Genome sequencing and application of Taiwanese macaque Macaca cyclopis.

Formosan macaque (Macaca cyclopis) is the only non-human primate in Taiwan Island. We performed de novo hybrid assembly for M. cyclopis using Illumina paired-end short reads, mate-pair reads and Nanopore long reads and obtained 5065 contigs with a N50 of 2.66 megabases. M. cyclopis contigs > = 10 kb were assigned to chromosomes using Indian rhesus macaque (Macaca mulatta mulatta) genome assembly Mmul_10 as reference, resulting in a draft of M. cyclopis genome of 2,846,042,475 bases, distributed in 21 chromosomes. The draft genome contains 23,462 transcriptional origins (genes), capable of expressing 716,231 exons in 59,484 transcripts. Genome-based phylogenetic study using the assembled M. cyclopis genome together with genomes of four other macaque species, human, orangutan and chimpanzee showed similar result as previously reported. However, the M. cyclopis species was found to diverge from Chinese M. mulatta lasiota about 1.8 million years ago. Fossil gene analysis detected the presence of gap and pol endogenous viral elements of simian retrovirus in all macaques tested, including M. fascicularis, M. m. mulatta and M. cyclopis. However, M. cyclopis showed ~ 2 times less in number and more uniform in chromosomal locations. The constrain in foreign genome disturbance, presumably due to geographical isolation, should be able to simplify genomics-related investigations, making M. cyclopis an ideal primate species for medical research.

Transcriptome-level assessment of the impact of deformed wing virus on honey bee larvae.

Deformed wing virus (DWV) prevalence is high in honey bee (Apis mellifera) populations. The virus infects honey bees through vertical and horizontal transmission, leading to behavioural changes, wing deformity, and early mortality. To better understand the impacts of viral infection in the larval stage of honey bees, artificially reared honey bee larvae were infected with DWV (1.55 × 1010 copies/per larva). No significant mortality occurred in infected honey bee larvae, while the survival rates decreased significantly at the pupal stage. Examination of DWV replication revealed that viral replication began at 2 days post inoculation (d.p.i.), increased dramatically to 4 d.p.i., and then continuously increased in the pupal stage. To better understand the impact of DWV on the larval stage, DWV-infected and control groups were subjected to transcriptomic analysis at 4 d.p.i. Two hundred fifty-five differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified. Of these DEGs, 168 genes were downregulated, and 87 genes were upregulated. Gene Ontology (GO) analysis showed that 141 DEGs (55.3%) were categorized into molecular functions, cellular components and biological processes. One hundred eleven genes (38 upregulated and 73 downregulated) were annotated by KO (KEGG Orthology) pathway mapping and involved metabolic pathways, biosynthesis of secondary metabolites and glycine, serine and threonine metabolism pathways. Validation of DEGs was performed, and the related gene expression levels showed a similar tendency to the DEG predictions at 4 d.p.i.; cell wall integrity and stress response component 1 (wsc1), cuticular protein and myo-inositol 2-dehydrogenase (iolG) were significantly upregulated, and small conductance calcium-activated potassium channel protein (SK) was significantly downregulated at 4 d.p.i. Related gene expression levels at different d.p.i. revealed that these DEGs were significantly regulated from the larval stage to the pupal stage, indicating the potential impacts of gene expression levels from the larval to the pupal stages. Taken together, DWV infection in the honey bee larval stage potentially influences the gene expression levels from larvae to pupae and reduces the survival rate of the pupal stage. This information emphasizes the consequences of DWV prevalence in honey bee larvae for apiculture.

Comparison of gut microbiota of healthy and diseased walking sticks, Phasmotaenia lanyuhensis.

