Sequence-specific DNA Binding Activity Research Articles

Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

A non-viral DNA delivery system consisting of multifunctional chimeric peptide fused with zinc-finger protein

Non-viral gene delivery systems have received sustained attention as a promising alternative to viral vectors for disease treatment and prevention in recent years. Numerous methods have been developed to enhance gene uptake and delivery in the cytoplasm; however, due to technical difficulties and delivery efficiency,these systems still face challenges in a range of biological applications, especially invivo. To alleviate this challenge, we devised a novel system for gene delivery based on a recombinant protein eTAT-ZF9-NLS, which consisted of a multifunctional chimeric peptide and a zinc-finger protein with sequence-specific DNA-binding activity. High transfection efficiency was observed in several mammalian cells after intracellular delivery of plasmid containing ZF9-binding sites mediated by eTAT-ZF9-NLS. Our new approach provides a novel transfection strategy and the transfection efficiency was confirmed both invitro and invivo, making it a preferential transfection reagent for possible gene therapy.

Altered m6A RNA methylation governs denervation-induced muscle atrophy by regulating ubiquitin proteasome pathway

BackgroundDenervation-induced muscle atrophy is complex disease involving multiple biological processes with unknown mechanisms. N6-methyladenosine (m6A) participates in skeletal muscle physiology by regulating multiple levels of RNA metabolism, but its impact on denervation-induced muscle atrophy is still unclear. Here, we aimed to explore the changes, functions, and molecular mechanisms of m6A RNA methylation during denervation-induced muscle atrophy.MethodsDuring denervation-induced muscle atrophy, the m6A immunoprecipitation sequencing (MeRIP-seq) as well as enzyme-linked immunosorbent assay analysis were used to detect the changes of m6A modified RNAs and the involved biological processes. 3-deazidenosine (Daa) and R-2-hydroxyglutarate (R-2HG) were used to verify the roles of m6A RNA methylation. Through bioinformatics analysis combined with experimental verification, the regulatory roles and mechanisms of m6A RNA methylation had been explored.ResultsThere were many m6A modified RNAs with differences during denervation-induced muscle atrophy, and overall, they were mainly downregulated. After 72 h of denervation, the biological processes involved in the altered mRNA with m6A modification were mainly related to zinc ion binding, ubiquitin protein ligase activity, ATP binding and sequence-specific DNA binding and transcription coactivator activity. Daa reduced overall m6A levels in healthy skeletal muscles, which reduced skeletal muscle mass. On the contrary, the increase in m6A levels mediated by R-2HG alleviated denervation induced muscle atrophy. The m6A RNA methylation regulated skeletal muscle mass through ubiquitin–proteasome pathway.ConclusionThis study indicated that decrease in m6A RNA methylation was a new symptom of denervation-induced muscle atrophy, and confirmed that targeting m6A alleviated denervation-induced muscle atrophy.

MP41-12 ASSOCIATION OF KIDNEY CANCER WITH BOTH NOVEL AND REPORTED MONOGENIC GENE MUTATIONS: RESULTS OF WHOLE EXOME SEQUENCING IN THE UK BIOBANK

