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The sequence upstream of the antibody variable region (antibody upstream sequence [AUS]) consists of a 5' untranslated region (5' UTR) and a preceding leader region. The sequence variations in AUS affect antibody engineering and PCR based antibody quantification and may also be implicated in mRNA transcription and translation. However, the diversity of AUSs remains elusive. Using 5' rapid amplification of cDNA ends and high-throughput antibody repertoire sequencing technique, we acquired full-length AUSs for human, rhesus macaque, cynomolgus macaque, mouse, and rat. We designed a bioinformatics pipeline and identified 3307 unique AUSs, corresponding to 3026 and 1457 unique sequences for 5' UTR and leader region, respectively. Comparative analysis indicated that 928 (63.69%) leader sequences are novel relative to those recorded in the international ImMunoGeneTics information system. Evolutionarily, leader sequences are more conserved than 5' UTR and seem to coevolve with their downstream V genes. Besides, single-nucleotide polymorphisms are position dependent for leader regions and may contribute to the functional reversal of the downstream V genes. Finally, the AUGs in AUSs were found to have little impact on gene expression. Taken together, our findings can facilitate primer design for capturing antibodies efficiently and provide a valuable resource for antibody engineering and molecule-level antibody studies.

Abnormal immune cell infiltration is associated with the pathogenesis of Crohn’s disease (CD). This study aimed to determine the diagnostic and predictive value of immune-related genes in CD. Seven Gene Expression Omnibus datasets that analyzed the gene expression in CD tissues were downloaded. Single-sample gene set enrichment analysis (ssGSEA) was used to estimate the infiltration of the immune cells in CD tissues. Immune-related genes were screened by overlapping the immune-related genes with differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was used to identify key immune-related DEGs. Diagnostic value of CD and predictive value of anti-TNFα therapy were analyzed. Immunohistochemical (IHC) assay was used to verify gene expression in CD tissues. There were significant differences among CD tissues, paired CD tissues, and normal intestinal tissues regarding the infiltration of immune cells. AQP9, CD27, and HVCN1 were identified as the key genes of the three sub-clusters in the PPI network. AQP9, CD27, and HVCN1 had mild to moderate diagnostic value in CD, and the diagnostic value of AQP9 was better than that of CD27 and HVCN1. AQP9 expression was decreased in CD after patients underwent anti-TNFα therapy, but no obvious changes were observed in non-responders. AQP9 had a moderate predictive value in patients who had undergone treatment. IHC assay confirmed that the expression of AQP9, CD27, and HVCN1 in CD tissues was higher than that in normal intestinal tissues, and AQP9, CD27 was correlated with the activity of CD. Immune-related genes, AQP9, CD27, and HVCN1 may act as auxiliary diagnostic indicators for CD, and AQP9 could serve as a promising predictive indicator in patients who underwent anti-TNF therapy.

ObjectiveFacilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC).DesignWe obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models.ResultsWe showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib.ConclusionIn conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

Background: Age-related macular degeneration (AMD) is a progressive and irreversible eye disease. The anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of neovascular AMD. However, the expense for such treatment is quite high. Methods: We used a traditional Chinese medicine ZQMT as an alternative therapeutic regimen for AMD. We employed two in vivo animal models mimicking dry and wet AMD respectively to assess the therapeutic efficacy of ZQMT on treating AMD-related retinopathy. AMD-related retinopathy in Crb1rd8 mice was evaluated from week 1 to 8 by fundus photography. Laser-induced choroidal neovascularization (CNV) was evaluated by fluorescein angiography and histopathology. Results: ZQMT increased CX3CR1 expression in murine CD4+ T cells either cultured in vitro or directly isolated from animals treated with ZQMT. We also performed both in vitro and in vivo studies to confirm that ZQMT has no apparent toxic effects. ZQMT alleviated AMD-related retinopathy in both Crb1rd8 and CNV models. Depletion of CCL2 and CX3CR1 in Crb1rd8 mice abolished the efficacy of ZQMT, suggesting that CCL2 and/or CX3CR1 may underlie the mechanisms of ZQMT in treating AMD-related retinopathy in mice. Conclusion: In summary, our study supports the protective roles of a traditional Chinese medicine ZQMT in AMD.

