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1.  Genome-wide in silico prediction of gene expression 
Bioinformatics  2012;28(21):2789-2796.
Motivation: Modelling the regulation of gene expression can provide insight into the regulatory roles of individual transcription factors (TFs) and histone modifications. Recently, Ouyang et al. in 2009 modelled gene expression levels in mouse embryonic stem (mES) cells using in vivo ChIP-seq measurements of TF binding. ChIP-seq TF binding data, however, are tissue-specific and relatively difficult to obtain. This limits the applicability of gene expression models that rely on ChIP-seq TF binding data.
Results: In this study, we build regression-based models that relate gene expression to the binding of 12 different TFs, 7 histone modifications and chromatin accessibility (DNase I hypersensitivity) in two different tissues. We find that expression models based on computationally predicted TF binding can achieve similar accuracy to those using in vivo TF binding data and that including binding at weak sites is critical for accurate prediction of gene expression. We also find that incorporating histone modification and chromatin accessibility data results in additional accuracy. Surprisingly, we find that models that use no TF binding data at all, but only histone modification and chromatin accessibility data, can be as (or more) accurate than those based on in vivo TF binding data.
Availability and implementation: All scripts, motifs and data presented in this article are available online at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3476338  PMID: 22954627
2.  Tree-Based Position Weight Matrix Approach to Model Transcription Factor Binding Site Profiles 
PLoS ONE  2011;6(9):e24210.
Most of the position weight matrix (PWM) based bioinformatics methods developed to predict transcription factor binding sites (TFBS) assume each nucleotide in the sequence motif contributes independently to the interaction between protein and DNA sequence, usually producing high false positive predictions. The increasing availability of TF enrichment profiles from recent ChIP-Seq methodology facilitates the investigation of dependent structure and accurate prediction of TFBSs. We develop a novel Tree-based PWM (TPWM) approach to accurately model the interaction between TF and its binding site. The whole tree-structured PWM could be considered as a mixture of different conditional-PWMs. We propose a discriminative approach, called TPD (TPWM based Discriminative Approach), to construct the TPWM from the ChIP-Seq data with a pre-existing PWM. To achieve the maximum discriminative power between the positive and negative datasets, the cutoff value is determined based on the Matthew Correlation Coefficient (MCC). The resulting TPWMs are evaluated with respect to accuracy on extensive synthetic datasets. We then apply our TPWM discriminative approach on several real ChIP-Seq datasets to refine the current TFBS models stored in the TRANSFAC database. Experiments on both the simulated and real ChIP-Seq data show that the proposed method starting from existing PWM has consistently better performance than existing tools in detecting the TFBSs. The improved accuracy is the result of modelling the complete dependent structure of the motifs and better prediction of true positive rate. The findings could lead to better understanding of the mechanisms of TF-DNA interactions.
PMCID: PMC3166302  PMID: 21912677
3.  Integrative genome-wide chromatin signature analysis using finite mixture models 
BMC Genomics  2012;13(Suppl 6):S3.
Regulation of gene expression has been shown to involve not only the binding of transcription factor at target gene promoters but also the characterization of histone around which DNA is wrapped around. Some histone modification, for example di-methylated histone H3 at lysine 4 (H3K4me2), has been shown to bind to promoters and activate target genes. However, no clear pattern has been shown to predict human promoters. This paper proposed a novel quantitative approach to characterize patterns of promoter regions and predict novel and alternative promoters. We utilized high-throughput data generated using chromatin immunoprecipitation methods followed by massively parallel sequencing (ChIP-seq) technology on RNA Polymerase II (Pol-II) and H3K4me2. Common patterns of promoter regions are modeled using a mixture model involving double-exponential and uniform distributions. The fitted model obtained were then used to search for regions displaying similar patterns over the entire genome to find novel and alternative promoters. Regions with high correlations with the common patterns are identified as putative novel promoters. We used this proposed algorithm, RNA-seq data and several transcripts databases to find alternative promoters in MCF7 (normal breast cancer) cell line. We found 7,235 high-confidence regions that display the identified promoter patterns. Of these, 4,167 regions (58%) can be mapped to RefSeq regions. 2,444 regions are in a gene body or overlap with transcripts (non-coding RNAs, ESTs, and transcripts that are predicted by RNA-seq data). Some of these maybe potential alternative promoters. We also found 193 regions that map to enhancer regions (represented by androgen and estrogen receptor binding sites) and other regulatory regions such as CTCF (CCCTC binding factor) and CpG island. Around 5% (431 regions) of these correlated regions do not overlap with any transcripts or regulatory regions suggesting that these might be potential new promoters or markers for other annotation which are currently undiscovered.
