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Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5′ capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA–DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

Notoginsenoside R1 (NGR1), the primary bioactive compound found in Panax notoginseng, is believed to have antihypertrophic and antiapoptotic properties, and has long been used to prevent and treat cardiovascular diseases. However, its potential role in prevention of diabetic cardiomyopathy remains unclear. The present study aimed to investigate the mechanism of NGR1 action in high glucose-induced cell injury. H9c2 cardiomyocytes were cultured in a high-glucose medium as an in-vitro model, and apoptotic cells were visualized using TUNEL staining. Expression of Nrf2 and HO-1 was measured using Western blotting or reverse transcription-quantitative PCR (RT-qPCR). The Nrf2 small interfering (si) RNA was transfected into cardiomyocytes using Opti-MEM containing Lipofectamine® RNAiMAX. NGR1 protected H9c2 cardiomyocytes from cell death, apoptosis and hypertrophy induced by high glucose concentration. Expression of auricular natriuretic peptide and brain natriuretic peptide was remarkably reduced in NGR1-treated H9C2 cells. Western blot analysis showed that high glucose concentration markedly inhibited AMPK, Nrf2 and HO-1, and this could be reversed by NGR1 treatment. However, the cardioprotective effect of NGR1 was attenuated by compound C, which reverses Nrf2 and HO-1 expression levels, suggesting that AMPK upregulates Nrf2 and HO-1 gene expression, protein synthesis and secretion. Transfection of H9C2 cells with Nrf2 siRNA markedly reduced the cardioprotective effect of NGR1 via reduced expression of HO-1. These results indicated that NGR1 attenuated high glucose-induced cell injury via AMPK/Nrf2 signaling and its downstream target, the HO-1 pathway. We conclude that the cardioprotective effects of NGR1 result from upregulation of AMPK/Nrf2 signaling and HO-1 expression in cardiomyocytes. Our findings suggest that NGR1 treatment might provide a novel therapy for diabetic cardiomyopathy.

CB1 receptor (CB1R) antagonism improves the deleterious effects of a high-fat diet (HFD) by reducing body fat mass and adipocyte cell size. Previous studies demonstrated that the beneficial effects of the CB1R antagonist rimonabant (RIM) in white adipose tissue (WAT) are partially due to an increase of mitochondria numbers and upregulation thermogenesis markers, suggesting an induction of WAT beiging. However, the molecular mechanism by which CB1R antagonism induces weight loss and WAT beiging is unclear. In this study, we probed for genes associated with beiging and explored longitudinal molecular mechanisms by which the beiging process occurs. HFD dogs received either RIM (HFD+RIM) or placebo (PL) (HFD+PL) for 16 wk. Several genes involved in beiging were increased in HFD+RIM compared with pre-fat, HFD, and HFD+PL. We evaluated lipolysis and its regulators including natriuretic peptide (NP) and its receptors ( NPRs), β-1 and β-3 adrenergic receptor ( β1R, β3R) genes. These genes were increased in WAT depots, accompanied by an increase in lipolysis in HFD+RIM. In addition, RIM decreased markers of inflammation and increased adiponectin receptors in WAT. We observed a small but significant increase in UCP1; therefore, we evaluated the newly discovered UCP1-independent thermogenesis pathway. We confirmed that SERCA2b and RYR2, the two key genes involved in this pathway, were upregulated in the WAT. Our data suggest that the upregulation of NPRs, β-1R and β-3R, lipolysis, and SERCA2b and RYR2 may be one of the mechanisms by which RIM promotes beiging and overall the improvement of metabolic homeostasis induced by RIM.

