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1.  ZNF217, a candidate breast cancer oncogene amplified at 20q13, regulates expression of the ErbB3 receptor tyrosine kinase in breast cancer cells 
Oncogene  2010;29(40):5500-5510.
Understanding the mechanisms underlying ErbB3 over-expression in breast cancer will facilitate the rational design of therapies to disrupt ErbB2-ErbB3 oncogenic function. While ErbB3 over-expression is frequently observed in breast cancer, the factors mediating its aberrant expression are poorly understood. In particular, the ErbB3 gene is not significantly amplified, raising the question as to how ErbB3 over-expression is achieved. In this study we demonstrate that the ZNF217 transcription factor, amplified at 20q13 in ~20% of breast tumors, regulates ErbB3 expression. Analysis of a panel of human breast cancer cell lines (n = 50) and primary human breast tumors (n=15) demonstrated a strong positive correlation between ZNF217 and ErbB3 expression. Ectopic expression of ZNF217 in human mammary epithelial cells induced ErbB3 expression while ZNF217 silencing in breast cancer cells resulted in decreased ErbB3 expression. While ZNF217 has previously been linked with transcriptional repression due to its close association with CtBP1/2 repressor complexes, our results demonstrate that ZNF217 also activates gene expression. We demonstrate that ZNF217 recruitment to the ErbB3 promoter is CtBP1/2-independent and that ZNF217 and CtBP1/2 play opposite roles in regulating ErbB3 expression. In addition, we identify ErbB3 as one of the mechanisms by which ZNF217 augments PI-3K/Akt signaling.
PMCID: PMC4256946  PMID: 20661224
ZNF217; ErbB3; CtBP2; 20q13; breast cancer
2.  Global loss of DNA methylation uncovers intronic enhancers in genes showing expression changes 
Genome Biology  2014;15(9):469.
Gene expression is epigenetically regulated by a combination of histone modifications and methylation of CpG dinucleotides in promoters. In normal cells, CpG-rich promoters are typically unmethylated, marked with histone modifications such as H3K4me3, and are highly active. During neoplastic transformation, CpG dinucleotides of CG-rich promoters become aberrantly methylated, corresponding with the removal of active histone modifications and transcriptional silencing. Outside of promoter regions, distal enhancers play a major role in the cell type-specific regulation of gene expression. Enhancers, which function by bringing activating complexes to promoters through chromosomal looping, are also modulated by a combination of DNA methylation and histone modifications.
Here we use HCT116 colorectal cancer cells with and without mutations in DNA methyltransferases, the latter of which results in a 95% reduction in global DNA methylation levels. These cells are used to study the relationship between DNA methylation, histone modifications, and gene expression. We find that the loss of DNA methylation is not sufficient to reactivate most of the silenced promoters. In contrast, the removal of DNA methylation results in the activation of a large number of enhancer regions as determined by the acquisition of active histone marks.
Although the transcriptome is largely unaffected by the loss of DNA methylation, we identify two distinct mechanisms resulting in the upregulation of distinct sets of genes. One is a direct result of DNA methylation loss at a set of promoter regions and the other is due to the presence of new intragenic enhancers.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0469-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4203885  PMID: 25239471
3.  Human ESC Self-Renewal Promoting microRNAs Induce Epithelial-Mesenchymal Transition in Hepatocytes by Controlling the PTEN and TGFβ Tumor Suppressor Signaling Pathways 
Molecular cancer research : MCR  2012;10(7):979-991.
The self-renewal capacity ascribed to embryonic stem cells (ESCs) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in hESCs and liver cancer cells (HCCs), but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11 and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFβ tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues, and induces epithelial-mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently up-regulated in human liver tumors, and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes.
