CAT vectors for analysis of eukaryotic promoters and enhancers

We have constructed two sets of plasmids for analysis of factors affecting mammalian gene expression. The pOCAT series contains a bacterial chloramphenicol-resistance expression unit (cat) and no eukaryotic promoter. The pUTKAT series contains the same cat unit under the control of the thymidine-kinase promoter of Herpes simplex virus. These plasmids are designed for testing effects of inserted regulatory elements on cat expression after transient transfection of mammalian cells in culture. We demonstrate here that the pOCAT series is useful for studying activities of inserted eukaryotic promoters, and the pUTKAT series is useful for studying activities of inserted eukaryotic enhancers.     

Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44⁺/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.     

Alterations of DNA methylome in human bladder cancer

Bladder cancer is the fourth most common cancer in men in the United States, and its recurrence rate is highest among all malignancies. The unmet need for improved strategies for early detection, treatment, and monitoring of the progression of this disease continues to translate into high mortality and morbidity. The quest for advanced diagnostic, therapeutic, and prognostic approaches for bladder cancer is a high priority, which can be achieved by understanding the molecular mechanisms of the initiation and progression of this malignancy. Aberrant DNA methylation in single or multiple cancer-related genes/loci has been found in human bladder tumors and cancer cell lines, and urine sediments, and correlated with many clinicopathological features of this disease, including tumor relapse, muscle-invasiveness, and survival. The present review summarizes the published research on aberrant DNA methylation in connection with human bladder cancer. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in this prime epigenetic field of research. Identifying the potentially reversible and ‘drugable’ aberrant DNA methylation events that initiate and promote bladder cancer development can highlight biological markers for early diagnosis, effective therapy and accurate prognosis of this malignancy.     

Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers

Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating “hit” during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan–Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.     

Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers

Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between constitutively and alternatively spliced exons. Representatives of these ESRs and two SR protein binding sites were shown, experimentally, to display variable regulatory effects on alternative splicing, depending on their relative locations in the exon. This finding signifies the delicate positional effect of ESRs on alternative splicing regulation.     

Metabolomic analysis of cancer cachexia reveals distinct lipid and glucose alterations

Cancer cachexia remains a challenging clinical problem with complex pathophysiology and unreliable diagnostic tools. A blood test to detect this metabolic derangement would aid in early treatment of these patients. A ¹H NMR-based metabolomics approach was used to determine the unique metabolic fingerprint of cachexia and to search for biomarkers in serum samples taken from an established murine model of cancer cachexia. Male CD2F1 mice received a subcutaneous flank injection of C26 adenocarcinoma cells to induce experimental cancer-related cachexia. Two molecular markers of muscle atrophy, upregulation of the E3 ubiquitin ligase Muscle Ring Finger 1 (MuRF1) and aberrant glycosylation of β-dystroglycan (β-DG), were used to confirm muscle wasting in the tumor-bearing mice. Serum samples were collected for metabolomic analysis during the development of the cachexia: at baseline, when the tumor was palpable, and when the mice demonstrated cachexia. The unsupervised statistical analysis demonstrated a distinct metabolic profile with the onset of cachexia. The critical metabolic changes associated with cachexia included increased levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), with decreased serum glucose levels. Regression analysis demonstrated a very high correlation of the presence of aberrant glycosylation of β-DG with the unique metabolic profile of cachexia. This study demonstrates for the first time that metabolomics has potential as a diagnostic tool in cancer cachexia, and in further elucidating simultaneous metabolic pathway alterations due to this syndrome. In addition, variations in VLDL and LDL deserve more investigation as surrogate serum biomarkers for cancer cachexia.     

An integrative pan-cancer-wide analysis of epigenetic enzymes reveals universal patterns of epigenomic deregulation in cancer

BackgroundOne of the most important recent findings in cancer genomics is the identification of novel driver mutations which often target genes that regulate genome-wide chromatin and DNA methylation marks. Little is known, however, as to whether these genes exhibit patterns of epigenomic deregulation that transcend cancer types.ResultsHere we conduct an integrative pan-cancer-wide analysis of matched RNA-Seq and DNA methylation data across ten different cancer types. We identify seven tumor suppressor and eleven oncogenic epigenetic enzymes which display patterns of deregulation and association with genome-wide cancer DNA methylation patterns, which are largely independent of cancer type. In doing so, we provide evidence that genome-wide cancer hyper- and hypo- DNA methylation patterns are independent processes, controlled by distinct sets of epigenetic enzyme genes. Using causal network modeling, we predict a number of candidate drivers of cancer DNA hypermethylation and hypomethylation. Finally, we show that the genomic loci whose DNA methylation levels associate most strongly with expression of these putative drivers are highly consistent across cancer types.ConclusionsThis study demonstrates that there exist universal patterns of epigenomic deregulation that transcend cancer types, and that intra-tumor levels of genome-wide DNA hypomethylation and hypermethylation are controlled by distinct processes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0699-9) contains supplementary material, which is available to authorized users.     