Research on gut microbiota of phytophagous insects has shown to be important for the physiological functions of insect hosts; however, little is known about the changes in gut microbiota when they are suffering from environmental stress or pathogen infections. During rearing of Phasmotaenia lanyuhensis (Phasmatodea: Phasmatidae), sluggish locomotion was usually followed by the death of the insect with a symptom of melanization in the front part of the abdomen. Therefore, the abnormal individuals were initially classified into moribund, light- and serious-symptom based on the level of abnormal physiological circumstances and melanization. The gut microbiota of these samples were further investigated by 16S metagenomic sequencing and the differences in bacterial abundance and structure of bacterial community were analyzed. A decrease in microbiota diversity was observed in the diseased P. lanyuhensis, with the abundance of phyla Proteobacteria and Firmicute relatively higher compared to those without symptom. Interestingly, principal component analysis based on the bacterial richness was correlated to the level of melanization symptom in the diseased P. lanyuhensis, suggested the change in bacterial microbiota involved in this abnormal circumstance. However, the factor that caused the initial alternation of microbiota remains to be identified. Additionally, the lack of bacterial diversity (i.e., absence of Meiothermus and Nubsella spp.) in P. lanyuhensis might reduce the fitness for surviving. This report provided the comprehensive microbiota analysis for P. lanyuhensis and concluded that either the relative abundance or the bacterial diversity of microbiota in the insect digestive system may influence the physiological functions of phytophagous insects.

Comprehensive Cohort Analysis of Mutational Spectrum in Early Onset Breast Cancer Patients.

Early onset breast cancer (EOBC), diagnosed at age ~40 or younger, is associated with a poorer prognosis and higher mortality rate compared to breast cancer diagnosed at age 50 or older. EOBC poses a serious threat to public health and requires in-depth investigation. We studied a cohort comprising 90 Taiwanese female patients, aiming to unravel the underlying mechanisms of EOBC etiopathogenesis. Sequence data generated by whole-exome sequencing (WES) and whole-genome sequencing (WGS) from white blood cell (WBC)-tumor pairs were analyzed to identify somatic missense mutations, copy number variations (CNVs) and germline missense mutations. Similar to regular breast cancer, the key somatic mutation-susceptibility genes of EOBC include TP53 (40% prevalence), PIK3CA (37%), GATA3 (17%) and KMT2C (17%), which are frequently reported in breast cancer; however, the structural protein-coding genes MUC17 (19%), FLG (16%) and NEBL (11%) show a significantly higher prevalence in EOBC. Furthermore, the top 2 genes harboring EOBC germline mutations, MUC16 (19%) and KRT18 (19%), encode structural proteins. Compared to conventional breast cancer, an unexpectedly higher number of EOBC susceptibility genes encode structural proteins. We suspect that mutations in structural proteins may increase physical permeability to environmental hormones and carcinogens and cause breast cancer to occur at a young age.

Long-read sequencing in deciphering human genetics to a greater depth.

Through four decades' development, DNA sequencing has inched into the era of single-molecule sequencing (SMS), or the third-generation sequencing (TGS), as represented by two distinct technical approaches developed independently by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Historically, each generation of sequencing technologies was marked by innovative technological achievements and novel applications. Long reads (LRs) are considered as the most advantageous feature of SMS shared by both PacBio and ONT to distinguish SMS from next-generation sequencing (NGS, or the second-generation sequencing) and Sanger sequencing (the first-generation sequencing). Long reads overcome the limitations of NGS and drastically improves the quality of genome assembly. Besides, ONT also contributes several unique features including ultra-long reads (ULRs) with read length above 300 kb and some close to 1 million bp, direct RNA sequencing and superior portability as made possible by pocket-sized MinION sequencer. Here, we review the history of DNA sequencing technologies and associated applications, with a special focus on the advantages as well as the limitations of ULR sequencing in genome assembly.

Application of cell-free DNA sequencing in characterization of bloodborne microbes and the study of microbe-disease interactions.

