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1.  CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription 
Cell  2015;163(7):1611-1627.
Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced ChIA-PET strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CTCF and RNAPII with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes towards CTCF-foci for coordinated transcription. Furthermore, we show that haplotype-variants and allelic-interactions have differential effects on chromosome configuration influencing gene expression and may provide mechanistic insights into functions associated with disease susceptibility. 3D-genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D-genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.
PMCID: PMC4734140  PMID: 26686651
2.  Bridging Tumor Genomics to Patient Outcomes through an Integrated Patient-Derived Xenograft (PDX) Platform 
Clinical lung cancer  2015;16(3):165-172.
New approaches to optimizing cancer drug development in both the laboratory and the clinic will be required to fully achieve the goal of individualized, precision cancer therapy. Improved preclinical models that more closely reflect the now recognized genomic complexity of human cancers are needed. Here we describe a collaborative research project integrating core resources of The Jackson Laboratory (JAX) Basic Science Cancer Center with genomics and clinical research facilities at the UC Davis Comprehensive Cancer Center (UCD) to establish a clinically and genomically-annotated patient-derived xenograft (PDX) platform designed to enhance new drug development and strategies for targeted therapies. Advanced stage non-small cell lung cancer (NSCLC) was selected for initial studies due to emergence of a number of “druggable” molecular targets, and recent recognition of substantial inter- and intra-patient tumor heterogeneity. Additionally, clonal evolution after targeted therapy interventions makes this tumor type ideal for investigation of this platform. Employing the immunodeficient NSG mouse, over 200 NSCLC tumor biopsies have been xenotransplanted. During the annotation process, patient tumors and subsequent PDXs are compared at multiple levels, including histomorphology, clinically applicable molecular biomarkers, global gene expression patterns, gene copy number variations and DNA/chromosomal alterations. NSCLC PDXs are grouped into panels of interest by oncogene subtype and/or histologic subtype. Multi-regimen drug testing, paired with pre- and post-therapy next generation sequencing (NGS) and timed tumor pharmacodynamics, enables determination of efficacy, signaling pathway alterations and mechanisms of sensitivity-resistance in individual models. This approach should facilitate derivation of new therapeutic strategies and the transition to individualized therapy.
PMCID: PMC4586173  PMID: 25838158
3.  Xenopus tropicalis Genome Re-Scaffolding and Re-Annotation Reach the Resolution Required for In Vivo ChIA-PET Analysis 
PLoS ONE  2015;10(9):e0137526.
Genome-wide functional analyses require high-resolution genome assembly and annotation. We applied ChIA-PET to analyze gene regulatory networks, including 3D chromosome interactions, underlying thyroid hormone (TH) signaling in the frog Xenopus tropicalis. As the available versions of Xenopus tropicalis assembly and annotation lacked the resolution required for ChIA-PET we improve the genome assembly version 4.1 and annotations using data derived from the paired end tag (PET) sequencing technologies and approaches (e.g., DNA-PET [gPET], RNA-PET etc.). The large insert (~10Kb, ~17Kb) paired end DNA-PET with high throughput NGS sequencing not only significantly improved genome assembly quality, but also strongly reduced genome “fragmentation”, reducing total scaffold numbers by ~60%. Next, RNA-PET technology, designed and developed for the detection of full-length transcripts and fusion mRNA in whole transcriptome studies (ENCODE consortia), was applied to capture the 5' and 3' ends of transcripts. These amendments in assembly and annotation were essential prerequisites for the ChIA-PET analysis of TH transcription regulation. Their application revealed complex regulatory configurations of target genes and the structures of the regulatory networks underlying physiological responses. Our work allowed us to improve the quality of Xenopus tropicalis genomic resources, reaching the standard required for ChIA-PET analysis of transcriptional networks. We consider that the workflow proposed offers useful conceptual and methodological guidance and can readily be applied to other non-conventional models that have low-resolution genome data.
PMCID: PMC4562602  PMID: 26348928
4.  Development and Characterization of Bladder Cancer Patient-Derived Xenografts for Molecularly Guided Targeted Therapy 
PLoS ONE  2015;10(8):e0134346.
The overarching goal of this project is to establish a patient-derived bladder cancer xenograft (PDX) platform, annotated with deep sequencing and patient clinical information, to accelerate the development of new treatment options for bladder cancer patients. Herein, we describe the creation, initial characterization and use of the platform for this purpose.