Regulatory factor X-5 (RFX5) represents a key transcription regulator of MHCII gene expression in the immune system. This study aims to explore the molecular mechanisms and biological significance of RFX5. Firstly, by analyzing ENCODE chromatin immunoprecipitation (ChIP)-seq in HepG2 and TCGA RNA-seq data, we discovered lysine-specific demethylase 4A (KDM4A), also named JMJD2A, to be a major downstream target gene of RFX5. Moreover, RFX5 was verified to bind directly to the KDM4A’s promoter region and sequentially promoted its transcription determined by the ChIP-PCR assay and luciferase assay. In addition, RFX5-dependent regulation of KDM4A was demonstrated in HCC. Compared with adjacent non-tumor tissues, the expression levels of KDM4A were significantly raised in HCC tumor tissues. Notably, elevated levels of KDM4A were strongly correlated with HCC patient prognosis. Functionally, KDM4A overexpression largely rescued the growth inhibitory effects of RFX5 deletion, highlighting KDM4A as a downstream effector of RFX5. Mechanistically, the RFX5-KDM4A pathway promoted the progression of the cell cycle from G0/G1 to S phase and was protective against cell apoptosis through regulation of p53 and its downstream genes in HCC. In conclusion, RFX5 could promote HCC progression via transcriptionally activating KDM4A expression.

Summary The bone marrow niche plays a critical role in controlling the fate of hematopoietic stem cells (HSCs) by integrating intrinsic and extrinsic signals. However, the molecular events in the HSC niche remain to be investigated. Here, we report that intercellular adhesion molecule-1 (ICAM-1) maintains HSC quiescence and repopulation capacity in the niche. ICAM-1-deficient mice (ICAM-1−/−) displayed significant expansion of phenotypic long-term HSCs with impaired quiescence, as well as favoring myeloid cell expansion. ICAM-1-deficient HSCs presented normal reconstitution capacity during serial transplantation; however, reciprocal transplantation experiments showed that ICAM-1 deficiency in the niche impaired HSC quiescence and repopulation capacity. In addition, ICAM-1 deletion caused failure to retain HSCs in the bone marrow and changed the expression profile of stroma cell-derived factors, possibly representing the mechanism for defective HSCs in ICAM-1−/− mice. Collectively, these observations identify ICAM-1 as a regulator in the bone marrow niche., Graphical Abstract, Highlights • ICAM-1 deficiency expands HSC−LT with impaired quiescence and repopulation • The defects characterizing HSC−LT in ICAM-1−/− mice are niche cell dependent • ICAM-1−/− niche brings about impaired bone marrow retention and homing of HSC−LT • ICAM-1 in human stroma cells might affect the progression of myelocytic leukemia, In this article, Jie Zhou and colleagues show that ICAM-1 expression in bone marrow niche contributes to the quiescence and repopulation capacity of HSCs. ICAM-1 deficiency changes the expression profile of stroma-derived factors, thus causing failure of HSC retention in the bone marrow. Clinical data indicate that ICAM-1 might participate in the progression of myelocytic leukemia.

C‐to‐U RNA editing is the conversion from cytidine to uridine at RNA level. In plants, the genes undergo C‐to‐U RNA modification are mainly chloroplast and mitochondrial genes. Case studies have identified the roles of C‐to‐U editing in various biological processes, but the functional consequence of the majority of C‐to‐U editing events is still undiscovered. We retrieved the deep sequenced transcriptome data in roots and shoots of Arabidopsis thaliana and profiled their C‐to‐U RNA editomes and gene expression patterns. We investigated the editing level and conservation pattern of these C‐to‐U editing sites. The levels of nonsynonymous C‐to‐U editing events are higher than levels of synonymous events. The fraction of nonsynonymous editing sites is higher than neutral expectation. Highly edited cytidines are more conserved at DNA level, and the gene expression levels are correlated with C‐to‐U editing levels. Our results demonstrate that the global C‐to‐U editome is shaped by natural selection and that many nonsynonymous C‐to‐U editing events are adaptive. The editing mechanism might be positively selected and maintained and could have profound effects on the modified RNAs.