PMCID: PMC4166560  PMID: 22622027
Human embryonic stem cells; microRNAs; hepatocellular carcinoma cells
4.  Using ChIPMotifs for De Novo Motif Discovery of OCT4 and ZNF263 Based on ChIP-Based High-Throughput Experiments 
DNA motifs are short sequences varying from 6 to 25 bp and can be highly variable and degenerated. One major approach for predicting transcription factor (TF) binding is using position weight matrix (PWM) to represent information content of regulatory sites; however, when used as the sole means of identifying binding sites suffers from the limited amount of training data available and a high rate of false-positive predictions. ChIPMotifs program is a de novo motif finding tool developed for ChIP-based high-throughput data, and W-ChIPMotifs is a Web application tool for ChIPMotifs. It composes various ab initio motif discovery tools such as MEME, MaMF, Weeder and optimizes the significance of the detected motifs by using bootstrap re-sampling error estimation and a Fisher test. Using these techniques, we determined a PWM for OCT4 which is similar to canonical OCT4 consensus sequence. In a separate study, we also use de novo motif discovery to suggest that ZNF263 binds to a 24-nt site that differs from the motif predicted by the zinc finger code in several positions.
PMCID: PMC4160035  PMID: 22130890
Motif; ChIP; Position weight matrix; OCT4; ZNF263
5.  Architecture of the human regulatory network derived from ENCODE data 
Nature  2012;489(7414):91-100.
Transcription factors (TFs) bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 TFs in 458 ChIP-Seq experiments. We found the combinatorial, co-association of TFs to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the TF binding into a hierarchy and integrated it with other genomic information (e.g. miRNA regulation), forming a dense meta-network. Factors at different levels have different properties: for instance, top-level TFs more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs -- e.g. noise-buffering feed-forward loops. Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (i.e., differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.
PMCID: PMC4154057  PMID: 22955619
6.  Using ChIP-Seq Technology to Generate High-Resolution Profiles of Histone Modifications 
The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within.
PMCID: PMC4151291  PMID: 21913086
Chromatin immunoprecipitation; ChIP-seq; Next generation sequencing; Epigenomics; Histone modifications; IP-Star; ChIP robot
7.  Transcription Factor Effector Domains 
Sub-cellular biochemistry  2011;52:261-277.
The last decade has seen an incredible breakthrough in technologies that allow histones, transcription factors (TFs), and RNA polymerases to be precisely mapped throughout the genome. From this research, it is clear that there is a complex interaction between the chromatin landscape and the general transcriptional machinery and that the dynamic control of this interface is central to gene regulation. However, the chromatin remodeling enzymes and general TFs cannot, on their own, recognize and stably bind to promoter or enhancer regions. Rather, they are recruited to cis regulatory regions through interaction with site-specific DNA binding TFs and/or proteins that recognize epigenetic marks such as methylated cytosines or specifically modified amino acids in histones. These “recruitment” factors are modular in structure, reflecting their ability to interact with the genome via one region of the protein and to simultaneously bind to other regulatory proteins via “effector” domains. In this chapter, we provide examples of common effector domains that can function in transcriptional regulation via their ability to (a) interact with the basal transcriptional machinery and general co-activators, (b) interact with other TFs to allow cooperative binding, and (c) directly or indirectly recruit histone and chromatin modifying enzymes.
PMCID: PMC4151296  PMID: 21557087
8.  Using ChIP-seq Technology to Identify Targets of Zinc Finger Transcription Factors 
Half of all human transcription factors are zinc finger proteins and yet very little is known concerning the biological role of the majority of these factors. In particular, very few genome-wide studies of the in vivo binding of zinc finger factors have been performed. Based on in vitro studies and other methods that allow selection of high affinity-binding sites in artificial conditions, a zinc finger code has been developed that can be used to compose a putative recognition motif for a particular zinc finger factor (ZNF). Theoretically, a simple bioinformatics analysis could then predict the genomic locations of all the binding sites for that ZNF. However, it is unlikely that all of the sequences in the human genome having a good match to a predicted motif are in fact occupied in vivo (due to negative influences from repressive chromatin, nucleosomal positioning, overlap of binding sites with other factors, etc). A powerful method to identify in vivo binding sites for transcription factors on a genome-wide scale is the chromatin immunoprecipitation (ChIP) assay, followed by hybridization of the precipitated DNA to microarrays (ChIP-chip) or by high throughput DNA sequencing of the sample (ChIP-seq). Such comprehensive in vivo binding studies would not only identify target genes of a particular zinc finger factor, but also provide binding motif data that could be used to test the validity of the zinc finger code. This chapter describes in detail the steps needed to prepare ChIP samples and libraries for high throughput sequencing using the Illumina GA2 platform and includes descriptions of quality control steps necessary to ensure a successful ChIP-seq experiment.