It is an important issue whether microorganisms can live harmoniously with normal cells in the cardiovascular system. The answer to the question will have enormous impact on medical microbiology. To address the issue, it is essential to identify and characterize the bloodborne microbes in an efficient and comprehensive manner. Due to microbial sequence complexity and the composition of significant number of unknown microbial species in the circulatory system, traditional approaches using cell culture, PCR, or microarray are not suitable for the purpose. Recent reports indicate that cell-free DNA (cfDNA) sequencing using next-generation sequencing (NGS) or single-molecule sequencing (SMS), together with bioinformatics approaches, possesses a strong potential enabling us to distinguish microbial species at the nucleotide level. Multiple studies using microbial cfDNA sequencing to identify microbes for septic patients have shown strong agreement with cell culture. Similar approaches have also been applied to reveal previously unidentified microorganisms or to demonstrate the feasibility of comprehensive assessment of bloodborne microorganisms for healthy and/or diseased individuals. SMS using either SMRT (single-molecule real-time) sequencing or Nanopore sequencing are providing new momentum to reinforce this line of investigation. Taken together, microbial cfDNA sequencing provides a novel opportunity allowing us to further understand the involvement of bloodborne microbes in development of diseases. Similar approaches should also be applicable to the study of metagenomics for sufficient and comprehensive analysis of microbial species living in various environments. This article reviews this line of research and discuss the methodological approaches that have been developed, or are likely to be developed in the future, which may have strong potential to facilitate cfDNA- and cfRNA-based studies of cancer and acute/chronic diseases, in the hope that a better understanding of the hidden microbes in the circulatory system will improve diagnosis, prevention and treatment of problematic diseases.

Genomic sequencing of Troides aeacus nucleopolyhedrovirus (TraeNPV) from golden birdwing larvae (Troides aeacus formosanus) to reveal defective Autographa californica NPV genomic features.

BACKGROUND: The golden birdwing butterfly (Troides aeacus formosanus) is a rarely observed species in Taiwan. Recently, a typical symptom of nuclear polyhedrosis was found in reared T. aeacus larvae. From the previous Kimura-2 parameter (K-2-P) analysis based on the nucleotide sequence of three genes in this isolate, polh, lef-8 and lef-9, the underlying virus did not belong to any known nucleopolyhedrovirus (NPV) species. Therefore, this NPV was provisionally named "TraeNPV". To understand this NPV, the nucleotide sequence of the whole TraeNPV genome was determined using next-generation sequencing (NGS) technology. RESULTS: The genome of TraeNPV is 125,477 bp in length with 144 putative open reading frames (ORFs) and its GC content is 40.45%. A phylogenetic analysis based on the 37 baculoviral core genes suggested that TraeNPV is a Group I NPV that is closely related to Autographa californica nucleopolyhedrovirus (AcMNPV). A genome-wide analysis showed that TraeNPV has some different features in its genome compared with other NPVs. Two novel ORFs (Ta75 and Ta139), three truncated ORFs (pcna, he65 and bro) and one duplicated ORF (38.7 K) were found in the TraeNPV genome; moreover, there are fewer homologous regions (hrs) than there are in AcMNPV, which shares eight hrs within the TraeNPV genome. TraeNPV shares similar genomic features with AcMNPV, including the gene content, gene arrangement and gene/genome identity, but TraeNPV lacks 15 homologous ORFs from AcMNPV in its genome, such as ctx, host cell-specific factor 1 (hcf-1), PNK/PNL, vp15, and apsup, which are involved in the auxiliary functions of alphabaculoviruses. CONCLUSIONS: Based on these data, TraeNPV would be clarified as a new NPV species with defective AcMNPV genomic features. The precise relationship between TraeNPV and other closely related NPV species were further investigated. This report could provide comprehensive information on TraeNPV for evolutionary insights into butterfly-infected NPV.

Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions.

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83-94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance.

Analysis of microbial sequences in plasma cell-free DNA for early-onset breast cancer patients and healthy females.