Enhancers generate bidirectional noncoding enhancer RNAs that may regulate gene expression. At present, mechanisms of eRNA functions are not fully understood. Here, we report an antisense eRNA PEARL that is transcribed from the protocadherin α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA-fragment editing, CRISPRi, and CRISPRa strategies, in conjunction with ChIRP, MeDIP, and DRIP experiments, we find that PEARL regulates Pcdhα expression by forming local R-loop in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancer and target promoters. These findings have important implications regarding mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

Rationale: The Polycomb group (PcG) protein EZH2 is implicated in cancer progression due to its frequent overexpression in many cancer types and therefore is a promising therapeutic target. Forkhead box transcription factor-1 (FOXO1) is a tumor suppressor that is often transcriptionally downregulated in human cancers such as prostate cancer although the underlying regulatory mechanisms remain elusive. Methods: Analysis of EZH2 ChIP-seq and ChIP-on-chip data in various cell types was performed. ChIP-qPCR, RT-qPCR, and western blot analyses were conducted to determine the mechanism by which EZH2 represses FOXO1 expression. Immunohistochemistry was employed to assess the correlation between EZH2 and FOXO1 protein expression in prostate cancer patient specimens. In vitro MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and animal experiments were performed to determine the anti-cancer efficacy of EZH2 inhibitor alone or in combination of docetaxel, a chemotherapy agent of the taxane family, and dependency of the efficacy on FOXO1 expression. Results: We demonstrated that EZH2 binds to the FOXO1 gene promoter. EZH2 represses FOXO1 gene expression at the transcriptional level. EZH2 protein level inversely correlated with FOXO1 protein expression in prostate cancer patient specimens. This repression requires the methyltransferase activity and the functional PRC2 complex. While effectively inducing loss of viability of PTEN-positive 22Rv1 prostate cancer cells, EZH2 inhibitor failed to inhibit growth of PTEN-negative C4-2 prostate cancer cells. Co-treatment with docetaxel overcame EZH2 inhibitor resistance in PTEN-negative cancer cells in vitro and in mice. This effect was largely mediated by docetaxel-induced nuclear localization and activation of FOXO1. Conclusions: This study identifies FOXO1 as a bona fide repression target of EZH2 and an essential mediator of EZH2 inhibition-induced cell death. Our findings suggest that EZH2 repression of FOXO1 can be targeted by EZH2 inhibitor as a monotherapy for PTEN-proficient cancers or in combination with taxane for treatment of cancers with PTEN mutation or deletion.

The Cydia pomonella granulovirus (CpGV) has been used as a biological control agent of codling moth (Cydia pomonella), a severe global pest on pome fruit. Despite the economic importance, our knowledge of its molecular biology is still limited and a detailed picture of its gene expression is still missing. Here, we sequenced the transcriptome of codling moth larvae infected with the Mexican isolate CpGV-M and analyzed the expression of viral genes at 12, 48, and 96 h post infection (hpi). The results showed that two genes (p6.9 and pp31/39K) related to DNA binding of virus production, were highly expressed at 48 and 96 hpi. From 48 to 96 hpi, the expression of genes associated with virus replication and dissemination decreased, whereas the expression of genes related to infectious virion production and per os infectivity increased. This study provides a comprehensive view of CpGV gene expression patterns in host larvae.

During development, gene expression is spatiotemporally regulated by long-distance chromatin interactions between distal enhancers and target promoters. However, how specificity of the interactions between enhancers and promoters is achieved remains largely unknown. As there are far more enhancers than promoters in mammalian genomes, the complexities of enhancer choice during gene regulation remain obscure. CTCF, the CCCTC-binding factor that directionally binds to a vast range of genomic sites known as CBSs (CTCF-binding sites), mediates oriented chromatin looping between a substantial set of distal enhancers and target promoters. To investigate mechanisms by which CTCF engages in enhancer choice, we used CRISPR/Cas9-based DNA-fragment editing to duplicate CBS-containing enhancers and promoters in the native genomic locus of the clustered Pcdhα genes. We found that the promoter is regulated by the proximal one among duplicated enhancers and that this choice is dependent on CTCF-mediated directional enhancer-promoter looping. In addition, gene expression is unaltered upon the switch of enhancers. Moreover, after promoter duplication, only the proximal promoter is chosen by CTCF-mediated directional chromatin looping to contact with the distal enhancer. Finally, we demonstrated that both enhancer activation and chromatin looping with the promoter are essential for gene expression. These findings have important implications regarding the role of CTCF in specific interactions between enhancers and promoters as well as developmental regulation of gene expression by enhancer switching.