Hybrids are the perfect materials to study cis regulatory elements because the two parental alleles are subjected to identical trans environments. There has been a debate on whether synonymous codon usage could affect gene expression. In vitro experiments found that luciferase genes with enhanced codon optimality showed elevated mRNA expression. However, the underlying mechanism is still unclear, and no direct evidence is observed to support this notion. By mapping the RNA-seq data of hybrids of Arabidopsis thaliana and Arabidopsis lyrata, we quantified the allele-specific reads and estimated the relative expression of orthologous genes. We focused on orthologous genes with dN = 0 and dS > 0, which means that they only differ in synonymous codon usage. We found that orthologous genes with higher codon optimality in A. thaliana tend to have higher expression levels of the A. thaliana allele. Codon usage bias could influence gene expression. This phenomenon is not only found in in vitro experiments but also supported by in vivo observations. Therefore, synonymous mutations could have a broad impact from multiple aspects and should not be automatically ignored in genomic studies. Key Message In Arabidopsis hybrids, alleles with higher codon optimality tend to have higher expression levels.

The structured vocabulary that describes gene function, the gene ontology (GO), serves as a powerful tool in biological research. One application of GO in computational biology calculates semantic similarity between two concepts to make inferences about the functional similarity of genes. A class of term similarity algorithms explicitly calculates the shared information (SI) between concepts then substitutes this calculation into traditional term similarity measures such as Resnik, Lin, and Jiang-Conrath. Alternative SI approaches, when combined with ontology choice and term similarity type, lead to many gene-to-gene similarity measures. No thorough investigation has been made into the behavior, complexity, and performance of semantic methods derived from distinct SI approaches. We apply bootstrapping to compare the generalized performance of 57 gene-to-gene semantic measures across six benchmarks. Considering the number of measures, we additionally evaluate whether these methods can be leveraged through ensemble machine learning to improve prediction performance. Results showed that the choice of ontology type most strongly influenced performance across all evaluations. Combining measures into an ensemble classifier reduces cross-validation error beyond any individual measure for protein interaction prediction. This improvement resulted from information gained through the combination of ontology types as ensemble methods within each GO type offered no improvement. These results demonstrate that multiple SI measures can be leveraged for machine learning tasks such as automated gene function prediction by incorporating methods from across the ontologies. To facilitate future research in this area, we developed the GO Graph Tool Kit (GGTK), an open source C++ library with Python interface (github.com/paulbible/ggtk). Keywords: Semantic similarity, Gene ontology, Function prediction, Machine learning, Protein–protein interaction, Gene expression

Highlight The roles of GmCIF1 and GmC/VIF1 as invertase inhibitors were examined in vitro in soybean (Glycine max), and post-translational elevation of extracellular invertase activity by silencing GmCIF1 expression was found to increase seed weight., Cell wall invertase (CWI) and vacuolar invertase (VI) play multiple functions in plant growth. As well as depending on transcriptional and post-transcriptional regulation, there is growing evidence that CWI and VI are also subject to post-translational control by small inhibitory proteins. Despite the significance of this, genes encoding inhibitors, their molecular and biochemical properties, and their potential roles in regulating seed production have not been well documented in soybean (Glycine max). In this study, two invertase inhibitor isoforms, GmCIF1 and GmC/VIF2, were characterized to possess inhibitory activities in vitro via heterologous expression. Transcript analyses showed that they were predominantly expressed in developing seeds and in response to ABA. In accordance with this, surveys of primary targets showed subcellular localizations to the apoplast in tobacco epidermis after expressing YFP-fusion constructs. Investigations using RNAi transgenic plants demonstrated marked elevations of CWI activities and improvements in seed weight in conjunction with higher accumulations of hexoses, starch, and protein in mature seeds. Further co-expression analyses of GmCIF1 with several putative CWI genes corroborated the notion that GmCIF1 modulation of CWI that affects seed weight is mainly contingent on post-translational mechanisms. Overall, the results suggest that post-translational elevation of CWI by silencing of GmCIF1 expression orchestrates the process of seed maturation through fine-tuning sucrose metabolism and sink strength.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