You have accessJournal of UrologyCME1 Apr 2023MP41-12 ASSOCIATION OF KIDNEY CANCER WITH BOTH NOVEL AND REPORTED MONOGENIC GENE MUTATIONS: RESULTS OF WHOLE EXOME SEQUENCING IN THE UK BIOBANK Jun Wei, Kristian Novakovic, Zhuqing Shi, Andrew Rifkin, S. Lilly Zheng, Brian Helfand, and Jianfeng Xu Jun WeiJun Wei More articles by this author , Kristian NovakovicKristian Novakovic More articles by this author , Zhuqing ShiZhuqing Shi More articles by this author , Andrew RifkinAndrew Rifkin More articles by this author , S. Lilly ZhengS. Lilly Zheng More articles by this author , Brian HelfandBrian Helfand More articles by this author , and Jianfeng XuJianfeng Xu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003279.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Pathogenic mutations in several cancer susceptibility genes have been reported in kidney cancer patients. However, their associations with kidney cancer have not been formally tested in well-characterized case-control studies and using the same sequencing and analytical pipeline. The objective of this study was to systematically evaluate association of kidney cancer with genes in the exome in a well-characterized prospective population-based case-control cohort. METHODS: A total of 187,966 subjects from the UK Biobank (UKB) with whole exome sequencing data were included in the analysis, including 1,217 kidney cancer patients (ICD10 C64). Pathogenic/likely pathogenic (P/LP) mutations were annotated based on criteria of the American College of Medical Genetics. The robust SKAT-O, a novel gene-based analysis that properly controls for type I error rates due to unbalanced case-control ratio, was used for association tests adjusting for age at recruitment, gender and genetic background. RESULTS: Among the 21 previously reported candidate genes, three were statistically significant at P<0.05, including VHL, SDHB, and CHEK2 (Table). In addition, associations with 9 novel genes (at P<0.0001) were found, including ZNF772, PRIMPOL, HES4, MOXD1, KRTAP10-9, ZNF827, ACAN, CDK20, and LRRFIP1. While these associations were only marginally significant after adjusting for multiple testing, they have potential roles in cancer development. For example, ZNF772 is predicted to enable DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity which are all known to be dysregulated in cancer. CONCLUSIONS: Identification of genes that influence kidney cancer may advance our understanding of disease etiology and identify potential therapeutic targets. Using the same sequencing method and mutation annotation pipeline in the largest population-based cohort to date, we confirmed several previously reported genes and identified several novel candidate genes for their association with kidney cancer. Confirmation in independent study populations is required for these novel candidate genes. Source of Funding: None. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e559 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jun Wei More articles by this author Kristian Novakovic More articles by this author Zhuqing Shi More articles by this author Andrew Rifkin More articles by this author S. Lilly Zheng More articles by this author Brian Helfand More articles by this author Jianfeng Xu More articles by this author Expand All Advertisement PDF downloadLoading ...

Exogenous c-di-GMP inhibited the biofilm formation of Vibrio splendidus.

Vibrio splendidus, a gram-negative bacterium that is ubiquitously present in marine environments, has been increasingly deemed an important opportunistic pathogen of marine animals. In this study, the biofilm formation of V. splendidus was quantitatively determined and morphologically characterized. Three stages of biofilm formation, including adhesion, aggregation and maturation were observed in the biofilm formed by V. splendidus. The inhibitory effect of exogenous bis (3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) on the biofilm formation from the scratch and preformed established biofilms of V. splendidus was determined. When 200μmol/L c-di-GMP was added, the quantity of biofilm decreased by 88.1% or 66.7% under the two conditions. To explore the preliminary mechanism of exogenous c-di-GMP on the biofilm formed by V. splendidus, proteomic analysis was performed. GO enrichment analysis showed that exogenous c-di-GMP upregulated biological processes, including the tricarboxylic acid cycle, oxidation‒reduction reactions and organonitrogen compound catabolism and significantly downregulated tRNA threonylcarbamoyladenosine modification, protein dephosphorylation, and lactate transmembrane transporter activity. Sequence-specific DNA binding activity was the most markedly downregulated molecular function. KEGG analysis showed that the valine, leucine and isoleucine degradation pathway was the most enriched pathway, followed by nitrogen metabolism, among the 20 upregulated pathways. Among the downregulated pathways, a nonribosomal peptide structure pathway and the streptomycine, polyketide sugar unit, acarbose and validamycin biosynthesis pathways were significantly enriched. Our present study provides basic data for the biofilm formation of V. splendidus and the preliminary inhibitory mechanism of exogenous c-di-GMP on the biofilm formation of V. splendidus.

DNA binding by polycomb-group proteins: searching for the link to CpG islands.

Polycomb group proteins predominantly exist in polycomb repressive complexes (PRCs) that cooperate to maintain the repressed state of thousands of cell-type-specific genes. Targeting PRCs to the correct sites in chromatin is essential for their function. However, the mechanisms by which PRCs are recruited to their target genes in mammals are multifactorial and complex. Here we review DNA binding by polycomb group proteins. There is strong evidence that the DNA-binding subunits of PRCs and their DNA-binding activities are required for chromatin binding and CpG targeting in cells. In vitro, CpG-specific binding was observed for truncated proteins externally to the context of their PRCs. Yet, the mere DNA sequence cannot fully explain the subset of CpG islands that are targeted by PRCs in any given cell type. At this time we find very little structural and biophysical evidence to support a model where sequence-specific DNA-binding activity is required or sufficient for the targeting of CpG-dinucleotide sequences by polycomb group proteins while they are within the context of their respective PRCs, either PRC1 or PRC2. We discuss the current knowledge and open questions on how the DNA-binding activities of polycomb group proteins facilitate the targeting of PRCs to chromatin.