Accurately predicting and testing the types of Pulmonary arterial hypertension (PAH) of each patient using cost-effective microarray-based expression data and machine learning algorithms could greatly help either identifying the most targeting medicine or adopting other therapeutic measures that could correct/restore defective genetic signaling at the early stage. Furthermore, the prediction model construction processes can also help identifying highly informative genes controlling PAH, leading to enhanced understanding of the disease etiology and molecular pathways. In this study, we used several different gene filtering methods based on microarray expression data obtained from a high-quality patient PAH dataset. Following that, we proposed a novel feature selection and refinement algorithm in conjunction with well-known machine learning methods to identify a small set of highly informative genes. Results indicated that clusters of small-expression genes could be extremely informative at predicting and differentiating different forms of PAH. Additionally, our proposed novel feature refinement algorithm could lead to significant enhancement in model performance. To summarize, integrated with state-of-the-art machine learning and novel feature refining algorithms, the most accurate models could provide near-perfect classification accuracies using very few (close to ten) low-expression genes., Author summary Pulmonary arterial hypertension (PAH) is a serious and progressive disease, with only a roughly 50% of 5-year survival rate even with best available therapies. Accurately detecting/differentiating different forms of PAH and developing drugs that could directly target at genes involved in PAH pathogenesis are essential. We proposed a computational approach using low-cost microarray data collected from a clinical trial and had accurately predicted each PAH group. In particular, we considered the fact that there might exist some low-expression genes that were usually discarded by researchers but might function collectively and significantly controlling the disease in each case. Therefore, we had developed different filtering algorithms that intentionally selected those low-expression genes for constructing prediction model. Using a few highly informative low-expression genes that had never been extensively investigated before, our systematic approach had produced models that could offer prefect accuracy in predicting PAH. Additionally, our analysis also found that the composition of gene factors controlling the PAH etiology under each form are quite different from each other.

International audience; The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237−780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.

Objective To study whether miR-200a and miR-200b target PTEN gene expression to regulate the endometrial cancer cell growth in vitro. Methods Endometrial cancer cells ECC-1 were cultured and transfected with the miR-200a and miR-200b mimics and inhibitors as well as the negative control mimics and inhibitors, and then the cell proliferation activity as well as the expression of PTEN and downstream genes in cells was determined; after transfection of miR-200a and miR-200b mimics as well as PTEN-3′UTR luciferase report gene plasmids, the fluorescence activity of luciferase reporter gene was determined. Results 12 h, 24 h and 48 h after transfection, the cell proliferation activity of miR-200a mimics group and miR-200b mimics group were significantly higher than those of NC mimics group while the cell proliferation activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly lower than those of NC inhibitor group; 48 h after transfection, PTEN expression in cells and PTEN-3′UTR luciferase reporter gene fluorescence activity of miR-200a mimics group and miR-200b mimics group were significantly lower than those of NC mimics group while p-PI3K and p-Akt expression were significantly higher than those of NC mimics group; PTEN expression in cells and PTEN-3′UTR luciferase reporter gene fluorescence activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly higher than those of NC inhibitor group while p-PI3K and p-Akt expression were significantly lower than those of NC inhibitor group. Conclusion miR-200a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.