BACKGROUND: Cell-free circulating DNA (cfDNA) is becoming a useful biopsy for noninvasive diagnosis of diseases. Microbial sequences in plasma cfDNA may provide important information to improve prognosis and treatment. We have developed a stringent method to identify microbial species via microbial cfDNA in the blood plasma of early-onset breast cancer (EOBC) patients and healthy females. Empirically, microbe-originated sequence reads were identified by mapping non-human PE reads in cfDNA libraries to microbial databases. Those mapped concordantly to unique microbial species were assembled into contigs, which were subsequently aligned to the same databases. Microbial species uniquely aligned were identified and compared across all individuals on MCRPM (Microbial CfDNA Reads Per Million quality PE reads) basis. RESULTS: The predominant microbial cfDNAs in all plasma samples examined are originated from bacteria and these bacteria were limited to only a few genera. Among those, Acinetobacter johnsonii XBB1 and low levels of Mycobacterium spp. were commonly found in all healthy females, but also present in an EOBC patient. Compared to those in healthy counterparts, bacterial species in EOBC patients are more diverse and more likely to present at high levels. Among these three EOBC patients tested, a patient who has record high titer (2,724 MCRPM) of Pseudomonas mendocina together with 8.82 MCRPM of Pannonibacter phragmitetus has passed away; another patient infected by multiple Sphingomonas species remains alive; while the third patient who has similar microbial species (Acinetobacter johnsonii XBB1) commonly seen in normal controls is having a normal life. CONCLUSIONS: Our preliminary data on the profiles of microbial cfDNA sequences suggested that it may have some prognostic value in cancer patients. Validation in larger number of patients is warranted.

T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids.

Body fluid DNA sequencing is a powerful noninvasive approach for the diagnosis of genetic defects, infectious agents and diseases. The success relies on the quantity and quality of the DNA samples. However, numerous clinical samples are either at low quantity or of poor quality due to various reasons. To overcome these problems, we have developed T oligo-primed polymerase chain reaction (TOP-PCR) for full-length nonselective amplification of minute quantity of DNA fragments. TOP-PCR adopts homogeneous "half adaptor" (HA), generated by annealing P oligo (carrying a phosphate group at the 5' end) and T oligo (carrying a T-tail at the 3' end), for efficient ligation to target DNA and subsequent PCR amplification primed by the T oligo alone. Using DNA samples from body fluids, we demonstrate that TOP-PCR recovers minute DNA fragments and maintains the DNA size profile, while enhancing the major molecular populations. Our results also showed that TOP-PCR is a superior method for detecting apoptosis and outperforms the method adopted by Illumina for DNA amplification.

Complete Taiwanese Macaque (Macaca cyclopis) Mitochondrial Genome: Reference-Assisted de novo Assembly with Multiple k-mer Strategy.

The Taiwanese (Formosan) macaque (Macaca cyclopis) is the only nonhuman primate endemic to Taiwan. This primate species is valuable for evolutionary studies and as subjects in medical research. However, only partial fragments of the mitochondrial genome (mitogenome) of this primate species have been sequenced, not mentioning its nuclear genome. We employed next-generation sequencing to generate 2 x 90 bp paired-end reads, followed by reference-assisted de novo assembly with multiple k-mer strategy to characterize the M. cyclopis mitogenome. We compared the assembled mitogenome with that of other macaque species for phylogenetic analysis. Our results show that, the M. cyclopis mitogenome consists of 16,563 nucleotides encoding for 13 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs. Phylogenetic analysis indicates that M. cyclopis is most closely related to M. mulatta lasiota (Chinese rhesus macaque), supporting the notion of Asia-continental origin of M. cyclopis proposed in previous studies based on partial mitochondrial sequences. Our work presents a novel approach for assembling a mitogenome that utilizes the capabilities of de novo genome assembly with assistance of a reference genome. The availability of the complete Taiwanese macaque mitogenome will facilitate the study of primate evolution and the characterization of genetic variations for the potential usage of this species as a non-human primate model for medical research.

Antibody variable domain interface and framework sequence requirements for stability and function by high-throughput experiments.

Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of ß strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site.

Loop-sequence features and stability determinants in antibody variable domains by high-throughput experiments.

Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function.

Global assessment of Antrodia cinnamomea-induced microRNA alterations in hepatocarcinoma cells.

Recent studies have demonstrated a potent anticancer potential of medicinal fungus Antrodia cinnamomea, especially against hepatocarcinoma. These studies, however, were performed with prolonged treatments, and the early anticancer events remain missing. To probe the early anticancer mechanisms of A. cinnamomea, we treated SK-Hep-1 liver cancer cell with A. cinnamomea fruiting body extract for 2 and 4 hours, sequenced RNA samples with next-generation sequencing approach, and profiled the genome-wide miRNA and mRNA transcriptomes. Results unmistakably associated the early anticancer effect of A. cinnamomea fruiting body extract with a global downregulation of miRNAs which occurred solely in the A. cinnamomea fruiting body extract-treated SK-Hep-1 cells. Moreover, the inhibitory effect of A. cinnamomea fruiting body extract upon cancer miRNAs imposed no discrimination against any particular miRNA species, with oncomirs miR-21, miR-191 and major oncogenic clusters miR-17-92 and miR-106b-25 among the most severely downregulated. Western blotting further indicated a decrease in Drosha and Dicer proteins which play a key role in miRNA biogenesis, together with an increase of XRN2 known to participate in miRNA degradation pathway. Transcriptome profiling followed by GO and pathway analyses indicated that A. cinnamomea induced apoptosis, which was tightly associated with a downregulation of PI3K/AKT and MAPK pathways. Phosphorylation assay further suggested that JNK and c-Jun were closely involved in the apoptotic process. Taken together, our data indicated that the anticancer effect of A. cinnamomea can take place within a few hours by targeting multiple proteins and the miRNA system. A. cinnamomea indiscriminately induced a global downregulation of miRNAs by simultaneously inhibiting the key enzymes involved in miRNA maturation and activating XRN2 protein involved in miRNA degradation. Collapsing of the miRNA system together with downregulation of cell growth and survival pathways and activation of JNK signaling unleash the extrinsic and intrinsic apoptosis pathways, leading to the cancer cell death.

Concordant and discordant regulation of target genes by miR-31 and its isoforms.

It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA) variants designated as "isomiR". Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M), which differ only slightly in their 5'- and/or 3'-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3'-untranslated region (UTR) of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.

Palindromic sequence impedes sequencing-by-ligation mechanism.

BACKGROUND: Current next-generation sequencing (NGS) platforms adopt two types of sequencing mechanisms: by synthesis or by ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed. METHODS: To test the effect of the palindromic region on sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from MmeI digestion of the amplified clone to generate a sequencing library for SOLiD 5500xl sequencer. RESULTS: Results showed that, traditional ABI sequencers, which adopt sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region. CONCLUSIONS: Taken together, we demonstrate that SOLiD machines, which employ sequencing-by-ligation mechanism, are unable to read through the palindromic region. On the other hand, sequencing-by-synthesis sequencers had no difficulty in doing so.

De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.

BACKGROUND: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARgamma binding sites in 3T3-L1 preadipocyte cells. METHODOLOGY/PRINCIPAL FINDINGS: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation. CONCLUSIONS/SIGNIFICANCE: We have identified in a genome-wide manner the binding sites of PPARgamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARgamma function in the context of adipocyte differentiation.

The use of multiple displacement amplification to amplify complex DNA libraries.

Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation. To reduce biases, large numbers of colonies are plated and scraped from solid-surface agar. This process is time consuming, tedious and limits scaling up. At the same time, multiple displacement amplification (MDA) has been recently developed as a method for in vitro amplification of DNA. However, MDA has no selection function for the removal of ligation multimers. We developed a novel method of briefly introducing ligation reactions into bacteria to select single insert DNA clones followed by MDA to amplify. We applied these methods to a Gene Identification Signatures with Paired-End diTags (GIS-PET) library, which is a complex transcriptome library created by pairing short tags from the 5' and 3' ends of cDNA fragments together, and demonstrated that this selection and amplification strategy is unbiased and efficient.