Methods and Findings
Twenty-two PDXs with annotated clinical information were established from uncultured unselected clinical bladder cancer specimens in immunodeficient NSG mice. The morphological fidelity was maintained in PDXs. Whole exome sequencing revealed that PDXs and parental patient cancers shared 92–97% of genetic aberrations, including multiple druggable targets. For drug repurposing, an EGFR/HER2 dual inhibitor lapatinib was effective in PDX BL0440 (progression-free survival or PFS of 25.4 days versus 18.4 days in the control, p = 0.007), but not in PDX BL0269 (12 days versus 13 days in the control, p = 0.16) although both expressed HER2. To screen for the most effective MTT, we evaluated three drugs (lapatinib, ponatinib, and BEZ235) matched with aberrations in PDX BL0269; but only a PIK3CA inhibitor BEZ235 was effective (p<0.0001). To study the mechanisms of secondary resistance, a fibroblast growth factor receptor 3 inhibitor BGJ398 prolonged PFS of PDX BL0293 from 9.5 days of the control to 18.5 days (p<0.0001), and serial biopsies revealed that the MAPK/ERK and PIK3CA-AKT pathways were activated upon resistance. Inhibition of these pathways significantly prolonged PFS from 12 day of the control to 22 days (p = 0.001). To screen for effective chemotherapeutic drugs, four of the first six PDXs were sensitive to the cisplatin/gemcitabine combination, and chemoresistance to one drug could be overcome by the other drug.
The PDX models described here show good correlation with the patient at the genomic level and known patient response to treatment. This supports further evaluation of the PDXs for their ability to accurately predict a patient’s response to new targeted and combination strategies for bladder cancer.
PMCID: PMC4535951  PMID: 26270481
5.  Patient-specific driver gene prediction and risk assessment through integrated network analysis of cancer omics profiles 
Nucleic Acids Research  2015;43(7):e44.
Extensive and multi-dimensional data sets generated from recent cancer omics profiling projects have presented new challenges and opportunities for unraveling the complexity of cancer genome landscapes. In particular, distinguishing the unique complement of genes that drive tumorigenesis in each patient from a sea of passenger mutations is necessary for translating the full benefit of cancer genome sequencing into the clinic. We address this need by presenting a data integration framework (OncoIMPACT) to nominate patient-specific driver genes based on their phenotypic impact. Extensive in silico and in vitro validation helped establish OncoIMPACT's robustness, improved precision over competing approaches and verifiable patient and cell line specific predictions (2/2 and 6/7 true positives and negatives, respectively). In particular, we computationally predicted and experimentally validated the gene TRIM24 as a putative novel amplified driver in a melanoma patient. Applying OncoIMPACT to more than 1000 tumor samples, we generated patient-specific driver gene lists in five different cancer types to identify modes of synergistic action. We also provide the first demonstration that computationally derived driver mutation signatures can be overall superior to single gene and gene expression based signatures in enabling patient stratification and prognostication. Source code and executables for OncoIMPACT are freely available from
PMCID: PMC4402507  PMID: 25572314
6.  Functional chromatin features are associated with structural mutations in cancer 
BMC Genomics  2014;15(1):1013.
Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers.
We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound.
Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1013) contains supplementary material, which is available to authorized users.
PMCID: PMC4253614  PMID: 25417144
Protein binding; Chromatin state; Structural mutations; Cancer
7.  Large-Scale Functional Organization of Long-Range Chromatin Interaction Networks 
Cell reports  2012;2(5):1207-1219.
Chromatin interactions play important roles in transcription regulation. To better understand the underlying evolutionary and functional constraints of these interactions, we implemented a systems approach to examine RNA polymerase-II-associated chromatin interactions in human cells. We found that 40% of the total genomic elements involved in chromatin interactions converged to a giant, scale-free-like, hierarchical network organized into chromatin communities. The communities were enriched in specific functions and were syntenic through evolution. Disease-associated SNPs from genome-wide association studies were enriched among the nodes with fewer interactions, implying their selection against deleterious interactions by limiting the total number of interactions, a model that we further reconciled using somatic and germline cancer mutation data. The hubs lacked disease-associated SNPs, constituted a nonrandomly interconnected core of key cellular functions, and exhibited lethality in mouse mutants, supporting an evolutionary selection that favored the nonrandom spatial clustering of the least-evolving key genomic domains against random genetic or transcriptional errors in the genome. Altogether, our analyses reveal a systems-level evolutionary framework that shapes functionally compartmentalized and error-tolerant transcriptional regulation of human genome in three dimensions.