It is acknowledged that the targeting ability of multivalent ligand-modified nanoparticles (MLNs) strongly depends on the ligand spatial presentation determined by ligand valency. However, the receptor overexpression level varies between different types or stages of tumors. Thus, it is essential to explore the influence of ligand valency on the targeting ability of MLNs to tumors with different levels of receptor overexpression. In this study, a dual-acting agent raltitrexed was used as a ligand to target the folate receptor (FR). Different copies of the raltitrexed-modified multivalent dendritic polyethyleneimine ligand cluster PRn (n = 2, 4, and 8) were conjugated onto magnetic nanoparticles to form multivalent magnetic NPs (MMNs) with different valences. The in vitro studies demonstrated that Fe-PR4 was the most effective valency in the treatment of high FR overexpressing KB cells with a decentralized receptor distribution, owing to the fact that Fe-PR2 was negative in statistical rebinding and Fe-PR8 could induce steric hindrance in the limited binding area. Instead, in moderate FR overexpressing HeLa cells with clustered receptor display, the extra ligands on Fe-PR8 would facilitate statistical rebinding more beneficially. Furthermore, in in vivo tumor inhibition and targeted magnetic resonance imaging (MRI) of KB tumors and another moderate FR expressing H22 tumor, similar results were obtained with the cell experiments. Overall, the optimizable treatment effect of Fe-PRn by modulating the ligand valency based on the overexpressing tumor receptor distribution behavior supports the potential of Fe-PRn as a nanomedicine for personalized theranostics.

CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the Pcdhα and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter usage. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.

G protein-coupled receptor 50 (GPR50), a risk factor for major depressive disorder and bipolar affective disorder, is expressed in both the developmental and adult brain. However, the function of GPR50 in the brain remains unknown. We here show GPR50 is expressed by neural progenitor cells (NPCs) in the ventricular zone of embryonic brain. Knockdown of GPR50 with a small interference RNA (siRNA) decreased self-renewal and neuronal differentiation, but not glial differentiation of NPCs. Moreover, overexpression of either full-length GPR50 or the intracellular domain of GPR50, rather than the truncated GPR50 in which the intracellular domain is deleted in, increased neuronal differentiation, indicating that GPR50 promotes neuronal differentiation of NPCs in an intracellular domain-dependent manner. We further described that the transcriptional activity of the intracellular domain of notch on Hes1 gene was repressed by overexpression of GPR50. In addition, decreased levels of transcription factor 7-like 2 (TCF7L2) mRNA was observed in GPR50 siRNA-transfected NPCs, suggesting that knockdown of GPR50 impairs wnt/β-catenin signaling. Moreover, the mRNA levels of neurogenin (Ngn) 1, Ngn2 and cyclin D1, the target genes of notch and wnt/β-catenin signalings, in NPCs were reduced by knockdown of GPR50. Therefore, GPR50 promotes self-renewal and neuronal differentiation of NPCs possibly through regulation of notch and wnt/β-catenin signalings.

Pituitary stalk interruption syndrome (PSIS) is a rare type of hypopituitarism manifesting various degrees of pituitary hormone deficiency. Although mutations have been identified in some familial cases, the underpinning mechanisms of sporadic patients with PSIS who are in a vast majority remain elusive, necessitating a comprehensive study using systemic approaches. We postulate that other genetic mechanisms may be responsible for the sporadic PSIS. To test this hypothesis, we conducted a study in 24 patients with PSIS of Han Chinese with no family history using whole‐exome sequencing (WES) and bioinformatic analysis. We identified a group of heterozygous mutations in 92% (22 of 24) of the patients, and these genes are mostly associated with Notch, Shh, Wnt signalling pathways. Importantly, 83% (20 of 24) of the patients had more than one mutation in those pathways suggesting synergy of compound mutations underpin the pathogenesis of sporadic PSIS.

MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. Here, we identified that miR-543 is up-regulated in gefitinib-resistant non-small cell lung cancer (NSCLC) patients comparing gefitinib-sensitive ones. It promotes NSCLC cell proliferation by negatively regulates its target gene PTEN. In NSCLC cell lines, CCK-8 proliferation assay indicated that the cell proliferation is promoted by miR-543 mimics. Transwell assay showed that miR-543 mimics promotes the invasion and migration of NSCLC cells. Luciferase assays confirmed that miR-543 directly binds to the 3'untranslated region of PTEN, and western blotting showed that miR-543 suppresses the expression of PTEN at the protein level. This study indicates that miR-543 promtes proliferation and invasion of NSCLC cell lines by PTEN. The miR-543 may represent a potential therapeutic target for gefitinib-resistant NSCLC intervention.