BCL11A promotes myeloid leukemogenesis by repressing PU.1 target genes

AbstractThe transcriptional repressor BCL11A is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase Trib1 in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells in vitro and in vivo. Chromatin immunoprecipitation sequencing analysis showed that, upon DNA binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components HDAC and LSD1 reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor pracinostat and the LSD1 inhibitor GSK2879552 resulted in growth inhibition in vitro and in vivo. High BCL11A expression is associated with worse prognosis in humans with AML. Blocking of BCL11A expression upregulates the expression of PU.1 target genes and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

Transcriptomic analysis of Allium sativum uncovers putative genes involved in photoperiodic pathway and hormone signaling under long day and short day conditions

Photoperiod is dominant environmental factor that controls plant growth and development. Even though research on plants response to photoperiod is significant in agriculture, molecular mechanisms of garlic in response to photoperiod remain largely unknown. In the current investigation, 3 months old garlic plants were treated with long day (LD) and short day (SD) for 10 and 20 days after treatment (DAT). Liquid chromatography–mass spectrometry (LC–MS) analysis of phytohormones exhibited that indole-3-acetic acid (IAA), zeatin riboside (ZR) and salicylic acid (SA) were observed maximum under LD at 10 DAT, whereas abscisic acid (ABA), gibberellic acid 3 (GA3), zeatin (ZT) and jasmonic acid (JA) were observed maximum under LD at 20 DAT. Transcriptome sequencing analysis was done to evaluate the transcriptional response to LD and SD. Differentially expressed genes (DEGs) were detected to have pathway enrichment. i.e., DNA binding transcription factor activity, transcription regulator activity, transferase activity, transferring hexosyl groups, and sequence specific-DNA binding activity, plant hormone signal transduction, circadian rhythm-plant, biosynthesis of amino acids, phenylpropanoid biosynthesis, and starch and sucrose metabolism. Furthermore, 28 and 40 DEGs were identified related to photoperiod and hormone signaling, respectively and their interaction in response to LD and SD were discussed in detail. Outcomes of current investigation might be useful to provide novel resources for garlic bulb formation in response to photoperiod.

The auxin response factor gene family in wheat (Triticum aestivum L.): Genome-wide identification, characterization and expression analyses in response to leaf rust

Auxin response factors (ARFs) are plant-specific transcription factors that bind explicitly to Auxin Response Elements and mediate downstream expression of auxin responsive genes involved at various stages of plant growth and development. This study identified 52 ARF genes located within the wheat genome. Based on phylogenetic analysis, the identified ARF proteins of wheat were classified into four classes (class I, II, III and IV). Synteny analysis revealed the orthologous relation with other monocots. The maximum numbers of predicted ARF genes were mapped on wheat chromosome 3B Ten conserved motifs of ARF was determined on all the identified sequences. Based on amino acid composition of the middle region of the ARF proteins, nine ARF proteins were transcription activators and the remaining were transcription repressors. The ARF genes of wheat contained 1–17 introns and deduced proteins of the genes having similar pattern of intron-exon distribution clustered within the same clade in the phylogenetic tree. TaARF1, TaARF4, TaARF7, TaARF34, TaARF39 were predicted to be targeted by the stress responsive miRNA Tae-miRNA160. The identified proteins were mostly localized within the nucleus and had sequence specific DNA binding activity. They were annotated in regulation of expression of early auxin responsive genes in auxin signaling pathway. Thirty-one TaARF proteins interacted and exhibited co-expressed regulation with other TaARF and Aux/IAA proteins. Increased expression of TaARF1 and TaARF23 at 120 h post inoculation (hpi) and 72 hpi in resistant and susceptible isolines respectively, specifies their roles in providing better resistance to the plant during leaf-rust infection.

A New Model Applied for Evaluating a Rhenium-diselenium Drug: Breast Cancer Cells Stimulated by Cytokines Induced from Polynuclear Cells by LPS.