PMCID: PMC4181841  PMID: 23103170
8.  Personalized Medicine: Does the Molecular Suit Fit? 
The Oncologist  2013;18(6):653-654.
PMCID: PMC4063388  PMID: 23814161
9.  Detection of Chromosomal Breakpoints in Patients with Developmental Delay and Speech Disorders 
PLoS ONE  2014;9(3):e90852.
Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).
PMCID: PMC3946304  PMID: 24603971
10.  HGV2009 Meeting: Bigger and Better Studies Provide More Answers and More Questions 
Human mutation  2010;31(7):10.1002/humu.21270.
The 11th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2009: Tallinn, Estonia, 11th–13th September 2009) provided a stimulating workshop environment where diverse academics and industry representatives explored the latest progress, challenges, and opportunities in relating genome variation to evolution, technology, health, and disease. Key themes included Genome-Wide Association Studies (GWAS), progress beyond GWAS, sequencing developments, and bioinformatics approaches to large-scale datasets.
PMCID: PMC3871991  PMID: 20506253
HGV2009; SNP; variation; GWAS; CNV
11.  Heterogeneous epigenetic regulation of TIMP3 in prostate cancer 
Epigenetics  2012;7(11):1279-1289.
Tissue inhibitor of metalloproteinase-3 (TIMP3) is a tumor suppressor gene frequently downregulated in prostate cancer. The mechanisms involved in TIMP3 transcriptional repression are not fully understood, but evidence suggests that promoter hypermethylation may not be the predominant epigenetic alteration in prostate cancer. To clarify this issue, we examined the contribution of both CpG site promoter methylation and histone modifications on TIMP3 downregulation. Using publicly available data sets, we confirmed that TIMP3 mRNA expression is decreased in prostate tumors relative to normal glands. Immunohistochemical analysis also showed decreased TIMP3 levels in high-grade primary tumors, but promoter hypermethylation was only detected in 6 of 28 (21%) high-grade specimens. Similarly, in prostate cancer cells, TIMP3 hypermethylation was only observed in DU145 cells. Treatment of DU145 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored TIMP3 expression, and this was significantly amplified by co-treating the cells with the HDAC inhibitor trichostatin A (TSA). Alternatively, in cells that did not exhibit aberrant TIMP3 methylation (LNCaP and PC3), TIMP3 expression could be upregulated by the combination of histone methylation inhibitor 3-Deazaneplanocin A (DZNep) and TSA. This reversal of transcriptional repression was associated with decreased H3K27me3 and increased H3K9ac histone marks at the TIMP3 promoter, as demonstrated by chromatin immunoprecipitation. Collectively, these results indicate that histone modifications can contribute to TIMP3 repression in the absence of promoter hypermethylation, and suggest that the combination of histone modifying agents could restore TIMP3 expression in prostate tumors harboring aberrant histone modifications at the TIMP3 promoter.
PMCID: PMC3499329  PMID: 23023649
prostate cancer; TIMP3; DNA methylation; histone modifications; epigenetic drugs
12.  Integrative genomics of gene and metabolic regulation by estrogen receptors α and β, and their coregulators 
To define how the estrogen receptors α and β control specific responses in breast cancer cells, genome-wide patterns of chromatin binding of the ERα and ERβ receptors and their coregulators, SRC3 and RIP140, were determined and integrated with gene expression data and functional analyses.
The closely related transcription factors, estrogen receptors ERα and ERβ, can elicit differential cellular responses.To understand the basis of this specificity, chromatin binding of ERs and key coregulators, and gene expression, were analyzed genome wide in human breast cancer cells containing ERα only, ERα+ERβ, and ERβ only.A clustering-based combinatorial analysis of ChIP-Seq and gene expression data was used to parse genes into groups, specifying their mode of functional regulation in a particular cell background.Through this analysis, RIP140 was identified as an ERβ-preferential cofactor regulating cell proliferation, apoptosis, and adipogenesis programs.A 20-gene ERβ and RIP140 signature was developed, which predicted outcome and disease-free survival in breast cancer patients.
The closely related transcription factors (TFs), estrogen receptors ERα and ERβ, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing breast cancer cells with ERα, ERβ, or both receptors as a model system to define the basis for differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and the use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ERβ and SRC3 with ERα. The receptors modified each other's transcriptional effect, and ERβ countered the proliferative drive of ERα through several novel mechanisms associated with specific binding-site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes, specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer.