Rheumatoid arthritis (RA) is a chronic, systemic and autoimmune disease with overexpression inflammation cytokines. The biological therapy targeting these inflammatory cytokines has been applied for the clinic. Drugs aimed at a single target are ineffective in some patients with RA. However, double-target and multi-target drugs have huge advantages in therapy. Interleukin-1β (IL-1β) and Interleukin-17A (IL-17A) are the keys to inflammatory factors in RA. The bispecific antibody(BsAb)against both human IL-1β and human IL-17A was formed and expressed in E. coli. The binding specificity and efficiency of the BsAb was tested by enzyme-linked immunosorbent assay, Western blotting and several cells experiments including THP-1, 3T3-L1 and L929 in vitro. Different doses of BsAb (5, 2, 0.8mg/kg) were compared in collagen-induced arthritis (CIA) mice, with Adalimumab and Dexamethasone as the positive control. The effects on mice were determined by the degree of arthritis severity, cytokines levels, the level of IgG against CII and rheumatoid factor level in serum, the transcription levels of relative cytokines in the spleen, and histological damage. Furthermore, the activation of NF-κB was analyzed by Western blotting. In conclusion, BsAb can bind with IL-1β and IL-17A to alleviate the severity of arthritis, to decrease the levels of inflammation cytokines in serum, to down-regulate the expression of IL-1β, IL-2, IL-6, IL-17A, TNF-α, IFN-γ, and MMP-3, to up-regulate the expression of IL-10, to relieve histological damage and to inhibit bone destruction. BsAb inhibited nuclear translocation of the p65 subunit and cytoplasm IκB-α degradation by blocking IL-1β and IL-17A, the upstream of NF-κB pathway. High doses of BsAb had a more beneficial effect on CIA mice than Adalimumab and Dexamethasone. Thus, these results indicate that BsAb can be regarded as an ideal candidate for RA therapy.

An efficient preparation of Periplaneta americana nymphae allergen, Cr PI (54 kDa) is described. It was expressed as a GST-tag fusion protein in Escherichia coli, strain BL21 (DE3). Expression of recombinant Cr PI (rCr PI), denaturation/ renaturation of the inclusion bodies and the effects of protein and L-arginine concentration on inclusion body aggregation were optimized. The fusion protein was purified by affinity chromatography and size exclusion chromatography, and Cr PI fusion protein was purified to > 95%. rCr PI bound strongly to IgE in the sera of individuals with cockroach allergies as shown by western blot and ELISA. Highly refolded and purified recombinant protein was obtained, providing a basis for the large-scale preparation of Cr PI allergen.

Three closely-linked clusters of protocadherin (Pcdh) genes (alpha, beta, and gamma) encoding more than 50 distinct mRNAs have been identified in humans and mice, and proposed to play important roles in neuronal connectivity in the CNS. The human and mouse Pcdh alpha and gamma clusters each span a region of about 300 kb genomic DNA, and are each organized into a tandem array of more than a dozen highly-similar "variable" exons, and three downstream "constant" exons. Little is known about the expression patterns of the alpha and gamma repertoires in the CNS. Here, we comprehensively analyzed the one megabase rat Pcdh genomic DNA sequences at the nucleotide level using various computational methods. We found that the clustered rat Pcdh genes display strict orthologous relationships with those of mice but not humans. Moreover, each rat Pcdh variable exon is preceded by a distinct promoter. We designed two complete sets of isoform-specific probes and extensively mapped the expression patterns for each member of the alpha and gamma repertoires in the adult rat CNS by non-isotopic in situ hybridization experiments. We found that most alpha and gamma mRNA isoforms are broadly expressed in similar patterns in subsets of cells (with some displaying interesting cortical layer-specific expression) throughout various CNS regions, including the olfactory bulb, cerebral cortex, hippocampus, cerebellum, and spinal cord. The broad expression of most alpha or gamma mRNAs throughout various regions of the CNS is consistent with the hypothesis that these genes may be used for neurons to establish their individuality and also provide the adhesive diversity required for complex synaptic connectivity in the mammalian CNS.

