Kuo Ping Chiu

The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells

Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference–mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.

A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome

The ability to derive a whole-genome map of transcription-factor binding sites (TFBS) is crucial for elucidating gene regulatory networks. Herein, we describe a robust approach that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of TFBS. We have applied this strategy to map p53 targets in the human genome. From a saturated sampling of over half a million PET sequences, we characterized 65,572 unique p53 ChIP DNA fragments and established overlapping PET clusters as a readout to define p53 binding loci with remarkable specificity. Based on this information, we refined the consensus p53 binding motif, identified at least 542 binding loci with high confidence, discovered 98 previously unidentified p53 target genes that were implicated in novel aspects of p53 functions, and showed their clinical relevance to p53-dependent tumorigenesis in primary cancer samples.

Whole-Genome Mapping of Histone H3 Lys4 and 27 Trimethylations Reveals Distinct Genomic Compartments in Human Embryonic Stem Cells

Epigenetic modifications are crucial for proper lineage specification and embryo development. To explore the chromatin modification landscapes in human ES cells, we profiled two histone modifications, H3K4me3 and H3K27me3, by ChIP coupled with the paired-end ditags sequencing strategy. H3K4me3 was found to be a prevalent mark and occurred in close proximity to the promoters of two-thirds of total human genes. Among the H3K27me3 loci identified, 56% are associated with promoters and the vast majority of them are comodified by H3K4me3. By deep-transcript digital counting, 80% of H3K4me3 and 36% of comodified promoters were found to be transcribed. Remarkably, we observed that different combinations of histone methylations are associated with genes from distinct functional categories. These global histone methylation maps provide an epigenetic framework that enables the discovery of novel transcriptional networks and delineation of different genetic compartments of the pluripotent cell genome.

Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection

Summary Background The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines. Methods We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04). Findings We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing. Interpretation Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations. Published online May 9, 2003 http://image.thelancet.com/extras/03art4454web.pdf

Global mapping of c-Myc binding sites and target gene networks in human B cells

The protooncogene MYC encodes the c-Myc transcription factor that regulates cell growth, cell proliferation, cell cycle, and apoptosis. Although deregulation of MYC contributes to tumorigenesis, it is still unclear what direct Myc-induced transcriptomes promote cell transformation. Here we provide a snapshot of genome-wide, unbiased characterization of direct Myc binding targets in a model of human B lymphoid tumor using ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET). Myc potentially occupies >4,000 genomic loci with the majority near proximal promoter regions associated frequently with CpG islands. Using gene expression profiles with ChIP-PET, we identified 668 direct Myc-regulated gene targets, including 48 transcription factors, indicating that Myc is a central transcriptional hub in growth and proliferation control. This first global genomic view of Myc binding sites yields insights of transcriptional circuitries and cis regulatory modules involving Myc and provides a substantial framework for our understanding of mechanisms of Myc-induced tumorigenesis.

Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation

We have developed a DNA tag sequencing and mapping strategy called gene identification signature (GIS) analysis, in which 5′ and 3′ signatures of full-length cDNAs are accurately extracted into paired-end ditags (PETs) that are concatenated for efficient sequencing and mapped to genome sequences to demarcate the transcription boundaries of every gene. GIS analysis is potentially 30-fold more efficient than standard cDNA sequencing approaches for transcriptome characterization. We demonstrated this approach with 116,252 PET sequences derived from mouse embryonic stem cells. Initial analysis of this dataset identified hundreds of previously uncharacterized transcripts, including alternative transcripts of known genes. We also uncovered several intergenically spliced and unusual fusion transcripts, one of which was confirmed as a trans-splicing event and was differentially expressed. The concept of paired-end ditagging described here for transcriptome analysis can also be applied to whole-genome analysis of cis-regulatory and other DNA elements and represents an important technological advance for genome annotation.

Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing™) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200 000 to 300 000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.

De-Novo Identification of PPARγ/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-γ (PPARγ). PPARγ 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 PPARγ have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARγ binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARγ 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 PPARγ 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 PPARγ and RXR and that they are functionally capable of driving PPARγ specific transcription. Our results strongly indicate that PPARγ is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARγ/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 PPARγ 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 PPARγ/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARγ 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 PPARγ and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARγ function in the context of adipocyte differentiation.

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.

PET-Tool: a software suite for comprehensive processing and managing of Paired-End diTag (PET) sequence data.

BackgroundWe recently developed the Paired End diTag (PET) strategy for efficient characterization of mammalian transcriptomes and genomes. The paired end nature of short PET sequences derived from long DNA fragments raised a new set of bioinformatics challenges, including how to extract PETs from raw sequence reads, and correctly yet efficiently map PETs to reference genome sequences. To accommodate and streamline data analysis of the large volume PET sequences generated from each PET experiment, an automated PET data process pipeline is desirable.ResultsWe designed an integrated computation program package, PET-Tool, to automatically process PET sequences and map them to the genome sequences. The Tool was implemented as a web-based application composed of four modules: the Extractor module for PET extraction; the Examiner module for analytic evaluation of PET sequence quality; the Mapper module for locating PET sequences in the genome sequences; and the ProjectManager module for data organization. The performance of PET-Tool was evaluated through the analyses of 2.7 million PET sequences. It was demonstrated that PET-Tool is accurate and efficient in extracting PET sequences and removing artifacts from large volume dataset. Using optimized mapping criteria, over 70% of quality PET sequences were mapped specifically to the genome sequences. With a 2.4 GHz LINUX machine, it takes approximately six hours to process one million PETs from extraction to mapping.ConclusionThe speed, accuracy, and comprehensiveness have proved that PET-Tool is an important and useful component in PET experiments, and can be extended to accommodate other related analyses of paired-end sequences. The Tool also provides user-friendly functions for data quality check and system for multi-layer data management.