PMCID: PMC3481451  PMID: 23134707
4.  Uncovering transcriptional interactions via an adaptive fuzzy logic approach 
BMC Bioinformatics  2009;10:400.
To date, only a limited number of transcriptional regulatory interactions have been uncovered. In a pilot study integrating sequence data with microarray data, a position weight matrix (PWM) performed poorly in inferring transcriptional interactions (TIs), which represent physical interactions between transcription factors (TF) and upstream sequences of target genes. Inferring a TI means that the promoter sequence of a target is inferred to match the consensus sequence motifs of a potential TF, and their interaction type such as AT or RT is also predicted. Thus, a robust PWM (rPWM) was developed to search for consensus sequence motifs. In addition to rPWM, one feature extracted from ChIP-chip data was incorporated to identify potential TIs under specific conditions. An interaction type classifier was assembled to predict activation/repression of potential TIs using microarray data. This approach, combining an adaptive (learning) fuzzy inference system and an interaction type classifier to predict transcriptional regulatory networks, was named AdaFuzzy.
AdaFuzzy was applied to predict TIs using real genomics data from Saccharomyces cerevisiae. Following one of the latest advances in predicting TIs, constrained probabilistic sparse matrix factorization (cPSMF), and using 19 transcription factors (TFs), we compared AdaFuzzy to four well-known approaches using over-representation analysis and gene set enrichment analysis. AdaFuzzy outperformed these four algorithms. Furthermore, AdaFuzzy was shown to perform comparably to 'ChIP-experimental method' in inferring TIs identified by two sets of large scale ChIP-chip data, respectively. AdaFuzzy was also able to classify all predicted TIs into one or more of the four promoter architectures. The results coincided with known promoter architectures in yeast and provided insights into transcriptional regulatory mechanisms.
AdaFuzzy successfully integrates multiple types of data (sequence, ChIP, and microarray) to predict transcriptional regulatory networks. The validated success in the prediction results implies that AdaFuzzy can be applied to uncover TIs in yeast.
PMCID: PMC2797023  PMID: 19961622
5.  Identifying Positioned Nucleosomes with Epigenetic Marks in Human from ChIP-Seq 
BMC Genomics  2008;9:537.
In vivo positioning and covalent modifications of nucleosomes play an important role in epigenetic regulation, but genome-wide studies of positioned nucleosomes and their modifications in human still remain limited.
This paper describes a novel computational framework to efficiently identify positioned nucleosomes and their histone modification profiles from nucleosome-resolution histone modification ChIP-Seq data. We applied the algorithm to histone methylation ChIP-Seq data in human CD4+ T cells and identified over 438,000 positioned nucleosomes, which appear predominantly at functionally important regions such as genes, promoters, DNase I hypersensitive regions, and transcription factor binding sites. Our analysis shows the identified nucleosomes play a key role in epigenetic gene regulation within those functionally important regions via their positioning and histone modifications.
Our method provides an effective framework for studying nucleosome positioning and epigenetic marks in mammalian genomes. The algorithm is open source and available at .
PMCID: PMC2596141  PMID: 19014516
6.  MYBS: a comprehensive web server for mining transcription factor binding sites in yeast 
Nucleic Acids Research  2007;35(Web Server issue):W221-W226.
Correct interactions between transcription factors (TFs) and their binding sites (TFBSs) are of central importance to gene regulation. Recently developed chromatin-immunoprecipitation DNA chip (ChIP-chip) techniques and the phylogenetic footprinting method provide ways to identify TFBSs with high precision. In this study, we constructed a user-friendly interactive platform for dynamic binding site mapping using ChIP-chip data and phylogenetic footprinting as two filters. MYBS (Mining Yeast Binding Sites) is a comprehensive web server that integrates an array of both experimentally verified and predicted position weight matrixes (PWMs) from eleven databases, including 481 binding motif consensus sequences and 71 PWMs that correspond to 183 TFs. MYBS users can search within this platform for motif occurrences (possible binding sites) in the promoters of genes of interest via simple motif or gene queries in conjunction with the above two filters. In addition, MYBS enables users to visualize in parallel the potential regulators for a given set of genes, a feature useful for finding potential regulatory associations between TFs. MYBS also allows users to identify target gene sets of each TF pair, which could be used as a starting point for further explorations of TF combinatorial regulation. MYBS is available at
PMCID: PMC1933147  PMID: 17537814
7.  HHMD: the human histone modification database 
Nucleic Acids Research  2009;38(Database issue):D149-D154.