Recent advances in G-quadruplex (G4) studies have confirmed that G4 structures exist in living cells and may have detrimental effects on various DNA transactions. How helicases resolve G4, however, has just begun to be studied and remains largely unknown. In the present paper, we use single-molecule fluorescence assays to probe Pif1-catalysed unfolding of G4 in a DNA construct resembling an ongoing synthesis of lagging strand stalled by G4. Strikingly, Pif1 unfolds and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and ‘acrobatic’ re-initiation of unfolding by the same monomer. Thus, Pif1 unfolds single G4 structures repetitively. Furthermore, it is found that Pif1 unfolds G4 sequentially in two large steps. Our study has revealed that, as a stable intermediate, G-triplex (G3) plays an essential role in this process. The repetitive unfolding activity may facilitate Pif1 disrupting the continuously reforming obstructive G4 structures to rescue a stalled replication fork. The proposed mechanism for step-wise unfolding of G4 is probably applicable to other helicases that resolve G4 structures for maintaining genome stability.

Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16−/− ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16−/− ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation. Embryonic stem cell differentiation requires activation of lineage-specific genes. Here the authors show that the histone H2A deubiquitinase Usp16 is required for relieving ubiquitinated H2A-mediated silencing of lineage-specific genes and embryonic stem cell differentiation.

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotency maintenance of mouse ES cells. Here, we report that Zfp322a is required for mES cell identity since depletion of Zfp322a directs mES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Pou5f1 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of mES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated mES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits mES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka's factors (OSKM). It can be even used in combination with Yamanaka's factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of mouse ES cells., Author Summary Embryonic stem (ES) cells are featured by their ability to self-renew and by their potential to differentiate into many different cell types. Recent studies have revealed that the unique properties of mouse ES cells are governed by a specific transcription regulatory network, including master regulators Oct4/Sox2/Nanog and other pluripotency factors. The importance of these factors was highlighted by the subsequent finding that combination of several transcription factors can reprogram differentiated fibroblasts back to pluripotent stem cells. Here, we report that Zfp322a is a novel factor which is required for mES cell identity. We revealed that Zfp322a can regulate the key pluripotency genes Pou5f1 and Nanog and functions as a repressor of MAPK/ERK pathway in mES cells, therefore preventing mES cell differentiation. Furthermore, we discovered that Zfp332a can promote the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Our results reveal that Zfp322a is a novel essential transcription factor which not only regulates ES cell pluripotency but also enhances iPSC formation.

SUMMARYInterleukin (IL)-13 and alternatively activated macrophages (AAMs) play an important role in liver granuloma and fibrosis of schistosomiasis. Paeoniflorin (PAE, C23H28O11) has been reported to have an anti-hepatic fibrosis effect in schistosomiasis; however, the mechanism has not been fully elucidated. In this study, we measured serum hyaluronic acid (HA) concentrations, liver granuloma diameter and volume density, fibrosis degree and expressions of IL-13, arginase-1 (ARG-1), nitric oxide synthase-2 (NOS-2), and phosphorylated signal transducer and activator of transcription 6 (p-STAT6) in mice liver of schistosomiasis. Then we detected expressions of specific biomarkers of AAMs and activity of Arg-1 in Kupffer cells (KCs) from infected and PAE-treated mice, or in KCs from uninfected mice, but exposed to rIL-13in vitro. Finally, we observed expression of IL-13 signalling molecules in KCs and secretion of IL-13 from lymphocytes of infected and PAE-treated mice. Our results showed that during schistosomiasis, IL-13 expression and secretion increased with liver macrophages activated alternatively. PAE not only directly inhibited alternative activation of macrophages via reducing the phosphorylations of janus-activated kinase 2 (JAK2) and/or STAT6, leading to reduction of AAMs-related markers and Arg-1 activity, but also indirectly suppressed alternative activation of macrophages through decreasing secretion of IL-13. PAE might be a promising prophylactic agent for hepatic granuloma and fibrosis of schistosomiasis japonica.