PMCID: PMC3964312  PMID: 23774759
coregulator usage; estrogen receptors α and β; gene regulation; metabolism; proliferation
14.  Extensive Promoter-centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation 
Cell  2012;148(1-2):84-98.
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 intra-genic, extra-genic and inter-genic 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 non-coding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into the transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.
PMCID: PMC3339270  PMID: 22265404
15.  PPARG Binding Landscapes in Macrophages Suggest a Genome-Wide Contribution of PU.1 to Divergent PPARG Binding in Human and Mouse 
PLoS ONE  2012;7(10):e48102.
Genome-wide comparisons of transcription factor binding sites in different species can be used to evaluate evolutionary constraints that shape gene regulatory circuits and to understand how the interaction between transcription factors shapes their binding landscapes over evolution.
We have compared the PPARG binding landscapes in macrophages to investigate the evolutionary impact on PPARG binding diversity in mouse and humans for this important nuclear receptor. Of note, only 5% of the PPARG binding sites were shared between the two species. In contrast, at the gene level, PPARG target genes conserved between both species constitute more than 30% of the target genes regulated by PPARG ligand in human macrophages. Moreover, the majority of all PPARG binding sites (55–60%) in macrophages show co-occupancy of the lineage-specification factor PU.1 in both species. Exploring the evolutionary dynamics of PPARG binding sites, we observed that PU.1 co-binding to PPARG sites appears to be important for possible PPARG ancestral functions such as lipid metabolism. Thus we speculate that PU.1 may have guided utilization of these species-specific PPARG conserved binding sites in macrophages during evolution.
We propose a model in which PU.1 sites may have served as “anchor” loci for the formation of new and functionally relevant PPARG binding sites throughout evolution. As PU.1 is an essential factor in macrophage biology, such an evolutionary mechanism would allow for the establishment of relevant PPARG regulatory modules in a PU.1-dependent manner and yet permit for nuanced regulatory changes in individual species.
PMCID: PMC3485280  PMID: 23118933
16.  Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons 
PLoS ONE  2012;7(9):e46152.
Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10–20 kb and compared their characteristics with short insert (1 kb) libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.
PMCID: PMC3461012  PMID: 23029419
17.  JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer 
We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer.
Through microarray informatics, Cox proportional hazards regression was used to analyze the correlation between gene expression and distant metastasis-free survival (DMFS) of patients in 14 independent breast cancer cohorts. JMJD6 emerged as a top candidate gene robustly associated with poor patient survival. Immunohistochemistry, siRNA-mediated silencing, and forced overexpression of JMJD6 in cell-based assays elucidated molecular mechanisms of JMJD6 action in breast cancer progression and shed light on the clinical breast cancer subtypes relevant to JMJD6 action.
JMJD6 was expressed at highest levels in tumors associated with worse outcomes, including ER- and basal-like, Claudin-low, Her2-enriched, and ER+ Luminal B tumors. High nuclear JMJD6 protein was associated with ER negativity, advanced grade, and poor differentiation in tissue microarrays. Separation of ER+/LN- patients that received endocrine monotherapy indicated that JMJD6 is predictive of poor outcome in treatment-specific subgroups. In breast cancer cell lines, loss of JMJD6 consistently resulted in suppressed proliferation but not apoptosis, whereas forced stable overexpression increased growth. In addition, knockdown of JMJD6 in invasive cell lines, such as MDA-MB231, decreased motility and invasion, whereas overexpression in MCF-7 cells slightly promoted motility but did not confer invasive growth. Microarray analysis showed that the most significant transcriptional changes occurred in cell-proliferation genes and genes of the TGF-β tumor-suppressor pathway. High proliferation was characterized by constitutively high cyclin E protein levels. The inverse relation of JMJD6 expression with TGF-β2 could be extrapolated to the breast cancer cohorts, suggesting that JMJD6 may affect similar pathways in primary breast cancer.
JMJD6 is a novel biomarker of tumor aggressiveness with functional implications in breast cancer growth and migration.
PMCID: PMC3446348  PMID: 22621393
18.  Cellular reprogramming by the conjoint action of ERα, FOXA1, and GATA3 to a ligand-inducible growth state 
Estrogen receptor α (ERα), FOXA1, and GATA3 form a functional enhanceosome in MCF-7 breast carcinoma cell that is significantly associated with active transcriptional features such as enhanced p300 co-activator and RNA Pol II recruitment as well as chromatin opening.The enhanceosome exerts significant impact and optimal transcriptional control in the regulation of E2-responsive genes.The presence of FOXA1 and GATA3 is indispensable in restoring the ERα growth-response machinery in the ERα-negative cells and recapitulating the appropriate expression cassette.