Histone modifications play important roles in chromatin remodeling, gene transcriptional regulation, stem cell maintenance and differentiation. Alterations in histone modifications may be linked to human diseases especially cancer. Histone modifications including methylation, acetylation and ubiquitylation probed by ChIP-seq, ChIP-chip and qChIP have become widely available. Mining and integration of histone modification data can be beneficial to novel biological discoveries. There has been no comprehensive data repository that is exclusive for human histone modifications. Therefore, we developed a relatively comprehensive database for human histone modifications. Human Histone Modification Database (HHMD, focuses on the storage and integration of histone modification datasets that were obtained from laboratory experiments. The latest release of HHMD incorporates 43 location-specific histone modifications in human. To facilitate data extraction, flexible search options are built in HHMD. It can be searched by histone modification, gene ID, functional categories, chromosome location and cancer name. HHMD also includes a user-friendly visualization tool named HisModView, by which genome-wide histone modification map can be shown. HisModView facilitates the acquisition and visualization of histone modifications. The database also has manually curated information of histone modification dysregulation in nine human cancers.
PMCID: PMC2808954  PMID: 19892823
8.  Identifying dispersed epigenomic domains from ChIP-Seq data 
Bioinformatics  2011;27(6):870-871.
Motivation: Post-translational modifications to histones have several well known associations with regulation of gene expression. While some modifications appear concentrated narrowly, covering promoters or enhancers, others are dispersed as epigenomic domains. These domains mark contiguous regions sharing an epigenomic property, such as actively transcribed or poised genes, or heterochromatically silenced regions. While high-throughput methods like ChIP-Seq have led to a flood of high-quality data about these epigenomic domains, there remain important analysis problems that are not adequately solved by current analysis tools.
Results: We present the RSEG method for identifying epigenomic domains from ChIP-Seq data for histone modifications. In contrast with other methods emphasizing the locations of ‘peaks’ in read density profiles, our method identifies the boundaries of domains. RSEG is also able to incorporate a control sample and find genomic regions with differential histone modifications between two samples.
Availability: RSEG, including source code and documentation, is freely available at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3051331  PMID: 21325299
9.  A clustering approach for identification of enriched domains from histone modification ChIP-Seq data 
Bioinformatics  2009;25(15):1952-1958.
Motivation: Chromatin states are the key to gene regulation and cell identity. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) is increasingly being used to map epigenetic states across genomes of diverse species. Chromatin modification profiles are frequently noisy and diffuse, spanning regions ranging from several nucleosomes to large domains of multiple genes. Much of the early work on the identification of ChIP-enriched regions for ChIP-Seq data has focused on identifying localized regions, such as transcription factor binding sites. Bioinformatic tools to identify diffuse domains of ChIP-enriched regions have been lacking.
Results: Based on the biological observation that histone modifications tend to cluster to form domains, we present a method that identifies spatial clusters of signals unlikely to appear by chance. This method pools together enrichment information from neighboring nucleosomes to increase sensitivity and specificity. By using genomic-scale analysis, as well as the examination of loci with validated epigenetic states, we demonstrate that this method outperforms existing methods in the identification of ChIP-enriched signals for histone modification profiles. We demonstrate the application of this unbiased method in important issues in ChIP-Seq data analysis, such as data normalization for quantitative comparison of levels of epigenetic modifications across cell types and growth conditions.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2732366  PMID: 19505939
10.  Epigenetic regulation of human cis-natural antisense transcripts 
Nucleic Acids Research  2012;40(4):1438-1445.