Lung adenocarcinoma is the most common subtype of non-small-cell lung cancer affecting people all over the globe. Recent studies have indicated that long non-coding RNAs (lncRNAs) possess the ability to regulate gene expression. Initially, we uncovered increased LINC00355 expressions in lung adenocarcinoma tissues and cells. Functionally, our findings demonstrated that LINC00355 silencing suppressed the proliferation in vitro and in vivo. In addition, we found that LINC00355 negatively regulated miR-195 in lung adenocarcinoma cells. Simultaneously, silencing LINC00355 by shRNA resulted in suppressed proliferation, colony formation and promoted cell cycle arrest and apoptosis via miR-195. Moreover, silencing LINC00355 by shRNA inhibited the cyclin E1 (CCNE1) gene expression via miR-195 in lung adenocarcinoma cells. Collectively, this study demonstrates the novel lncRNA LINC00355 in regulatory network of CCNE1 via miR-195 in lung adenocarcinoma, highlighting LINC00355 as a new target for the treatment of lung adenocarcinoma.

Background & aims It has yet to be firmly established whether host IFNL3 (IL28B) genotype influences interferon responsiveness in patients with chronic hepatitis B. We investigated associations between single-nucleotide polymorphisms (SNPs) in the IFNL3 region and response to peginterferon alfa-2a in 701 patients enrolled in three large, randomized, international studies. Methods Responses were defined as hepatitis B surface antigen (HBsAg) loss and/or hepatitis B e antigen (HBeAg) seroconversion plus hepatitis B virus (HBV) DNA < 2000 IU/ml in HBeAg-positive patients, and HBsAg loss and/or HBV DNA < 2000 IU/ml in HBeAg-negative patients (24 weeks after end of treatment). Associations between treatment response and the number of copies of the poor-response allele at three SNPs (rs8099917, rs12980275, rs12979860) were explored with logistic regression models in Asian and white patients. Results The HBeAg-positive and -negative populations comprised 465 (92% Asian, 50% HBV genotype C) and 236 (79% Asian, 41% HBV genotype C) patients, respectively, and had respective response rates of 26% and 47%. The IFNL3 genotype was strongly associated with ethnicity. There was no association between IFNL3 genotype and treatment response in HBeAg-positive or -negative patients. Independent predictors of treatment response were: sex, HBV DNA level and alanine aminotransferase level in HBeAg-positive Asian patients; age in HBeAg-negative Asian patients; and HBV DNA in HBeAg-negative white patients. Conclusions This is the largest analysis to date of associations between IFNL3 genotype and peginterferon response in patients with chronic hepatitis B. The data suggest that IFNL3 polymorphism is not a major determinant of the response to peginterferon alfa-2a in either HBeAg-positive or HBeAg-negative patients.

Motivation Mass spectrometry (MS) based quantification of proteins/peptides has become a powerful tool in biological research with high sensitivity and throughput. The accuracy of quantification, however, has been problematic as not all peptides are suitable for quantification. Several methods and tools have been developed to identify peptides that response well in mass spectrometry and they are mainly based on predictive models, and rarely consider the linearity of the response curve, limiting the accuracy and applicability of the methods. An alternative solution is to select empirically superior peptides that offer satisfactory MS response intensity and linearity in a wide dynamic range of peptide concentration. Results We constructed a reference database for proteome quantification based on experimental mass spectrum response curves. The intensity and dynamic range of over 2 647 773 transitions from 121 318 peptides were obtained from a set of dilution experiments, covering 11 040 gene products. These transitions and peptides were evaluated and presented in a database named SCRIPT-MAP. We showed that the best-responder (BR) peptide approach for quantification based on SCRIPT-MAP database is robust, repeatable and accurate in proteome-scale protein quantification. This study provides a reference database as well as a peptides/transitions selection method for quantitative proteomics. Availability and implementation SCRIPT-MAP database is available at http://www.firmiana.org/responders/. Supplementary information Supplementary data are available at Bioinformatics online.

Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn's disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.

Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.