BackgroundThe Tientsin Albino 2 (TA2) mouse is an inbred strain originating from the Kunming strain. It has a high incidence of spontaneous breast cancer without the need for external inducers or carcinogens. Until now, the mechanism of carcinogenesis has remained unclear. In this study, we investigate differential gene expression, especially the expression of decorin, EGFR and cyclin D1, during mammary gland epithelial cell carcinogenesis in TA2 mice.MethodsGene expression profiles of spontaneous breast cancer and matched normal mammary gland tissues in TA2 mice were ascertained using an Affymetrix Mouse 430 2.0 array. Twelve mammary tissue samples from five month-old female TA2 mice (Group A), as well as 28 samples from mammary (Group B) and cancer tissues (Group C) of spontaneous breast cancer-bearing TA2 mice, were subsequently used to detect the expression of decorin, EGFR and cyclin D1 by real-time PCR and immunohistochemical methods.ResultsSeveral imprinted genes, oncogenes and tumor suppressor genes were differentially expressed between normal mammary gland tissues and breast cancer tissues of TA2 mice. The imprinted gene decorin and the oncogene EGFR were down-regulated in tumor tissues, while the oncogene cyclin D1 was up-regulated. Immunohistochemistry showed that samples in Group A showed high decorin expression more frequently than those in Group B (P< 0.05). More tissue samples in Group B than Group A were positive for nuclear EGFR, and tissue samples in Group B more frequently showed high nuclear EGFR expression than those in Group A or Group C (P< 0.05). The labeling index for cyclin D1 in Group C was significantly higher than in Group B. Mammary tissues of Group A expressed the highest level of decorin mRNA (P< 0.05), and mammary tissues of Group B expressed the highest level of EGFR mRNA (P< 0.05), while cancer tissues expressed the highest level of cyclin D1 mRNA (P< 0.05).ConclusionsThe expression of decorin, EGFR and cyclin D1 in mammary epithelial cells changes with increasing age. The abnormal expression of them may partly contribute to the genesis of spontaneous breast cancer in TA2 mice.

To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation.With gift plasmid encoding Sox2 gene as a template, Sox2 K247R was obtained by overlapping extension PCR, followed by construction of pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R. After enzyme digestion analysis and DNA sequencing, these two constructs were transfected or co-transfected with pCMV-Myc-SUMO1 into 293FT cells by lipofectin method. Western blot was employed to analyze expression and SUMO ylation of Sox2.It was revealed that eukaryotic expression vectors were constructed with correct sequence, where in mutant Sox2, the AAG codon was switched to CGG codon. Western blot results showed that good expression of both wt and mut Sox2, of which the latter could not be modified by SUMO1.Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.

We describe here a streamlined procedure for targeting vector construction, which often is a limiting factor for gene targeting (knockout) technology. This procedure combines various highly efficient recombination-based cloning methods in bacteria, consisting of three steps. First step is the use of Red-pathway-mediated recombination (recombineering) to capture a genomic fragment into a Gateway-compatible vector. Second, the vector is modified by recombineering to include a positive selection gene neo, from a variety of modular reagents. Finally, through a simple in vitro Gateway recombination, the modified genomic fragment is switched into a vector that contains negative selection cassettes, as well as unique sites for linearization. To demonstrate the usefulness of this protocol, we report targeted disruptions of members of the cadherin gene family, focusing on those that have not been previously studied at the molecular genetic level. This protocol needs 2 weeks to construct a targeting vector, and several vectors can be easily handled simultaneously using common laboratory setup.