Mammalian genomes encode numerous cis-natural antisense transcripts (cis-NATs). The extent to which these cis-NATs are actively regulated and ultimately functionally relevant, as opposed to transcriptional noise, remains a matter of debate. To address this issue, we analyzed the chromatin environment and RNA Pol II binding properties of human cis-NAT promoters genome-wide. Cap analysis of gene expression data were used to identify thousands of cis-NAT promoters, and profiles of nine histone modifications and RNA Pol II binding for these promoters in ENCODE cell types were analyzed using chromatin immunoprecipitation followed by sequencing (ChIP-seq) data. Active cis-NAT promoters are enriched with activating histone modifications and occupied by RNA Pol II, whereas weak cis-NAT promoters are depleted for both activating modifications and RNA Pol II. The enrichment levels of activating histone modifications and RNA Pol II binding show peaks centered around cis-NAT transcriptional start sites, and the levels of activating histone modifications at cis-NAT promoters are positively correlated with cis-NAT expression levels. Cis-NAT promoters also show highly tissue-specific patterns of expression. These results suggest that human cis-NATs are actively transcribed by the RNA Pol II and that their expression is epigenetically regulated, prerequisites for a functional potential for many of these non-coding RNAs.
PMCID: PMC3287164  PMID: 22371288
11.  Genome-wide histone acetylation data improve prediction of mammalian transcription factor binding sites 
Bioinformatics  2010;26(17):2071-2075.
Motivation: Histone acetylation (HAc) is associated with open chromatin, and HAc has been shown to facilitate transcription factor (TF) binding in mammalian cells. In the innate immune system context, epigenetic studies strongly implicate HAc in the transcriptional response of activated macrophages. We hypothesized that using data from large-scale sequencing of a HAc chromatin immunoprecipitation assay (ChIP-Seq) would improve the performance of computational prediction of binding locations of TFs mediating the response to a signaling event, namely, macrophage activation.
Results: We tested this hypothesis using a multi-evidence approach for predicting binding sites. As a training/test dataset, we used ChIP-Seq-derived TF binding site locations for five TFs in activated murine macrophages. Our model combined TF binding site motif scanning with evidence from sequence-based sources and from HAc ChIP-Seq data, using a weighted sum of thresholded scores. We find that using HAc data significantly improves the performance of motif-based TF binding site prediction. Furthermore, we find that within regions of high HAc, local minima of the HAc ChIP-Seq signal are particularly strongly correlated with TF binding locations. Our model, using motif scanning and HAc local minima, improves the sensitivity for TF binding site prediction by ∼50% over a model based on motif scanning alone, at a false positive rate cutoff of 0.01.
Availability: The data and software source code for model training and validation are freely available online at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2922897  PMID: 20663846
12.  Understanding Variation in Transcription Factor Binding by Modeling Transcription Factor Genome-Epigenome Interactions 
PLoS Computational Biology  2013;9(12):e1003367.
Despite explosive growth in genomic datasets, the methods for studying epigenomic mechanisms of gene regulation remain primitive. Here we present a model-based approach to systematically analyze the epigenomic functions in modulating transcription factor-DNA binding. Based on the first principles of statistical mechanics, this model considers the interactions between epigenomic modifications and a cis-regulatory module, which contains multiple binding sites arranged in any configurations. We compiled a comprehensive epigenomic dataset in mouse embryonic stem (mES) cells, including DNA methylation (MeDIP-seq and MRE-seq), DNA hydroxymethylation (5-hmC-seq), and histone modifications (ChIP-seq). We discovered correlations of transcription factors (TFs) for specific combinations of epigenomic modifications, which we term epigenomic motifs. Epigenomic motifs explained why some TFs appeared to have different DNA binding motifs derived from in vivo (ChIP-seq) and in vitro experiments. Theoretical analyses suggested that the epigenome can modulate transcriptional noise and boost the cooperativity of weak TF binding sites. ChIP-seq data suggested that epigenomic boost of binding affinities in weak TF binding sites can function in mES cells. We showed in theory that the epigenome should suppress the TF binding differences on SNP-containing binding sites in two people. Using personal data, we identified strong associations between H3K4me2/H3K9ac and the degree of personal differences in NFκB binding in SNP-containing binding sites, which may explain why some SNPs introduce much smaller personal variations on TF binding than other SNPs. In summary, this model presents a powerful approach to analyze the functions of epigenomic modifications. This model was implemented into an open source program APEG (Affinity Prediction by Epigenome and Genome,
Author Summary
We developed a model-based approach to systematically analyze the epigenomic functions in modulating transcription factor-DNA binding. We postulated the existence of TF-specific epigenomic motifs, which could explain why some TFs appeared to have different DNA binding motifs derived from in vivo and in vitro experiments. The theoretical results suggested that the epigenome can modulate transcriptional noise and boost the cooperativity of weak TF binding sites. A preliminary analysis of the existing data suggested that epigenomic boost of binding affinities in weak TF binding sites could be a widespread regulatory mechanism in mES cells. Moreover, using personal data, we identified strong associations between H3K4me2/H3K9ac and the degree of individual differences in NFκB binding in SNP-containing binding sites, suggesting the theoretical mechanism for epigenome to attenuate the TF binding differences on SNP-containing binding sites in two individuals may contribute to link genomic variation to phenotypic variation. Thus, this model presents a powerful approach to analyze the functions of epigenomic modifications.