Regulatory factor X-5 (RFX5) was previously characterized as an essential and highly specific regulator of major histocompatibility class II (MHCII) gene expression in the immune system. We found that RFX5 is significantly upregulated in hepatocellular carcinoma (HCC) tumors and cell lines compared with non-tumor tissues in mRNA expression levels, but it fails to induce the expression of MHCII. However, RFX5 can strongly bind to the tripeptidyl peptidase 1 (TPP1) promoter region and then increase its transcriptional activity. We also found that manipulation the expression of RFX5 can significantly affect the expression of TPP1 in HepG2, which suggested that RFX5 can transcriptionally activate TPP1 in HCC. Moreover, TPP1 is overexpressed in HCC tissues and significantly correlated with poor prognosis of HCC patients, suggesting that it may have potential biological implications in HCC.

Members of the CYCLOIDEA2 (CYC2) clade of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF transcription factor genes are widely involved in controlling floral zygomorphy, a key innovation in angiosperm evolution, depending on their persistently asymmetric expression in the corresponding floral domains. However, it is unclear how this asymmetric expression is maintained throughout floral development. Selecting Primulina heterotricha as a model, we examined the expression and function of two CYC2 genes, CYC1C and CYC1D. We analyzed the role of their promoters in protein-DNA interactions and transcription activation using electrophoresis mobility shift assays, chromatin immunoprecipitation, and transient gene expression assays. We find that CYC1C and CYC1D positively autoregulate themselves and cross-regulate each other. Our results reveal a double positive autoregulatory feedback loop, evolved for a pair of CYC2 genes to maintain their expression in developing flowers. Further comparative genome analyses, together with the available expression and function data of CYC2 genes in the core eudicots, suggest that this mechanism might have led to the independent origins of floral zygomorphy, which are associated with plant-insect coevolution and the adaptive radiation of angiosperms.

Signal Transducers and Activators of Transcription (STAT) 5A/B regulate cytokine-inducible genes upon binding to GAS motifs. It is not known what percentage of genes with GAS motifs bind to and are regulated by STAT5. Moreover, it is not clear whether genome-wide STAT5 binding is modulated by its concentration. To clarify these issues we established genome-wide STAT5 binding upon growth hormone (GH) stimulation of wild-type (WT) mouse embryonic fibroblasts (MEFs) and MEFs overexpressing STAT5A more than 20-fold. Upon GH stimulation, 23 827 and 111 939 STAT5A binding sites were detected in WT and STAT5A overexpressing MEFs, respectively. 13 278 and 71 561 peaks contained at least one GAS motif. 1586 and 8613 binding sites were located within 2.5 kb of promoter sequences, respectively. Stringent filtering revealed 78 genes in which the promoter/upstream region (-10 kb to +0.5 kb) was recognized by STAT5 both in WT and STAT5 overexpressing MEFs and 347 genes that bound STAT5 only in overexpressing cells. Genome-wide expression analyses identified that the majority of STAT5-bound genes was not under GH control. Up to 40% of STAT5-bound genes were not expressed. For the first time we demonstrate the magnitude of opportunistic genomic STAT5 binding that does not translate into transcriptional activation of neighboring genes.

Background The complement activation molecule C5a has been found in the eye and is implicated in the pathogenesis of ocular inflammatory diseases. In this study, the authors sought to investigate C5a9s effects on human retinal pigment epithelial (RPE) cells and peripheral blood mononuclear cells (PBMCs), and on the interaction between RPE cells and PBMCs. Methods Arising retinal pigment epithelia cell line-19 and PBMCs isolated from healthy donors were used in this study. Western blot, real-time PCR and cell surface receptor staining were used to detect C5a receptor expression. Real-time PCR was used to detect cytokine mRNA expression. A thiazolyl blue tetrazolium bromide assay was used to detect cell viability. Cells were stained with Annexin V and 7-aminoactinomycin D for an apoptosis assay. Cell proliferation was measured using a tritiated thymidine incorporation assay. Results C5a receptors were present on RPE cells, and receptor expression was increased by pro-inflammatory cytokines. C5a suppressed RPE cells9 production of transforming growth factor β2, an important immunosuppressive agent in the eye. In addition, the viability of RPE cells was decreased in the presence of C5a, and this effect was not due to apoptosis. C5a increased proliferation of PBMCs and upregulated their production of pro-inflammatory cytokines. Finally, C5a decreased RPE cells9 ability to suppress immune cell proliferation. Conclusion The results provide a direct link between complement activation and intraocular inflammation. This line of information may help to understand the mechanism of the pathogenesis of intraocular inflammatory diseases. Moreover, the authors show that a close, reciprocal interaction between the innate immune system and the adaptive immune system may be involved in the development of such diseases.