PMCID: PMC4151297  PMID: 20680851
Zinc fingers; chromatin immunoprecipitation; ChIP-seq; next generation sequencing
9.  Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation 
Nucleic Acids Research  2014;42(16):10856-10868.
Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.
PMCID: PMC4176344  PMID: 25122745
10.  Global analysis of ZNF217 chromatin occupancy in the breast cancer cell genome reveals an association with ERalpha 
BMC Genomics  2014;15(1):520.
The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells.
ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival.
Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-520) contains supplementary material, which is available to authorized users.
PMCID: PMC4082627  PMID: 24962896
Breast cancer; ZNF217; ERalpha; GATA3; FOXA1; ChIP-seq; RNA-seq; Endocrine resistance
11.  Differential regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros 
Nature immunology  2013;14(10):1073-1083.
C2H2 zinc fingers are found in several transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for stable DNA binding, suggesting that different fingers regulate different genes and functions. Mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the phenotypes of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis and finger 4 was selectively required for tumor suppression. The distinct phenotypes suggest that only a small number of Ikaros target genes are critical for each of its biological functions. Subdivision of phenotypes and targets by mutagenesis of individual fingers will facilitate efforts to understand how members of this prevalent family regulate development, immunity and disease.
PMCID: PMC3800053  PMID: 24013668
12.  Renewable, recombinant antibodies to histone post-translational modifications 
Nature methods  2013;10(10):10.1038/nmeth.2605.
Variability in the quality of antibodies to histone post-translational modifications (PTMs) presents widely recognized hindrance in epigenetics research. Here, by using antibody engineering technologies we produced recombinant antibodies directed to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and their high specificity enabled us to identify positive and negative correlations among histone PTMs.
PMCID: PMC3828030  PMID: 23955773
13.  Comprehensive Functional Annotation of 77 Prostate Cancer Risk Loci 
PLoS Genetics  2014;10(1):e1004102.
Genome-wide association studies (GWAS) have revolutionized the field of cancer genetics, but the causal links between increased genetic risk and onset/progression of disease processes remain to be identified. Here we report the first step in such an endeavor for prostate cancer. We provide a comprehensive annotation of the 77 known risk loci, based upon highly correlated variants in biologically relevant chromatin annotations— we identified 727 such potentially functional SNPs. We also provide a detailed account of possible protein disruption, microRNA target sequence disruption and regulatory response element disruption of all correlated SNPs at . 88% of the 727 SNPs fall within putative enhancers, and many alter critical residues in the response elements of transcription factors known to be involved in prostate biology. We define as risk enhancers those regions with enhancer chromatin biofeatures in prostate-derived cell lines with prostate-cancer correlated SNPs. To aid the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancers, as well as the transcription factor TCF7L2. We analyzed in depth three variants in risk enhancers, two of which show significantly altered androgen sensitivity in LNCaP cells. This includes rs4907792, that is in linkage disequilibrium () with an eQTL for NUDT11 (on the X chromosome) in prostate tissue, and rs10486567, the index SNP in intron 3 of the JAZF1 gene on chromosome 7. Rs4907792 is within a critical residue of a strong consensus androgen response element that is interrupted in the protective allele, resulting in a 56% decrease in its androgen sensitivity, whereas rs10486567 affects both NKX3-1 and FOXA-AR motifs where the risk allele results in a 39% increase in basal activity and a 28% fold-increase in androgen stimulated enhancer activity. Identification of such enhancer variants and their potential target genes represents a preliminary step in connecting risk to disease process.