Estrogen tightly regulates the levels of circulating gonadotropins, but a direct effect of estrogen receptor alpha (ERalpha) on the mammalian LHbeta gene has remained poorly defined. We demonstrate here that ERalpha can associate with the LHbeta promoter through interactions with Sf-1 and Pitx1 without requiring an estrogen response element (ERE). We show that gonadotropin-releasing hormone (GnRH) promotes ERalpha ubiquitylation and also degradation while stimulating expression of ubc4. GnRH also increases the association and lengthens the cycling time of ERalpha on the LHbeta promoter. The ERalpha association and transactivation of the LHbeta gene, as well as ERalpha degradation, are increased following ubc4 overexpression, while the effects of GnRH are abated following ubc4 knockdown. Our results indicate that ERalpha ubiquitylation and subsequent transactivation of the LHbeta gene can be induced by increasing the levels of the E2 enzyme as a result of signaling by an extracellular hormone, thus providing a new form of cross talk in hormonally stimulated regulation of gene expression.

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+) T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+) T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/-) mice, we found that CD4(+) T cells and ICOS(+/+) T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+)Foxp3 (+) ICOS(+) T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+) Tregs promoted survival in animals that received ICOS(-/-) T cells. Direct comparison of ICOS(-/-) Tregs to ICOS(+/+) Tregs found defects in vitro but no differences in the ability of ICOS(-/-) Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

Exon trapping from a bacterial artificial chromosome (BAC 78138) mapping to the 19q13.3 glioma tumor suppressor candidate region yielded two exons that recognized a 3.6-kb transcript on Northern blot. Screening of a human fetal brain cDNA library with these exons identified three novel genes, designated EHD2, EHD3, and EHD4, which are homologous to the recently characterized human EHD1 (testilin/HPAST) and its mouse homolog Ehd1, as well as to homologs in Drosophila (Past1) and Caenorhabditis elegans. Alignment of the predicted peptide sequences revealed striking similarities, with multiple conserved regions that include a nucleotide-binding consensus site at the N-terminus, a bipartite nuclear localization signal, and an eps15 homology (EH) protein-binding domain with an EF-hand motif at the C-terminus. The genes are specifically expressed, with EHD2 highly expressed in heart, EHD3 in brain and heart, and EHD4 in heart and pancreas. EHD2 was confirmed to originate from BAC 78138 at 19q13.3; radiation hybrid mapping localized EHD3 and EHD4 to 2p21 and 15q11.1, respectively; EHD1 has been previously mapped to 11q13. The three EHD1 paralogs therefore represent novel members of a family of human EH domain-containing proteins that may play a role in endocytosis and signaling. Mutation analysis of the five coding exons of EHD2 in gliomas failed to detect any tumor-specific alterations, thus indicating that EHD2 is an unlikely candidate for the 19q tumor suppressor gene.

SummaryCTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.

Objective To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E. coli) under optimistic conditions, obtain and identify protein expressed, analyze the structure and characteristics of the protein using bioinformatics methods for future applications. Methods Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction, and was cloned into the pET-30a vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3), and then purified and identified by western blotting. The essential physical-chemical properties of the protein were predicated by bioinformatics tools, including subcellular location, secondary structure, domains, antigenic epitopes, etc. Tertiary structure of the protein based on homology modeling was established, while multi-sequence homological alignment and phylogenetic analysis were proformed. Results The recombinant protein was obtained in soluble fraction from expression system in E. coli BL21(DE3) carrying pET30- Rv3265c plasmid, and Rv3265c gene was expressed correctly. Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices, located outside of membrane. Secondary structure analysis revealed it contained α-helix, extended strand and random coil, 46.8%, 14.6%, 38.6%, respectively. Furthermore, it possessed six potential antigenic epitopes, one glycosyl transferase domain. A simple three-dimensional model of this protein was constructed by Swiss-model sever. Both sequences and structures were conservative and especial either in gene or in protein. Conclusions Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine. The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.