Epigenetic alteration is a common trait of human cancers. Chemical modifications on DNA and core histone proteins are the major mechanisms for epigenetic regulation. Recently, emerging evidence suggested that reversible chemical modifications on RNA also play a critical role in epigenetic control of gene expression. N6-methyladenosine (m6A) is the most abundant modification found in mammalian mRNA. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. In this study, we investigated the expression of m6A methyltransferases and demethylases in human hepatocellular carcinoma (HCC). We found the major m6A methyltransferase METTL3 was remarkably up-regulated in HCC. High METTL3 expression was associated with poor overall and disease-free survival in HCC patients. Consistent with the METTL3 overexpression, we found that mRNA m6A level was significantly elevated in human HCC. To investigate the roles of METTL3 in human HCC, we employed lentiviral-based shRNA and CRISPR/Cas9 systems to inactivation METTL3 in HCC cell lines. We showed that knockdown and knockout of METTL3 drastically suppressed HCC proliferation and migration in vitro and abolished HCC tumorigenicity and lung metastasis in vivo. In contrast, overexpression of METTL3 by CRISPR/dCas9 SAM system promoted HCC growth. Using RNA-seq and m6A-Seq, we identified tumor suppressor gene SOCS2 as a novel target of METTL3. We detected m6A modification in SOCS2 mRNA and the m6A modification was diminished upon METTL3 knockdown. m6A modification promoted SOCS2 mRNA degradation. We demonstrated that knockdown of METTL3, mutation of m6A modification sites, and treatment of demethylating agent augmented SOCS2 mRNA expression in HCC cells. In addition, knockdown of YTHDF2, an m6A reader protein, also rescued SOCS2 mRNA expression in HCC cells. The above findings together demonstrated that up-regulation of METTL3 lead to m6A modification of SOCS2, which in turn promotes SOCS2 mRNA degradation through YTHDF2 dependent mechanism. Our findings provided a proof-of-concept model to demonstrate the importance of aberrant m6A modification in epigenetic silencing of tumor suppressor genes. Citation Format: Mengnuo Chen, Lai Wei, Cheuk-Ting Law, Felice H. Tsang, Jialing Shen, Carol L. Cheng, Long-Hin Tsang, Daniel W. Ho, David K. Chiu, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4321.

IFNs selectively regulate gene expression through several signaling pathways. The present study explored the involvement of STAT3 in the IFN-induced expression of the gene encoding the CXCL11 chemokine. The CXCL11 gene was induced in IFN-sensitive Daudi cells, but not in an IFN-resistant DRST3 subline with a defective STAT3 signaling pathway. Although the IFN-stimulated gene ISG15 was induced to a similar extent in Daudi and DRST3 cells, expression of wild-type STAT3 in DRST3 cells restored the IFN inducibility of CXCL11. Reconstitution of STAT3 knockout mouse embryonic fibroblasts with wild-type STAT3, or STAT3 with the canonical STAT3 dimerization site at Y705 mutated, restored IFN inducibility of the CXCL11 gene. These data indicate that CXCL11 gene induction by IFN is STAT3 dependent, but that phosphorylation of Y705 of STAT3 is not required. Chromatin immunoprecipitation assays demonstrated that IFN treatment of Daudi and DRST3 cells induced STAT3 binding to the CXCL11 promoter. Chromatin immunoprecipitation assays also revealed that NF-κB family member p65 and IFN regulatory factor (IRF)1 were bound to CXCL11 promoter upon IFN treatment of Daudi cells. In contrast, IFN induced the binding of p50 and IRF2 to the CXCL11 promoter in DRST3 cells. The profile of promoter binding was indistinguishable in IFN-sensitive Daudi cells and DRST3 cells reconstituted with wild-type STAT3. Thus, STAT3 also plays a role in the recruitment of the transcriptional activators p65 and IRF1, and the displacement of the transcriptional repressors p50 and IRF2 from the CXCL11 promoter also appears to regulate the induction of CXCL11 gene transcription.