New anticancer drugs are usually tested on cancer cells in culture in a standard medium. We stimulated immune polynuclear cells by lipopolysaccharides to obtain an enriched medium (EM) containing inflammatory cytokines more closely reflecting the tumor microenvironment and tested a rhenium-diselenium (Re-diSe) drug in this new model. Concentrations of cytokines were compared with a control medium (CM). Human-derived breast cancer cells were grown in culture either in CM or EM with or without Re-diSe. Assays of tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), intereukin 1 beta (IL1β), transforming growth factor-beta (TGFβ), insulin growth factor 1 (IGF1) and vascular epidermal growth factor A (VEGFA) were performed by enzyme-linked immunosorbent assays. The production of reactive oxygen species (ROS) was determined by 2,7-dichlorofluorescein test. The cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests. Concentrations of TNFα, IL6 and Il1β were observed to be significantly higher in EM than in CM. There was no difference for TGFβ, IGF1 and VEGFA. The cells were sensitive to Re-diSe, with reduced concentrations of TGFβ, IGF1, VEGFA and ROS, but the half-maximal inhibitory concentration was significantly higher in EM than in CM. The efficacy of the Re-diSe drug was confirmed in this model of aggressive cancer.

Interactome of the yeast RNA polymerase III transcription machinery constitutes several chromatin modifiers and regulators of the genes transcribed by RNA polymerase II

Eukaryotic transcription is a highly regulated fundamental life process. A large number of regulatory proteins and complexes, many of them with sequence-specific DNA-binding activity are known to influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes genes encoding small, stable, non-translated RNAs. The pol III transcription is precisely regulated under various stress conditions. We used pol III transcription complex (TC) components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits and mass spectrometry to identify their potential interactors in vivo. A large interactome constituting chromatin modifiers, regulators and factors of transcription by pol I and pol II supports the possibility of a crosstalk between the three transcription machineries. The association of proteins and complexes involved in various basic life processes like ribogenesis, RNA processing, protein folding and degradation, DNA damage response, replication and transcription underscores the possibility of the pol III TC serving as a signaling hub for communication between the transcription and other cellular physiological activities under normal growth conditions. We also found an equally large number of proteins and complexes interacting with the TC under nutrient starvation condition, of which at least 25% were non-identical under the two conditions. The data reveal the possibility of a large number of signaling cues for pol III transcription against adverse conditions, necessary for an efficient co-ordination of various cellular functions.

Transcriptomic analyses identify key differentially expressed genes and clinical outcomes between triple-negative and non-triple-negative breast cancer.

PurposeThere are significant differences in the biological behavior between triple-negative breast cancer (TNBC) and non-triple-negative breast cancer (non-TNBC). In the present study, we identify key differential genes and clinical outcomes between TNBC and non-TNBC.Materials and methodsTranscriptomic analyses used GEO datasets (GSE76275), gene ontology, KEGG pathway analysis and cBioPortal. Quantitative RT-PCR analysis (qRT-PCR) was used to validate the differentially expressed genes. We used the KM Plotter Online Tool and 240 patients with TNBC tissue microarray to assay the prognostic value of HORMAD1.ResultsThe upregulated differentially expressed genes were enriched in transcription factor activity, sequence-specific DNA binding and nucleic acid binding transcription factor activity. Only 16 genes were upregulated when further screened for fold change >4-fold change. HORMAD1 and SOX8 exhibited high frequencies of change of greater than 10% (HORMAD1 was close to 20%). qRT-PCR results indicated that HORMAD1 and SOX8 mRNA levels were significantly upregulated in TNBC samples. In KM Plotter Online Tool, high HORMAD1 was associated with worse outcome. In our tissue microarray (including 240 TNBC tissues), IHC analysis revealed that 29.7% (55/240) of the tumor samples exhibited high HORMAD1 expression and 70.3% (185/240) of the tumor samples exhibited low HORMAD1 expression levels. Meanwhile, high HORMAD1 group has a bad prognosis.ConclusionThe status of transcriptional activation is an important difference between TNBC and non-TNBC. HORMAD1 is a key differential gene associated with poor outcome in TNBC. Epigenetic therapy and agents targeting cancer/testis antigens might potentially help to customize therapies of TNBC.

Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus

BackgroundChicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules.ResultsIn this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA.ConclusionsThe results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes.