PMCID: PMC3854512  PMID: 24339764
13.  Optimizing the GATA-3 position weight matrix to improve the identification of novel binding sites 
BMC Genomics  2012;13:416.
The identifying of binding sites for transcription factors is a key component of gene regulatory network analysis. This is often done using position-weight matrices (PWMs). Because of the importance of in silico mapping of tentative binding sites, we previously developed an approach for PWM optimization that substantially improves the accuracy of such mapping.
The present work implements the optimization algorithm applied to the existing PWM for GATA-3 transcription factor and builds a new di-nucleotide PWM. The existing available PWM is based on experimental data adopted from Jaspar. The optimized PWM substantially improves the sensitivity and specificity of the TF mapping compared to the conventional applications. The refined PWM also facilitates in silico identification of novel binding sites that are supported by experimental data. We also describe uncommon positioning of binding motifs for several T-cell lineage specific factors in human promoters.
Our proposed di-nucleotide PWM approach outperforms the conventional mono-nucleotide PWM approach with respect to GATA-3. Therefore our new di-nucleotide PWM provides new insight into plausible transcriptional regulatory interactions in human promoters.
PMCID: PMC3481455  PMID: 22913572
Transcription factor; Binding sites; GATA-3; Human promoter; Position weight matrix; Optimization
14.  A regression analysis of gene expression in ES cells reveals two gene classes that are significantly different in epigenetic patterns 
BMC Bioinformatics  2011;12(Suppl 1):S50.
To understand the gene regulatory system that governs the self-renewal and pluripotency of embryonic stem cells (ESCs) is an important step for promoting regenerative medicine. In it, the role of several core transcription factors (TFs), such as Oct4, Sox2 and Nanog, has been intensively investigated, details of their involvement in the genome-wide gene regulation are still not well clarified.
We constructed a predictive model of genome-wide gene expression in mouse ESCs from publicly available ChIP-seq data of 12 core TFs. The tag sequences were remapped on the genome by various alignment tools. Then, the binding density of each TF is calculated from the genome-wide bona fide TF binding sites. The TF-binding data was combined with the data of several epigenetic states (DNA methylation, several histone modifications, and CpG island) of promoter regions. These data as well as the ordinary peak intensity data were used as predictors of a simple linear regression model that predicts absolute gene expression. We also developed a pipeline for analyzing the effects of predictors and their interactions.
Through our analysis, we identified two classes of genes that are either well explained or inefficiently explained by our model. The latter class seems to be genes that are not directly regulated by the core TFs. The regulatory regions of these gene classes show apparently distinct patterns of DNA methylation, histone modifications, existence of CpG islands, and gene ontology terms, suggesting the relative importance of epigenetic effects. Furthermore, we identified statistically significant TF interactions correlated with the epigenetic modification patterns.
Here, we proposed an improved prediction method in explaining the ESC-specific gene expression. Our study implies that the majority of genes are more or less directly regulated by the core TFs. In addition, our result is consistent with the general idea of relative importance of epigenetic effects in ESCs.
PMCID: PMC3044308  PMID: 21342583
15.  Global Mapping of Transcription Factor Binding Sites by Sequencing Chromatin Surrogates: a Perspective on Experimental Design, Data Analysis, and Open Problems 
Statistics in Biosciences  2012;5(1):156-178.
Mapping genome-wide binding sites of all transcription factors (TFs) in all biological contexts is a critical step toward understanding gene regulation. The state-of-the-art technologies for mapping transcription factor binding sites (TFBSs) couple chromatin immunoprecipitation (ChIP) with high-throughput sequencing (ChIP-seq) or tiling array hybridization (ChIP-chip). These technologies have limitations: they are low-throughput with respect to surveying many TFs. Recent advances in genome-wide chromatin profiling, including development of technologies such as DNase-seq, FAIRE-seq and ChIP-seq for histone modifications, make it possible to predict in vivo TFBSs by analyzing chromatin features at computationally determined DNA motif sites. This promising new approach may allow researchers to monitor the genome-wide binding sites of many TFs simultaneously. In this article, we discuss various experimental design and data analysis issues that arise when applying this approach. Through a systematic analysis of the data from the Encyclopedia Of DNA Elements (ENCODE) project, we compare the predictive power of individual and combinations of chromatin marks using supervised and unsupervised learning methods, and evaluate the value of integrating information from public ChIP and gene expression data. We also highlight the challenges and opportunities for developing novel analytical methods, such as resolving the one-motif-multiple-TF ambiguity and distinguishing functional and non-functional TF binding targets from the predicted binding sites.