Gene expression QTL (eQTL) mapping can suggest candidate regulatory relationships between genes. Recent advances in mammalian phenotype annotation such as mammalian phenotype ontology (MPO) enable systematic analysis of the phenotypic spectrum subserved by many genes. In this study we combined eQTL mapping and phenotypic spectrum analysis to predict gene regulatory relationships. Five pairs of genes with similar phenotypic effects and potential regulatory relationships suggested by eQTL mapping were identified. Lines of evidence supporting some of the predicted regulatory relationships were obtained from biological literature. A particularly notable example is that promoter sequence analysis and real-time PCR assays support the predicted regulation of protein kinase C epsilon (Prkce) by cAMP responsive element binding protein 1 (Creb1). Our results show that the combination of gene eQTL mapping and phenotypic spectrum analysis may provide a valuable approach to uncovering gene regulatory relations underlying mammalian phenotypes.

Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors. IFN-alpha/beta activates another important transcription factor, nuclear factor-kappaB (NF-kappaB), but its role in IFN-mediated activity is poorly understood. Sensitivity to the antiviral and gene-inducing effects of IFN was examined in normal fibroblasts and in NF-kappaB knockout fibroblasts from p50- and p65-null mice. Antiviral assays demonstrated that NF-kappaB knockout fibroblasts were sensitized to the antiviral action of IFN. Moreover, analysis of IFN-stimulated gene expression by real-time PCR demonstrated selective effects of NF-kappaB on gene expression. Our results demonstrate that a subset of IFN-stimulated genes is regulated through an NF-kappaB-dependent pathway and that NF-kappaB may regulate the sensitivity of cells to IFN-mediated antiviral activity.

2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.

Recognition of virus infection by innate pattern recognition receptors (PRRs), including membrane-associated toll-like receptors (TLR) and cytoplasmic RIG-I-like receptors (RLR), activates cascades of signal transduction pathways leading to production of type I interferons (IFN) and proinflammatory cytokines that orchestrate the elimination of the viruses. Although it has been demonstrated that PRR-mediated innate immunity plays an essential role in defending virus from infection, it also occasionally results in overwhelming production of proinflammatory cytokines that cause severe inflammation, blood vessel leakage and tissue damage. In our efforts to identify small molecules that selectively enhance PRR-mediated antiviral, but not the detrimental inflammatory response, we discovered a compound, RO 90-7501 ('2'-(4-Aminophenyl)-[2,5'-bi-1H-benzimidazol]-5-amine), that significantly promoted both TLR3 and RLR ligand-induced IFN-β gene expression and antiviral response, most likely via selective activation of p38 mitogen-activated protein kinase (MAPK) pathway. Our results thus imply that pharmacological modulation of PRR signal transduction pathways in favor of the induction of a beneficial antiviral response can be a novel therapeutic strategy.

The prognosis of hepatocellular carcinoma (HCC) after surgery is poor due to its high recurrence rate. In order to unfold the mechanism of different recurrent-free survival (RFS) times following resection, expression profiling of tumor tissues from 32 HCC patients with different RFS time were used to identify differential expression of individual genes and signaling pathway components correlated with RFS time. Quantitative RT-PCR, Western blotting, and immunohistochemistry were used to validate the expression of selected genes. Up-regulation of several immune related genes and pathways, especially HLA II-related antigen presenting pathways, significantly correlated with longer RFS time. The expression of MHCII molecules were found to be mainly located in either CD68+ cells or CD45+ cells, and their expression significantly correlated with the expression of CIITA (HLA II genes transactivator) in the tumor. The results suggest that the high expression level of CIITA and MHCII molecules in hepatocellular carcinoma tissue is an effective prognostic marker for longer RFS time in HCC.