Estrogen receptor α (ERα) is a ligand-inducible hormone nuclear receptor that has important physiology and pathology roles in reproduction, cancer, and cardiovascular biology. The regulation of ERα involves its binding to the DNA recognition sequence also known as estrogen-response elements (EREs) and recruits a variety of co-activators, corepressors, and chromatin remodeling enzymes to initiate transcription machinery. In our previous (Lin et al, 2007) and recent (Joseph et al, 2010) studies, we have identified high confidence ERα binding sites in MCF-7 human mammary carcinoma cells. With known motif scanning and de novo motif detection, we identified that FOXA1 and GATA3 motifs were commonly enriched around ERα binding sites. Moreover, numerous microarray studies have documented the co-expression of ERα, FOXA1, and GATA3 in primary breast tumors (Badve et al, 2007; Wilson and Giguere, 2008). This evidence suggests that these three transcription factors (TFs) may cluster on DNA binding sites and contribute to the breast cancer phenotype. However, there is little understanding as to the nature of their coordinated interaction at the genome level or the biological consequences of their detailed interaction.
We mapped the genome-wide binding profiles of ERα, FOXA1, and GATA3 using the massive parallel chromatin immunoprecipitation-sequencing (ChIP-seq) approach. We observed that ERα, FOXA1, and GATA3 colocalized in a coordinated manner where ∼30% of all ERα binding sites were overlapped with FOXA1 and GATA3 bindings upon estrogen (E2) stimulation. Moreover, we found that the ERα+FOXA1+GATA3 conjoint sites were associated with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Such results indicate that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. And such enhanceosome binding sites appear to regulate the genes driving core ERα function.
To further validate that ERα+FOXA1+GATA3 co-binding represents an optimal configuration for E2-mediated transcriptional activation, we have performed luciferase reporter assays on GREB1 locus that actively engages ERα enhanceosome sites in gene regulation (Figure 5C). The presence of ERα induced the GREB1 luciferase activity to ∼246% (as compared with the control construct). The individual presence of FOXA1 and GATA3 or combination of both only produced subtle changes to the GREB1 luciferase activity. The combination of ERα+FOXA1 and ERα+GATA3 has increased the luciferase activity to ∼330%. Interestingly, the assemblage of ERα+FOXA1+GATA3 provided the optimal ER responsiveness to 370%. This suggests that ERα provides the fundamental gene regulatory module but that FOXA1 and GATA3 incrementally improve ERα-regulated transcriptional induction.
It is known that ERα is a ligand-activated TF that mediates the proliferative effects of E2 in breast cancer cells. Garcia et al (1992) showed inhibited growth in MDA-MB-231 cells with forced expression of ERα upon E2 treatment. The rationale for these different outcomes has remained elusive. We posited that these higher order regulatory mechanisms of ERα function such as the formation and composition of enhanceosomes may explain the establishment of transcriptional regulatory cassettes favoring either growth enhancement or growth repression.
To test this hypothesis, we stably transfected the MDA-MB-231 cells with individual ERα, FOXA1, GATA3, or in combinations (Figure 6A). We observed inhibited growth in cells with enforced expression of ERα or FOXA1. There was unaltered growth in cells with expression of GATA3. Co-expression of ERα+FOXA1 or ERα+GATA3 exhibited inhibition of cell proliferation as compared with control cells. However, the co-expression of ERα together with FOXA1 and GATA3 resulted in marked induction of cell proliferation under E2 stimulation. We have recapitulated this cellular reprogramming in another ERα-negative breast cancer cell line, BT-549 and observed similar E2-responsive growth induction in the ERα+FOXA1+GATA3-expressing BT-549 cells. This suggests that only with the full activation of conjoint binding sites by the three TFs will the proliferative phenotype associated with ligand induced ERα be manifest.