Genome-wide mapping of RELA(p65) binding identifies E2F1 as a transcriptional activator recruited by NF-kappaB upon TLR4 activation.

NF-kappaB is a key mediator of inflammation. Here, we mapped the genome-wide loci bound by the RELA subunit of NF-kappaB in lipopolysaccharide (LPS)-stimulated human monocytic cells, and together with global gene expression profiling, found an overrepresentation of the E2F1-binding motif among RELA-bound loci associated with NF-kappaB target genes. Knockdown of endogenous E2F1 impaired the LPS inducibility of the proinflammatory cytokines CCL3(MIP-1alpha), IL23A(p19), TNF-alpha, and IL1-beta. Upon LPS stimulation, E2F1 is rapidly recruited to the promoters of these genes along with p50/RELA heterodimer via a mechanism that is dependent on NF-kappaB activation. Together with the observation that E2F1 physically interacts with p50/RELA in LPS-stimulated cells, our findings suggest that NF-kappaB recruits E2F1 to fully activate the transcription of NF-kappaB target genes. Global gene expression profiling subsequently revealed a spectrum of NF-kappaB target genes that are positively regulated by E2F1, further demonstrating the critical role of E2F1 in the Toll-like receptor 4 pathway.

Pathway aberrations of murine melanoma cells observed in Paired-End diTag transcriptomes.

BACKGROUND: Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. However, little is known about melanoma pathway aberrations. Here we applied the robust Gene Identification Signature Paired End diTag (GIS-PET) approach to investigate the melanoma transcriptome and characterize the global pathway aberrations. METHODS: GIS-PET technology directly links 5' mRNA signatures with their corresponding 3' signatures to generate, and then concatenate, PETs for efficient sequencing. We annotated PETs to pathways of KEGG database and compared the murine B16F1 melanoma transcriptome with three non-melanoma murine transcriptomes (Melan-a2 melanocytes, E14 embryonic stem cells, and E17.5 embryo). Gene expression levels as represented by PET counts were compared across melanoma and melanocyte libraries to identify the most significantly altered pathways and investigate the expression levels of crucial cancer genes. RESULTS: Melanin biosynthesis genes were solely expressed in the cells of melanocytic origin, indicating the feasibility of using the PET approach for transcriptome comparison. The most significantly altered pathways were metabolic pathways, including upregulated pathways: purine metabolism, aminophosphonate metabolism, tyrosine metabolism, selenoamino acid metabolism, galactose utilization, nitrobenzene degradation, and bisphenol A degradation; and downregulated pathways: oxidative phosphorylation, ATPase synthesis, TCA cycle, pyruvate metabolism, and glutathione metabolism. The downregulated pathways concurrently indicated a slowdown of mitochondrial activities. Mitochondrial permeability was also significantly altered, as indicated by transcriptional activation of ATP/ADP, citrate/malate, Mg++, fatty acid and amino acid transporters, and transcriptional repression of zinc and metal ion transporters. Upregulation of cell cycle progression, MAPK, and PI3K/Akt pathways were more limited to certain region(s) of the pathway. Expression levels of c-Myc and Trp53 were also higher in melanoma. Moreover, transcriptional variants resulted from alternative transcription start sites or alternative polyadenylation sites were found in Ras and genes encoding adhesion or cytoskeleton proteins such as integrin, beta-catenin, alpha-catenin, and actin. CONCLUSION: The highly correlated results unmistakably point to a systematic downregulation of mitochondrial activities, which we hypothesize aims to downgrade the mitochondria-mediated apoptosis and the dependency of cancer cells on angiogenesis. Our results also demonstrate the advantage of using the PET approach in conjunction with KEGG database for systematic pathway analysis.