Electronic Supplementary Material
The online version of this article (doi:10.1007/s12561-012-9066-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3677239  PMID: 23762209
Transcription factor binding sites; DNase-seq; ChIP-seq; FAIRE-seq; Next-generation sequencing; Motif
16.  The chromatin architectural proteins HMGD1 and H1 bind reciprocally and have opposite effects on chromatin structure and gene regulation 
BMC Genomics  2014;15:92.
Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins.
Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1 in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. We find that the ratio of HMGD1 to H1 binding is a better predictor of gene activity than either protein by itself, which suggests that reciprocal binding between these proteins is important for gene regulation. Using knockdown experiments, we show that HMGD1 and H1 affect the occupancy of the other protein, change nucleosome repeat length and modulate gene expression.
Collectively, our data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and their linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation.
PMCID: PMC3928079  PMID: 24484546
Chromatin structure; Transcriptional regulation; Histone H1; High mobility group protein; Nucleosome repeat length
17.  Integrative analysis of genomic, functional and protein interaction data predicts long-range enhancer-target gene interactions 
Nucleic Acids Research  2010;39(7):2492-2502.
Multicellular organismal development is controlled by a complex network of transcription factors, promoters and enhancers. Although reliable computational and experimental methods exist for enhancer detection, prediction of their target genes remains a major challenge. On the basis of available literature and ChIP-seq and ChIP-chip data for enhanceosome factor p300 and the transcriptional regulator Gli3, we found that genomic proximity and conserved synteny predict target genes with a relatively low recall of 12–27% within 2 Mb intervals centered at the enhancers. Here, we show that functional similarities between enhancer binding proteins and their transcriptional targets and proximity in the protein–protein interactome improve prediction of target genes. We used all four features to train random forest classifiers that predict target genes with a recall of 58% in 2 Mb intervals that may contain dozens of genes, representing a better than two-fold improvement over the performance of prediction based on single features alone. Genome-wide ChIP data is still relatively poorly understood, and it remains difficult to assign biological significance to binding events. Our study represents a first step in integrating various genomic features in order to elucidate the genomic network of long-range regulatory interactions.
PMCID: PMC3074119  PMID: 21109530
18.  ChIP-Seq: advantages and challenges of a maturing technology 
Nature reviews. Genetics  2009;10(10):669-680.
Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications, or nucleosomes. Enabled by the tremendous progress in next-generation sequencing technology, ChIP-Seq offers higher resolution, less noise, and greater coverage than its array-based predecessor ChIP-chip. With the decreasing cost of sequencing, ChIP-Seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this review, we describe the benefits as well as the challenges in harnessing this technique, with an emphasis on issues related to experimental design and data analysis. ChIP-Seq experiments generate large quantities of data, and effective computational analysis will be critical for uncovering biological mechanisms.
PMCID: PMC3191340  PMID: 19736561
19.  Three Distinct Patterns of Histone H3Y41 Phosphorylation Mark Active Genes 
Cell Reports  2012;2(3):470-477.
The JAK2 tyrosine kinase is a critical mediator of cytokine-induced signaling. It plays a role in the nucleus, where it regulates transcription by phosphorylating histone H3 at tyrosine 41 (H3Y41ph). We used chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq) to define the genome-wide pattern of H3Y41ph in human erythroid leukemia cells. Our results indicate that H3Y41ph is located at three distinct sites: (1) at a subset of active promoters, where it overlaps with H3K4me3, (2) at distal cis-regulatory elements, where it coincides with the binding of STAT5, and (3) throughout the transcribed regions of active, tissue-specific hematopoietic genes. Together, these data extend our understanding of this conserved and essential signaling pathway and provide insight into the mechanisms by which extracellular stimuli may lead to the coordinated regulation of transcription.