To assess the nature of this transcriptional reprogramming, we asked the question if the reprogrammed MDA-MB-231 cells display any similarity in the expression profile of the ERα-positive breast cancer cell line, MCF-7 (Figure 6C). We combined the E2-regulated genes from these differently transfected MDA-MB-231 cells, and compared their expressions in these MDA-MB-231-transfected cells and MCF-7 cells. Strikingly, we found that the expression profiles of ERα+FOXA1+GATA3-expressing MDA-MB-231 cells display a good correlation (R=0.42) with the E2-induced expression profile of MCF-7. We did not observe such correlation between the expression profiles of MDA-MB-231 transfected with ERα only (R=−0.21). Furthermore, we observed that there is marginal induced expression of luminal marker genes and reduced expression of basal genes in the ERα+FOXA1+GATA3-expressing MDA-MB-231 as compared with the vector control cells. This suggests that the enhanceosome component is competent to partially reprogramme the basal cells to resemble the luminal cells.
Taken together, we have uncovered the genomics impact as well as the functional importance of an enhanceosome comprising ERα, FOXA1, and GATA3 in the estrogen responsiveness of ERα-positive breast cancer cells. This enhanceosome exerts significant combinatorial control of the transcriptional network regulating growth and proliferation of ERα-positive breast cancer cells. Most importantly, we show that the transfection of the enhanceosome component was necessary to reprogramme the ERα-negative cells to restore the estrogen-responsive growth and to transcriptionally induce a basal to luminal transition.
Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.
PMCID: PMC3202798  PMID: 21878914
enhanceosome; estrogen receptor α; FOXA1; GATA3; synthetic phenotypes
19.  Personalized Cancer Medicine 2011: Toward Individualized Cancer Treatments. XV International Fritz Bender Symposium Held on February 21 to 23, 2011 at Matrix, Biopolis, Singapore 
Translational Oncology  2011;4(4):199-202.
At the symposium, approaches to individualized cancer medicine were considered, from basic sciences (genetics, epigenetics, biological tumor signatures) to clinical investigations, including strategies about how best to undertake the clinical development of targeted agents.
PMCID: PMC3140006  PMID: 21804914
20.  Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer 
EMBO Molecular Medicine  2011;3(8):451-464.
Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER− cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.
PMCID: PMC3377086  PMID: 21656687
breast cancer; cancer therapy; distant metastasis-free survival; expression microarrays; oncogenes
21.  PanSNPdb: The Pan-Asian SNP Genotyping Database 
PLoS ONE  2011;6(6):e21451.
The HUGO Pan-Asian SNP consortium conducted the largest survey to date of human genetic diversity among Asians by sampling 1,719 unrelated individuals among 71 populations from China, India, Indonesia, Japan, Malaysia, the Philippines, Singapore, South Korea, Taiwan, and Thailand. We have constructed a database (PanSNPdb), which contains these data and various new analyses of them. PanSNPdb is a research resource in the analysis of the population structure of Asian peoples, including linkage disequilibrium patterns, haplotype distributions, and copy number variations. Furthermore, PanSNPdb provides an interactive comparison with other SNP and CNV databases, including HapMap3, JSNP, dbSNP and DGV and thus provides a comprehensive resource of human genetic diversity. The information is accessible via a widely accepted graphical interface used in many genetic variation databases. Unrestricted access to PanSNPdb and any associated files is available at:
PMCID: PMC3121791  PMID: 21731755
22.  Molecular conservation of estrogen-response associated with cell cycle regulation, hormonal carcinogenesis and cancer in zebrafish and human cancer cell lines 
BMC Medical Genomics  2011;4:41.
The zebrafish is recognized as a versatile cancer and drug screening model. However, it is not known whether the estrogen-responsive genes and signaling pathways that are involved in estrogen-dependent carcinogenesis and human cancer are operating in zebrafish. In order to determine the potential of zebrafish model for estrogen-related cancer research, we investigated the molecular conservation of estrogen responses operating in both zebrafish and human cancer cell lines.
Microarray experiment was performed on zebrafish exposed to estrogen (17β-estradiol; a classified carcinogen) and an anti-estrogen (ICI 182,780). Zebrafish estrogen-responsive genes sensitive to both estrogen and anti-estrogen were identified and validated using real-time PCR. Human homolog mapping and knowledge-based data mining were performed on zebrafish estrogen responsive genes followed by estrogen receptor binding site analysis and comparative transcriptome analysis with estrogen-responsive human cancer cell lines (MCF7, T47D and Ishikawa).