Mutational analysis reveals distinct features of the Nox4-p22 phox complex

The integral membrane protein p22(phox) forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22(phox) constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22(phox) essential for formation and localization of the Nox4-p22(phox) complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22(phox)-deficient and promotes cell surface expression of the Nox4-p22(phox) heterodimer. Expression of p22(phox) truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22(phox) interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22(phox), which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22(phox) mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22(phox) association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22(phox) subunit with Nox4, a feature which could provide the basis for selective inhibitor development.     

Comparative proteomic analysis reveals similar and distinct features of proteins in dry and wet stigmas

Angiosperm stigma supports compatible pollen germination and tube growth, resulting in fertilization and seed production. Stigmas are mainly divided into two types, dry and wet, according to the absence or presence of exudates on their surfaces. Here, we used 2DE and MS to identify proteins specifically and preferentially expressed in the stigmas of maize (Zea Mays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed from the surface of the tobacco stigma. We found that the specifically and preferentially expressed proteins in maize and tobacco stigmas share similar distributions in functional categories. However, these proteins showed important difference between dry and wet stigmas in a few aspects, such as protein homology in "signal transduction" and "lipid metabolism," relative expression levels of proteins containing signal peptides and proteins in "defense and stress response." These different features might be related to the specific structures and functions of dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed. Our results provide important information on functions of proteins in dry and wet stigmas and reveal aspects of conservation and divergence between dry and wet stigmas at the proteomic level.     

Comparative proteomic analysis of myotube caveolae after milli-calpain deregulation

Caveolae are specialised RAFTs (detergent-resistant membrane microdomains enriched in cholesterol and glycosphingolipids). Caveolin, the main caveolae protein, is essential to the organisation of proteins and lipids, and interacts with numerous mediating proteins through a 'Caveolin Scalfolding Domain'. Consequently, caveolae play a major role in signal transduction and appear to be veritable signalling platforms. In muscle cells, caveolae are essential for fusion and differentiation, and are also implicated in a type of muscular dystrophy (LGMD1C). In a preceding work, we demonstrated the presence of active milli-calpain (m-calpain) in myotube caveolae. Calpains are calcium-dependent proteases involved in several cellular processes, including myoblast fusion and migration, PKC-mediated intracellular signalling and remodelling of the cytoskeleton. For the first time, we have proved the cholesterol-dependent localisation of m-calpain in the caveolae of C(2)C(12) myotubes. Calpain-dependent caveolae involvement in myoblast fusion was also strongly suggested. Furthermore, eight differentially expressed caveolae associated proteins were identified by 2-DE and LC-MS/MS analyses using an m-calpain antisense strategy. This proteomic study also demonstrates the action of m-calpain on vimentin, desmin and vinculin in myotube caveolae and suggests m-calpain's role in several mitochondrial pathways.     

Comparative genomic analysis reveals distinct genotypic features of the emerging pathogen Haemophilus influenzae type f

Background

The incidence of invasive disease caused by encapsulated Haemophilus influenzae type f (Hif) has increased in the post-H. influenzae type b (Hib) vaccine era. We previously annotated the first complete Hif genome from a clinical isolate (KR494) that caused septic shock and necrotizing myositis. Here, the full genome of Hif KR494 was compared to sequenced reference strains Hib 10810, capsule type d (Hid) Rd Kw20, and finally nontypeable H. influenzae 3655. The goal was to identify possible genomic characteristics that may shed light upon the pathogenesis of Hif.

Results

The Hif KR494 genome exhibited large regions of synteny with other H. influenzae, but also distinct genome rearrangements. A predicted Hif core genome of 1390 genes was shared with the reference strains, and 6 unique genomic regions comprising half of the 191 unique coding sequences were revealed. The majority of these regions were inserted genetic fragments, most likely derived from the closely-related Haemophilus spp. including H. aegyptius, H. haemolyticus and H. parainfluenzae. Importantly, the KR494 genome possessed several putative virulence genes that were distinct from non-type f strains. These included the sap2 operon, aef3 fimbriae, and genes for kanamycin nucleotidyltranserase, iron-utilization proteins, and putative YadA-like trimeric autotransporters that may increase the bacterial virulence. Furthermore, Hif KR494 lacked a hisABCDEFGH operon for de novo histidine biosynthesis, hmg locus for lipooligosaccharide biosynthesis and biofilm formation, the Haemophilus antibiotic resistance island and a Haemophilus secondary molybdate transport system. We confirmed the histidine auxotrophy and kanamycin resistance in Hif by functional experiments. Moreover, the pattern of unique or missing genes of Hif KR494 was similar in 20 Hif clinical isolates obtained from different years and geographical areas. A cross-species comparison revealed that the Hif genome shared more characteristics with H. aegyptius than Hid and NTHi.

Conclusions

The genomic comparative analyses facilitated identification of genotypic characteristics that may be related to the specific virulence of Hif. In relation to non-type f H. influenzae strains, the Hif genome contains differences in components involved in metabolism and survival that may contribute to its invasiveness.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-38) contains supplementary material, which is available to authorized users.