Graphical Abstract
► Histone H3Y41 phosphorylation is associated with actively transcribed genes ► H3Y41ph correlates with H3K4me3 at the TSS of a subset of active genes ► H3Y41ph and STAT5 binding are coincident at some JAK2/STAT5 target genes ► H3Y41ph blankets the entire transcribed region of active tissue-specific genes
JAK2 tyrosine kinase, a critical mediator of cytokine-induced signaling, plays a role in the nucleus, where it regulates transcription by phosphorylating histone H3 at tyrosine 41 (H3Y41ph). Using Chip-seq, Göttgens, Kouzarides, and colleagues now show that H3Y41ph marks specific sets of genes stimulated by this signaling pathway and that it blankets lineage-specific hematopoietic genes. Notably, at certain genes and enhancers, H3Y41ph coincides with STAT5 binding. These data provide insight into the mechanisms by which extracellular stimuli may lead to the coordinated regulation of transcription.
PMCID: PMC3607218  PMID: 22999934
20.  Application of ChIP-Seq and related techniques to the study of immune function 
Immunity  2011;34(6):830-842.
Behaviors observed at the cellular level such as development and acquisition of effector functions by immune cells result from transcriptional changes. The biochemical mediators of transcription are sequence specific transcription factors (TFs), chromatin modifying enzymes, and chromatin, the complex of DNA and histone proteins. Covalent modification of DNA and histones, also termed epigenetic modification, influences the accessibility of target sequences for transcription factors on chromatin and the expression of linked genes required for immune functions. Genome-wide techniques such as ChIP-Seq have described the entire “cistrome” of transcription factors involved in specific developmental steps of B and T cells and started to define specific immune responses in terms of the binding profiles of critical effectors and epigenetic modification patterns. Current data suggest that both promoters and enhancers are prepared for action at different stages of activation by epigenetic modification through distinct transcription factors in different cells.
PMCID: PMC3137373  PMID: 21703538
21.  Discovering transcription factor regulatory targets using gene expression and binding data 
Bioinformatics  2011;28(2):206-213.
Motivation: Identifying the target genes regulated by transcription factors (TFs) is the most basic step in understanding gene regulation. Recent advances in high-throughput sequencing technology, together with chromatin immunoprecipitation (ChIP), enable mapping TF binding sites genome wide, but it is not possible to infer function from binding alone. This is especially true in mammalian systems, where regulation often occurs through long-range enhancers in gene-rich neighborhoods, rather than proximal promoters, preventing straightforward assignment of a binding site to a target gene.
Results: We present EMBER (Expectation Maximization of Binding and Expression pRofiles), a method that integrates high-throughput binding data (e.g. ChIP-chip or ChIP-seq) with gene expression data (e.g. DNA microarray) via an unsupervised machine learning algorithm for inferring the gene targets of sets of TF binding sites. Genes selected are those that match overrepresented expression patterns, which can be used to provide information about multiple TF regulatory modes. We apply the method to genome-wide human breast cancer data and demonstrate that EMBER confirms a role for the TFs estrogen receptor alpha, retinoic acid receptors alpha and gamma in breast cancer development, whereas the conventional approach of assigning regulatory targets based on proximity does not. Additionally, we compare several predicted target genes from EMBER to interactions inferred previously, examine combinatorial effects of TFs on gene regulation and illustrate the ability of EMBER to discover multiple modes of regulation.
Availability: All code used for this work is available at
Supplementary Information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3259433  PMID: 22084256
22.  Identifying ChIP-seq enrichment using MACS 
Nature protocols  2012;7(9):10.1038/nprot.2012.101.
Model-based Analysis of ChIP-seq (MACS) is a computational algorithm that identifies genome-wide locations of transcription/chromatin factor binding or histone modification from ChIP-seq data. MACS consists of four steps: removing redundant reads, adjusting read position, calculating peak enrichment, and estimating the empirical false discovery rate. In this protocol, we provide a detailed demonstration of how to install MACS and how to use it to analyze three common types of ChIP-seq datasets with different characteristics: the sequence-specific transcription factor FoxA1, the histone modification mark H3K4me3 with sharp enrichment, and the H3K36me3 mark with broad enrichment. We also explain how to interpret and visualize the results of MACS analyses. The algorithm requires approximately 3 GB of RAM and 1.5 hours of computing time to analyze a ChIP-seq dataset containing 30 million reads, an estimate that increases with sequence coverage. MACS is open-source and is available from
PMCID: PMC3868217  PMID: 22936215
MACS; ChIP-seq; peak calling; transcription factor; histone modification
23.  Clustered ChIP-Seq-defined transcription factor binding sites and histone modifications map distinct classes of regulatory elements 
BMC Biology  2011;9:80.
