Wing-Kin Sung

Integration of External Signaling Pathways with the Core Transcriptional Network in Embryonic Stem Cells

Transcription factors (TFs) and their specific interactions with targets are crucial for specifying gene-expression programs. To gain insights into the transcriptional regulatory networks in embryonic stem (ES) cells, we use chromatin immunoprecipitation coupled with ultra-high-throughput DNA sequencing (ChIP-seq) to map the locations of 13 sequence-specific TFs (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12). These factors are known to play different roles in ES-cell biology as components of the LIF and BMP signaling pathways, self-renewal regulators, and key reprogramming factors. Our study provides insights into the integration of the signaling pathways into the ES-cell-specific transcription circuitries. Intriguingly, we find specific genomic regions extensively targeted by different TFs. Collectively, the comprehensive mapping of TF-binding sites identifies important features of the transcriptional regulatory networks that define ES-cell identity.

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.

An oestrogen-receptor-α-bound human chromatin interactome

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor α (ER-α) in the human genome. We found that most high-confidence remote ER-α-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-α functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.

Extensive Promoter-centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

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.

CTCF-mediated functional chromatin interactome in pluripotent cells

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. However, little is known about CTCF-associated higher-order chromatin structures at a global scale. Here we applied chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, we identified 1,480 cis- and 336 trans-interacting loci with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive cross-talk between promoters and regulatory elements. This highly complex nuclear organization offers insights toward the unifying principles that govern genome plasticity and function.

Exploiting indirect neighbours and topological weight to predict protein function from protein--protein interactions

Motivation: Most approaches in predicting protein function from protein--protein interaction data utilize the observation that a protein often share functions with proteins that interacts with it (its level-1 neighbours). However, proteins that interact with the same proteins (i.e. level-2 neighbours) may also have a greater likelihood of sharing similar physical or biochemical characteristics. We speculate that functional similarity between a protein and its neighbours from the two different levels arise from two distinct forms of functional association, and a protein is likely to share functions with its level-1 and/or level-2 neighbours. We are interested in finding out how significant is functional association between level-2 neighbours and how they can be exploited for protein function prediction. Results: We made a statistical study on recent interaction data and observed that functional association between level-2 neighbours is clearly observable. A substantial number of proteins are observed to share functions with level-2 neighbours but not with level-1 neighbours. We develop an algorithm that predicts the functions of a protein in two steps: (1) assign a weight to each of its level-1 and level-2 neighbours by estimating its functional similarity with the protein using the local topology of the interaction network as well as the reliability of experimental sources and (2) scoring each function based on its weighted frequency in these neighbours. Using leave-one-out cross validation, we compare the performance of our method against that of several other existing approaches and show that our method performs relatively well. Contact: g0306417@nus.edu.sg

Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

Assemblathon 1: A competitive assessment of de novo short read assembly methods

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.

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.