Ribosomal protein (RP) genes must be coordinately expressed for proper assembly of the ribosome yet the mechanisms that control expression of RP genes in metazoans are poorly understood. Recently, TATA-binding protein-related factor 2 (TRF2) rather than the TATA-binding protein (TBP) was found to function in transcription of RP genes in Drosophila. Unlike TBP, TRF2 lacks sequence-specific DNA binding activity, so the mechanism by which TRF2 is recruited to promoters is unclear. We show that the transcription factor M1BP, which associates with the core promoter region, activates transcription of RP genes. Moreover, M1BP directly interacts with TRF2 to recruit it to the RP gene promoter. High resolution ChIP-exo was used to analyze in vivo the association of M1BP, TRF2 and TFIID subunit, TAF1. Despite recent work suggesting that TFIID does not associate with RP genes in Drosophila, we find that TAF1 is present at RP gene promoters and that its interaction might also be directed by M1BP. Although M1BP associates with thousands of genes, its colocalization with TRF2 is largely restricted to RP genes, suggesting that this combination is key to coordinately regulating transcription of the majority of RP genes in Drosophila.

Whole-exome sequencing to identify HOMEZ mutation as a prognostic marker in esophageal squamous cell carcinoma treated with concurrent chemoradiotherapy.

e15523 Background: The outcomes of inoperable esophageal squamous carcinoma (ESCC) patients treated with concurrent chemoradiotherapy remain poor. The discovery of novel biomarkers for predicting prognosis could allow for more accurate risk stratification and identification of high-risk patients. Although previous studies have elucidated some genomic alterations could be predictors of survival in these patients, none has focused on comprehensive genetic analysis using high-throughput platforms. Methods: Whole-exome sequencing was done in 96 inoperable ESCC patients treated with concurrent chemoradiotherapy from our phase Ⅲ clinical trial (NCT00686114). The prognostic marker was developed using a Random Survival Forest supervised classification methods based on log-rank splitting rules. The independence of the marker in survival prediction was evaluated by multivariable Cox regression analysis. The marker was further validated for the prognostic utility in an independent cohort (n = 150). The biological function of the genomic alteration was predicted by GO annotations. Results: A HOMEZ mutation (chr14:23744800_ACATCAT/ACAT) was detected in 54.1% of patients with ESCC received concurrent chemoradiotherapy. We identified the mutation was significantly associated with overall survival (hazard ratio [HR] 1.842; 95% confidence interval [CI] 1.080-3.142; P = 0.025) and loco-regional free survival (HR 3.008; 95% CI 1.302-6.947; P = 0.010). Multivariable Cox regression analysis showed that the mutation was also an independent prognostic factors for patients with ESCC (mutation type vs. wild type, overall survival: HR 2.161; 95% CI 1.241-3.764; p = 0·007; loco-regional free survival: HR 3.576; 95% CI 1.509-8.473; p = 0.004). GO annotations showed that HOMEZ tended to be frequently related to transcription factor activity, sequence-specific DNA binding and transcription corepressor activity. Conclusions: HOMEZ mutation is a novel independent prognostic factor for inoperable ESCC patients treated with concurrent chemoradiotherapy, and may be a potential therapeutic target.

Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding

The MDMX oncoprotein is an important regulator of tumor suppressor p53 activity during embryonic development. Despite sequence homology to the ubiquitin E3 ligase MDM2, MDMX depletion activates p53 without significant increase in p53 level, implicating a degradation-independent mechanism. We present evidence that MDMX inhibits the sequence-specific DNA binding activity of p53. This function requires the cooperation between MDMX and CK1α, and phosphorylation of S289 on MDMX. Depletion of MDMX or CK1α increases p53 DNA binding without stabilization of p53. A proteolytic fragment release assay revealed that in the MDMX-p53 complex, the MDMX acidic domain and RING domain interact stably with the p53 DNA binding domain. These interactions are referred to as secondary interactions because they only occur after the canonical-specific binding between the MDMX and p53 N termini, but exhibit significant binding stability in the mature complex. CK1α cooperates with MDMX to inhibit p53 DNA binding by further stabilizing the MDMX acidic domain and p53 core domain interaction. These results suggest that secondary intermolecular interaction is important in p53 regulation by MDMX, which may represent a common phenomenon in complexes containing multidomain proteins.

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.