Our transcriptome analysis captured multiple estrogen-responsive genes and signaling pathways that increased cell proliferation, promoted DNA damage and genome instability, and decreased tumor suppressing effects, suggesting a common mechanism for estrogen-induced carcinogenesis. Comparative analysis revealed a core set of conserved estrogen-responsive genes that demonstrate enrichment of estrogen receptor binding sites and cell cycle signaling pathways. Knowledge-based and network analysis led us to propose that the mechanism involving estrogen-activated estrogen receptor mediated down-regulation of human homolog HES1 followed by up-regulation cell cycle-related genes (human homologs E2F4, CDK2, CCNA, CCNB, CCNE), is highly conserved, and this mechanism may involve novel crosstalk with basal AHR. We also identified mitotic roles of polo-like kinase as a conserved signaling pathway with multiple entry points for estrogen regulation.
The findings demonstrate the use of zebrafish for characterizing estrogen-like environmental carcinogens and anti-estrogen drug screening. From an evolutionary perspective, our findings suggest that estrogen regulation of cell cycle is perhaps one of the earliest forms of steroidal-receptor controlled cellular processes. Our study provides first evidence of molecular conservation of estrogen-responsiveness between zebrafish and human cancer cell lines, hence demonstrating the potential of zebrafish for estrogen-related cancer research.
PMCID: PMC3114699  PMID: 21575170
zebrafish; microarray; estrogen; anti-estrogen ICI 182,780; estrogen-responsive genes; signaling pathways; carcinogenesis; human cancer cell lines; molecular conservation; model organism
23.  GWAS of Follicular Lymphoma Reveals Allelic Heterogeneity at 6p21.32 and Suggests Shared Genetic Susceptibility with Diffuse Large B-cell Lymphoma 
PLoS Genetics  2011;7(4):e1001378.
Non-Hodgkin lymphoma (NHL) represents a diverse group of hematological malignancies, of which follicular lymphoma (FL) is a prevalent subtype. A previous genome-wide association study has established a marker, rs10484561 in the human leukocyte antigen (HLA) class II region on 6p21.32 associated with increased FL risk. Here, in a three-stage genome-wide association study, starting with a genome-wide scan of 379 FL cases and 791 controls followed by validation in 1,049 cases and 5,790 controls, we identified a second independent FL–associated locus on 6p21.32, rs2647012 (ORcombined = 0.64, Pcombined = 2×10−21) located 962 bp away from rs10484561 (r2<0.1 in controls). After mutual adjustment, the associations at the two SNPs remained genome-wide significant (rs2647012:ORadjusted = 0.70, Padjusted = 4×10−12; rs10484561:ORadjusted = 1.64, Padjusted = 5×10−15). Haplotype and coalescence analyses indicated that rs2647012 arose on an evolutionarily distinct haplotype from that of rs10484561 and tags a novel allele with an opposite (protective) effect on FL risk. Moreover, in a follow-up analysis of the top 6 FL–associated SNPs in 4,449 cases of other NHL subtypes, rs10484561 was associated with risk of diffuse large B-cell lymphoma (ORcombined = 1.36, Pcombined = 1.4×10−7). Our results reveal the presence of allelic heterogeneity within the HLA class II region influencing FL susceptibility and indicate a possible shared genetic etiology with diffuse large B-cell lymphoma. These findings suggest that the HLA class II region plays a complex yet important role in NHL.
Author Summary
Earlier studies have established a marker rs10484561, in the HLA class II region on 6p21.32, associated with increased follicular lymphoma (FL) risk. Here, in a three-stage genome-wide association study of 1,428 FL cases and 6,581 controls, we identified a second independent FL–associated marker on 6p21.32, rs2647012, located 962 bp away from rs10484561. The associations at two SNPs remained genome-wide significant after mutual adjustment. Haplotype and coalescence analyses indicated that rs2647012 arose on an evolutionarily distinct lineage from that of rs10484561 and tags a novel allele with an opposite, protective effect on FL risk. Moreover, in an analysis of the top 6 FL–associated SNPs in 4,449 cases of other NHL subtypes, rs10484561 was associated with risk of diffuse large B-cell lymphoma. Our results reveal the presence of allelic heterogeneity at 6p21.32 in FL risk and suggest a shared genetic etiology with the common diffuse large B-cell lymphoma subtype.
PMCID: PMC3080853  PMID: 21533074
24.  Genetic variation of ESR1 and its co-activator PPARGC1B is synergistic in augmenting the risk of estrogen receptor-positive breast cancer 
Given the role of estrogen in breast carcinogenesis and the modification of estrogen receptor (ER) activity by its biochemical cofactors, we hypothesize that genetic variation within ER cofactor genes alters cellular response to estrogen exposure and consequently modifies the risk for ER-positive breast cancer.