To construct a eukaryotic expression vector for expressing hepatitis B virus (HBV) recombinant HBsAg-EGFP fusion protein and obtain a stable transfected Chang Liver cell line.The coding region of HBsAg gene of HBV was amplified by PCR and was digested by BamH I/EcoR I . This fragment was inserted into pEGFPN1 with T4 ligase and transformed E-coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into Chang Liver cell by Lipofectamine 2000 cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level HBsAg-EGFP fusion protein was obtained.The eukaryotic expression vector named pEGFPN1-HBsAg was successfully constructed and the stable transfected Chang Liver cell line expressing pEGFPN1-HBsAg fusion protein was obtained.The stable transfected Chang Liver cell line could express pEGFPN1-HBsAg fusion protein, could be used to screen the proteins differentially expressed in HBsAg expression Chang Liver cells, which brought some new clues for studying the potential molecular mechanism of HBsAg protein.

Type I interferons (IFNs) regulate diverse cellular functions by modulating the expression of IFN-stimulated genes (ISGs) through the activation of the well established signal transduction pathway of the Janus Kinase (JAK) and signal transducers and activators of transcription (STAT) proteins. Although the JAK-STAT signal transduction pathway is critical in mediating IFN's antiviral and antiproliferative activities, other signaling pathways are activated by IFNs and regulate cellular response to IFN. The NF-kappaB transcription factor regulates the expression of genes involved in cell survival and immune responses. We have identified a novel IFN mediated signal pathway that leads to NF-kappaB activation and demonstrate that a subset of ISGs that play key roles in cellular response to IFN is regulated by NF-kappaB. This review focuses on the IFN-induced NF-kappaB activation pathway and the role of NF-kappaB in ISG expression, antiviral activity and apoptosis, and the therapeutic application of IFN in cancer and infectious disease.

We previously showed that cRel, ΔNp63, and TAp73, members of NF-kappaB and TP53 transcription factor (TF) families, together regulate broad biological processes critical for inflammation and cancer survival. However, the underlying molecular mechanisms explaining the global cross-regulation of transcriptome and interactive signal network have not been revealed. Here, we demonstrate that inflammatory cytokine TNF-α induces a dynamic, genome-wide increase in cRel and ΔNp63 chromatin occupancy after replacing TAp73 binding activity, using Illumina Next Generation ChIP-sequencing and fitting the data into a dynamic Poisson distribution model integrated in MACS. Globally, significant binding peaks (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4193. doi:1538-7445.AM2012-4193

Background Successful strategies for QTL gene identification benefit from combined experimental and bioinformatic approaches. Unique design aspects of the BXD recombinant inbred line mapping panel allow use of archived gene microarray expression data to filter likely from unlikely candidates. This prompted us to propose a simple five-filter protocol for candidate nomination. To filter more likely from less likely candidates, we required candidate genes near to the QTL to have mRNA abundance that correlated with the phenotype among the BXD lines as well as differed between the parental lines C57BL/6J and DBA/2J. We also required verification of mRNA abundance by an independent method, and finally we required either differences in protein levels or confirmed DNA sequence differences. Results QTL mapping of mouse forebrain weight in 34 BXD RI lines found significant association on chromosomes 1 and 11, with each C57BL/6J allele increasing weight by more than half a standard deviation. The intersection of gene lists that were within ± 10 Mb of the strongest associated location, that had forebrain mRNA abundance correlated with forebrain weight among the BXD, and that had forebrain mRNA abundance differing between C57BL/6J and DBA/2J, produced two candidates, Tnni1 (troponin 1) and Asb3 (ankyrin repeat and SOCS box-containing protein 3). Quantitative RT-PCR confirmed the direction of an increased expression in C57BL/6J genotype over the DBA/2J genotype for both genes, a difference that translated to a 2-fold difference in Asb3 protein. Although Tnni1 protein differences could not be confirmed, a 273 bp indel polymorphism was discovered 1 Kb upstream of the transcription start site. Conclusion Delivery of well supported candidate genes following a single quantitative trait locus mapping experiment is difficult. However, by combining available gene expression data with QTL mapping, we illustrated a five-filter protocol that nominated Asb3 and Tnni1 as candidates affecting increased mouse forebrain weight. We recommend our approach when (1) investigators are working with phenotypic differences between C57BL/6J and DBA/2J, and (2) gene expression data are available on http://www.genenetwork.org that relate to the phenotype of interest. Under these circumstances, measurement of the phenotype in the BXD lines will likely also deliver excellent candidate genes.