Transcription factor binding to DNA requires both an appropriate binding element and suitably open chromatin, which together help to define regulatory elements within the genome. Current methods of identifying regulatory elements, such as promoters or enhancers, typically rely on sequence conservation, existing gene annotations or specific marks, such as histone modifications and p300 binding methods, each of which has its own biases.
Herein we show that an approach based on clustering of transcription factor peaks from high-throughput sequencing coupled with chromatin immunoprecipitation (Chip-Seq) can be used to evaluate markers for regulatory elements. We used 67 data sets for 54 unique transcription factors distributed over two cell lines to create regulatory element clusters. By integrating the clusters from our approach with histone modifications and data for open chromatin, we identified general methylation of lysine 4 on histone H3 (H3K4me) as the most specific marker for transcription factor clusters. Clusters mapping to annotated genes showed distinct patterns in cluster composition related to gene expression and histone modifications. Clusters mapping to intergenic regions fall into two groups either directly involved in transcription, including miRNAs and long noncoding RNAs, or facilitating transcription by long-range interactions. The latter clusters were specifically enriched with H3K4me1, but less with acetylation of lysine 27 on histone 3 or p300 binding.
By integrating genomewide data of transcription factor binding and chromatin structure and using our data-driven approach, we pinpointed the chromatin marks that best explain transcription factor association with different regulatory elements. Our results also indicate that a modest selection of transcription factors may be sufficient to map most regulatory elements in the human genome.
PMCID: PMC3239327  PMID: 22115494
transcription factor; ChIP-Seq; histone modification; chromatin
24.  Identification of co-occurring transcription factor binding sites from DNA sequence using clustered position weight matrices 
Nucleic Acids Research  2011;40(5):e38.
Accurate prediction of transcription factor binding sites (TFBSs) is a prerequisite for identifying cis-regulatory modules that underlie transcriptional regulatory circuits encoded in the genome. Here, we present a computational framework for detecting TFBSs, when multiple position weight matrices (PWMs) for a transcription factor are available. Grouping multiple PWMs of a transcription factor (TF) based on their sequence similarity improves the specificity of TFBS prediction, which was evaluated using multiple genome-wide ChIP-Seq data sets from 26 TFs. The Z-scores of the area under a receiver operating characteristic curve (AUC) values of 368 TFs were calculated and used to statistically identify co-occurring regulatory motifs in the TF bound ChIP loci. Motifs that are co-occurring along with the empirical bindings of E2F, JUN or MYC have been evaluated, in the basal or stimulated condition. Results prove our method can be useful to systematically identify the co-occurring motifs of the TF for the given conditions.
PMCID: PMC3300004  PMID: 22187154
25.  Empirical methods for controlling false positives and estimating confidence in ChIP-Seq peaks 
BMC Bioinformatics  2008;9:523.
High throughput signature sequencing holds many promises, one of which is the ready identification of in vivo transcription factor binding sites, histone modifications, changes in chromatin structure and patterns of DNA methylation across entire genomes. In these experiments, chromatin immunoprecipitation is used to enrich for particular DNA sequences of interest and signature sequencing is used to map the regions to the genome (ChIP-Seq). Elucidation of these sites of DNA-protein binding/modification are proving instrumental in reconstructing networks of gene regulation and chromatin remodelling that direct development, response to cellular perturbation, and neoplastic transformation.
Here we present a package of algorithms and software that makes use of control input data to reduce false positives and estimate confidence in ChIP-Seq peaks. Several different methods were compared using two simulated spike-in datasets. Use of control input data and a normalized difference score were found to more than double the recovery of ChIP-Seq peaks at a 5% false discovery rate (FDR). Moreover, both a binomial p-value/q-value and an empirical FDR were found to predict the true FDR within 2–3 fold and are more reliable estimators of confidence than a global Poisson p-value. These methods were then used to reanalyze Johnson et al.'s neuron-restrictive silencer factor (NRSF) ChIP-Seq data without relying on extensive qPCR validated NRSF sites and the presence of NRSF binding motifs for setting thresholds.
The methods developed and tested here show considerable promise for reducing false positives and estimating confidence in ChIP-Seq data without any prior knowledge of the chIP target. They are part of a larger open source package freely available from
PMCID: PMC2628906  PMID: 19061503

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