We genotyped 790 tagging SNPs within 60 ER cofactor genes in 1,257 cases and 1,464 controls from Sweden and in 2,215 cases and 1,265 controls from Finland, and tested their associations with either ER-positive or ER-negative breast cancer.
Seven SNPs showed consistent association with ER-positive breast cancer in the two independent samples, and six of them were located within PPARGC1B, encoding an ER co-activator, with the strongest association at rs741581 (odds ratio = 1.41, P = 4.84 × 10-5) that survived Bonferroni correction for multiple testing in the combined ER-positive breast cancer sample (Pcorrected = 0.03). Moreover, we also observed significant synergistic interaction (Pinteraction = 0.008) between the genetic polymorphisms within PPARGC1B and ESR1 in ER-positive breast cancer. By contrast, no consistent association was observed in ER-negative breast cancer. Furthermore, we found that administration of estrogen in the MCF-7 cell line induced PPARGC1B expression and enhanced occupancies of ER and RNA polymerase II within the region of SNP association, suggesting the upregulation of PPARGC1B expression by ESR1 activation.
Our study revealed that DNA polymorphisms of PPARGC1B, coding a bona fide ER co-activator, are associated with ER-positive breast cancer risk. The feed-forward transcriptional regulatory loop between PPARGC1B and ESR1 further augments their protein interaction, which provides a plausible mechanistic explanation for the synergistic genetic interaction between PPARGC1B and ESR1 in ER-positive breast cancer. Our study also highlights that biochemically and genomically informed candidate gene studies can enhance the discovery of interactive disease susceptibility genes.
PMCID: PMC3109578  PMID: 21269472
25.  Integrative model of genomic factors for determining binding site selection by estrogen receptor-α 
Besides the estrogen receptor response elements (EREs), estrogen receptor-α binding is augmented by FOXA1 co-binding, the presence of the histone mark, histone 3 monomethylated at the lysine 4 position and the presence of open chromatin.The major determinant of ER binding is the strength of the ERE.The differences in estrogen receptor-binding profiles between breast cancer cell lines appear to be at sites with less ‘attractive' EREs but modulated by the non-sequence factors.
A major question in transcription factor (TF) biology is why a TF binds to only a small fraction of motif eligible binding sites in the genome. Estrogen receptor-α (ERα) binds only to 2% of optimal sequence eligible sites. Using ERα as a model ligand-inducible TF, we sought to explicitly define parameters that determine TF-binding site selection on a genomic scale in an inducible system that minimizes confounding chromatin effects by the TF itself. By examining 12 separate genetic and epigenetic parameters, we find that an energetically favorable estrogen response element (ERE) motif sequence, evidence of occupancy of a ‘pioneering' TF FOXA1, the presence of the enhancer mark, H3K4me1 and an open chromatin configuration at the pre-ligand state provide specificity for ER binding. These factors can model estrogen-induced ER binding with high accuracy (ROC-AUC=0.95 using random genomic regions as background). Moreover, when ERα and FOXA1 binding as well as H3K4me1 and FAIRE localization (measure of open chromatin) was assessed in another estrogen-responsive cell line, T47D, this model was shown to be highly predictive for ER binding (ROC-AUC=0.86). Variance in binding site selection between MCF-7 and T47D appeared to reside in sites with suboptimal ERE-binding motifs but modulated by the chromatin configuration. Taken together, these results suggest definable interplay between sequence motifs and local chromatin characteristics in determining TF-binding site selection.
A major question in transcription factor (TF) biology is why a TF binds to only a small fraction of motif eligible binding sites in the genome. Using the estrogen receptor-α as a model system, we sought to explicitly define parameters that determine TF-binding site selection. By examining 12 genetic and epigenetic parameters, we find that an energetically favorable estrogen response element (ERE) motif sequence, co-occupancy by the TF FOXA1, the presence of the H3K4me1 mark and an open chromatin configuration in the pre-ligand state provide specificity for ER binding. These factors can model estrogen-induced ER binding with high accuracy (ROC-AUC=0.95 and 0.88 using different genomic backgrounds). Moreover, when assessed in another estrogen-responsive cell line, this model was highly predictive for ERα binding (ROC-AUC=0.86). Variance in binding site selection between MCF-7 and T47D resides in sites with suboptimal ERE motifs, but modulated by the chromatin configuration. These results suggest a definable interplay between sequence motifs and local chromatin in selecting TF binding.
PMCID: PMC3018168  PMID: 21179027
chromatin; DNA binding; modeling; recognition motifs; transcription factor

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