Common BRAF(V600E)-directed pathway mediates widespread epigenetic silencing in colorectal cancer and melanoma

During cancer development, it is well established that many genes, including tumor suppressor genes, are hypermethylated and transcriptionally repressed, a phenomenon referred to as epigenetic silencing. In general, the factors involved in, and the mechanistic basis of, epigenetic silencing during cancer development are not well understood. We have recently described an epigenetic silencing pathway, directed by the oncogenic B-Raf proto-oncogene (BRAF) variant BRAF(V600E), that mediates widespread epigenetic silencing in colorectal cancer (CRC). Notably, the BRAF(V600E) mutation is also present in 50-70% of melanomas. Here, we show that the same pathway we identified in CRC also directs epigenetic silencing of a similar set of genes in BRAF-positive melanoma. In both CRC and melanoma, BRAF(V600E) promotes epigenetic silencing through up-regulation of v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), a transcriptional repressor with sequence-specific DNA-binding activity. The elevated concentration of MAFG drives DNA binding on the promoter. Promoter-bound MAFG recruits a set of corepressors that includes its heterodimeric partner BTB and CNC homology 1, basic leucine zipper transcription factor 1 (BACH1), the chromatin remodeling factor chromodomain helicase DNA-binding protein 8 (CHD8), and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. Our results reveal a common BRAF(V600E)-directed transcriptional regulatory pathway that mediates epigenetic silencing in unrelated solid tumors and provide strong support for an instructive model of oncoprotein-directed epigenetic silencing.

P300 Acetylates STAT3 and Increases Its Constitutive Transcriptional Activity in CLL Cells

Abstract Introduction: Signal transducer and activator of transcription (STAT)-3 is thought to promote oncogenesis by modulating the expression of genes that are required for tumor cell survival1. Following translation STAT3 may undergo several modifications such as phosphorylation or acetylation which modulate its activity. These modifications are typically cytokine-dependent but may also be cytokine-independent or constitutive. In chronic lymphocytic leukemia (CLL) STAT3 is constitutively phosphorylated (p) on serine-727 residues2. Serine pSTAT3 forms heterodimers, shuttles to the nucleus and induces transcription of STAT3 targeted genes. The transcriptional activity of STAT3 is also increased if STAT3 undergoes acetylation3. The conserved bormodomain motif of the acetyltransferase p300 binds and acetylates lysine residues of several proteins including STAT34. Whether STAT3 is acetylated in CLL cells is currently unknown. We sought to determine whether p300 acetylates STAT3 and affects its transcriptional activity in CLL cells. Methods and Results: Using western immunoblotting of the peripheral blood (PB) lymphocytes from 16 CLL patients we found that STAT3 is constitutively phosphorylated on lysine 685 residues. As previously reported2,5 we also detected serine pSTAT3 in all samples. Flow cytometry analysis revealed that a significant number of CD19+ CLL cells are constitutively phosphorylated on serine 727 and acetylated on lysine 685 residues. Immunoprecipitation studies confirmed these data. Protein extract of PB from 8 CLL patients were immunoprecipitated either with STAT3 or serine pSTAT3 antibodies and acetyl-STAT3 was detected in all immunoprecipitates, suggesting that the activated form of STAT3 is also acetylated in CLL cells. Because previous studies suggested that p300 acetylates STAT3 we obtained PB samples from 5 CLL patients and using western immunobloting and detected high levels of p300 in all patients but not in B lymphocytes from 2 healthy individuals. Then, to determine whether p300 affects the transcriptional activity of STAT3, we transfected CLL cells with p300 small-interfering (si) RNA. With a transfection efficiency of 30%, p300 transcripts and protein levels and acetyl-STAT3 levels were significantly reduced. Transfection with p300-siRNA reduced STAT3-DNA binding, as assessed by an electormobility shift assay (EMSA), decrease the levels of the STAT3-target gene p21 as assessed by chromatin immunoprecipitation (ChIP), and the levels of several STAT3-induced genes including STAT3, VEGF-c, c-Myc and p21, as assessed by RT-PCR. In addition, using Annexin V/PI we detected high apoptosis rates in p300-siRNA- transfected cells. Taken together these data suggest that the transcriptional activity of STAT3 is enhanced by p300 and STAT3 acetylation provides the cells with survival advantage. Conclusion: Our findings suggest that the aberrant expression of p300 induces acetylation and increases the transcriptional activity of STAT3 in CLL. Whether targeting STAT3 acetylation could be effective therapeutic modality in CLL remains to be determined.