279115 polymorphisms. The groups were not significantly different regarding the promoter methylation of BAX gene. Nonetheless, the MM status of BCL2 promoter had a significantly higher frequency in the PE group and was associated with 2.7-fold higher risk of PE (OR=2.7, 95% CI=1.3-5.6; P = 0.01). The relative mRNA expression of BCL2 was decreased in the placentas of PE women (P < 0.0001). The expression of BAX gene was not significantly different between the two groups. There was no association between placental BAX rs4645878 and BCL2 rs2279115 polymorphisms and mRNA expression levels. In silico analysis indicated that BAX rs4645878 and BCL2 rs2279115 polymorphisms were located in the core recognition site of different transcription factors and these substitutions of wild allele resulted in the loss and/ or change of these binding sites and subsequently may alter BCL2 and BAX expression. This study showed that the BAX and BCL2 polymorphisms and BAX promoter methylation were not associated with PE risk. The BCL2 promoter methylation was associated with lower BCL2 expression and higher PE susceptibility.

Epigenetically upregulated oncoprotein PLCE1 drives esophageal carcinoma angiogenesis and proliferation via activating the PI-PLC\u03b5-NF-\u03baB signaling pathway and VEGF-C/ Bcl-2 expression

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Neovascularization during tumorigenesis supplies oxygen and nutrients to proliferative tumor cells, and serves as a conduit for migration. Targeting oncogenes involved in angiogenesis is needed to treat organ-confined and locally advanced ESCC. Although the phospholipase C epsilon-1 (PLCE1) gene was originally identified as a susceptibility gene for ESCC, how PLCE1 is involved in ESCC is unclear.MethodsMatrix-assisted laser desorption ionization time-of-flight mass spectrometry were used to measure the methylation status of the PLCE1 promoter region. To validate the underlying mechanism for PLCE1 in constitutive activation of the NF-κB signaling pathway, we performed studies using in vitro and in vivo assays and samples from 368 formalin-fixed esophageal cancer tissues and 215 normal tissues with IHC using tissue microarrays and the Cancer Genome Atlas dataset.ResultsWe report that hypomethylation-associated up-regulation of PLCE1 expression was correlated with tumor angiogenesis and poor prognosis in ESCC cohorts. PLCE1 can activate NF-κB through phosphoinositide-phospholipase C-ε (PI-PLCε) signaling pathway. Furthermore, PLCE1 can bind p65 and IκBα proteins, promoting IκBα-S32 and p65-S536 phosphorylation. Consequently, phosphorylated IκBα promotes nuclear translocation of p50/p65 and p65, as a transcription factor, can bind vascular endothelial growth factor-C and bcl-2 promoters, enhancing angiogenesis and inhibiting apoptosis in vitro. Moreover, xenograft tumors in nude mice proved that PLCE1 can induce angiogenesis, inhibit apoptosis, and increase tumor aggressiveness via the NF-κB signaling pathway in vivo.ConclusionsOur findings not only provide evidence that hypomethylation-induced PLCE1 confers angiogenesis and proliferation in ESCC by activating PI-PLCε-NF-κB signaling pathway and VEGF-C/Bcl-2 expression, but also suggest that modulation of PLCE1 by epigenetic modification or a selective inhibitor may be a promising therapeutic approach for the treatment of ESCC.

Dietary pomegranate supplement alleviates murine pancreatitis by modulating Nrf2-p21 interaction and controlling apoptosis to survival switch

Dietary supplementation of polyphenol-rich pomegranate extract (POMx) has been shown to have anti-oxidant and anti-inflammatory activities. Here, we evaluate the efficacy of POMx in mitigating pancreatitis in mice and provide a mechanistic outline of the process. Age-matched male Swiss albino mice were injected with Lipopolysaccharide (LPS) and given POMx supplement alone or in combination with LPS. After 4 weeks of treatment histological scoring for pancreatic edema and vacuolization was performed. Serum insulin levels were estimated and the glucose tolerance test (IPGTT) data revealed that POMx reduced inflammation induced hyperglycemia in mice. Analysis of TLR4, IκB expression, and NF-κB nuclear translocation, and concentrations of IL-6 and TNFα showed that POMx is able to modulate the molecular instigators of inflammatory responses. Annexin V assay indicated that POMx protects against inflammation-mediated apoptosis in the pancreas. Expression profile of SAPK/JNK pathway, p53, Bax, Bcl-2 and Caspase-3 validate an apoptotic to survival shift in POMx treatment group. Co-immunoprecipitation studies show that POMx stabilizes p21 and Nrf2 interaction and increases its nuclear translocation. The study also proves that the nuclear fraction of Nrf2 is able to bind to the Bcl-2 promoter and activate an anti-apoptotic program. The findings of our study underline an anti-inflammatory, anti-oxidative and anti-apoptotic role of POMx and provide a mechanistic idea of how POMx confers protection during pancreatitis.