Author Summary
In the following work we provide a complete summary annotation of functional hypotheses relating to risk identified by genome wide association studies of prostate cancer. In addition, we present new genome-wide profiles for H3K27-acetylation and TCF7L2 binding in LNCaP cells. We also introduce the concept of a risk enhancer, and characterize two novel androgen-sensitive enhancers whose activity is specifically affected by prostate-cancer risk SNPs. Our findings represent a preliminary approach to systematic identification of causal variation underlying cancer risk in the prostate.
PMCID: PMC3907334  PMID: 24497837
14.  DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape 
Nature genetics  2013;45(7):10.1038/ng.2649.
Transposable element (TE) derived sequences comprise half of our genome and DNA methylome, and are presumed densely methylated and inactive. Examination of the genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues revealed unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the tissue type and their expression correlated strongly with hypomethylation of the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks including H3K4me1 and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and exhibited evidence for evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks, and have acquired tissue-specific epigenetic regulation.
PMCID: PMC3695047  PMID: 23708189
15.  LOcating Non-Unique matched Tags (LONUT) to Improve the Detection of the Enriched Regions for ChIP-seq Data 
PLoS ONE  2013;8(6):e67788.
One big limitation of computational tools for analyzing ChIP-seq data is that most of them ignore non-unique tags (NUTs) that match the human genome even though NUTs comprise up to 60% of all raw tags in ChIP-seq data. Effectively utilizing these NUTs would increase the sequencing depth and allow a more accurate detection of enriched binding sites, which in turn could lead to more precise and significant biological interpretations. In this study, we have developed a computational tool, LOcating Non-Unique matched Tags (LONUT), to improve the detection of enriched regions from ChIP-seq data. Our LONUT algorithm applies a linear and polynomial regression model to establish an empirical score (ES) formula by considering two influential factors, the distance of NUTs to peaks identified using uniquely matched tags (UMTs) and the enrichment score for those peaks resulting in each NUT being assigned to a unique location on the reference genome. The newly located tags from the set of NUTs are combined with the original UMTs to produce a final set of combined matched tags (CMTs). LONUT was tested on many different datasets representing three different characteristics of biological data types. The detected sites were validated using de novo motif discovery and ChIP-PCR. We demonstrate the specificity and accuracy of LONUT and show that our program not only improves the detection of binding sites for ChIP-seq, but also identifies additional binding sites.
PMCID: PMC3692479  PMID: 23825685
16.  ZBTB33 binds unmethylated regions of the genome associated with actively expressed genes 
DNA methylation and repressive histone modifications cooperate to silence promoters. One mechanism by which regions of methylated DNA could acquire repressive histone modifications is via methyl DNA-binding transcription factors. The zinc finger protein ZBTB33 (also known as Kaiso) has been shown in vitro to bind preferentially to methylated DNA and to interact with the SMRT/NCoR histone deacetylase complexes. We have performed bioinformatic analyses of Kaiso ChIP-seq and DNA methylation datasets to test a model whereby binding of Kaiso to methylated CpGs leads to loss of acetylated histones at target promoters.
Our results suggest that, contrary to expectations, Kaiso does not bind to methylated DNA in vivo but instead binds to highly active promoters that are marked with high levels of acetylated histones. In addition, our studies suggest that DNA methylation and nucleosome occupancy patterns restrict access of Kaiso to potential binding sites and influence cell type-specific binding.
We propose a new model for the genome-wide binding and function of Kaiso whereby Kaiso binds to unmethylated regulatory regions and contributes to the active state of target promoters.
PMCID: PMC3663758  PMID: 23693142
DNA methylation; Zinc finger proteins; Histone modifications; Transcription factor binding; Epigenetics; Transcriptional regulation
19.  Thematic Minireview Series on Results from the ENCODE Project: Integrative Global Analyses of Regulatory Regions in the Human Genome 
The Journal of Biological Chemistry  2012;287(37):30885-30887.
PMCID: PMC3438920  PMID: 22451669
Chromatin Histone Modification; Chromatin Immunoprecipitation (ChIP); Chromatin Regulation; Gene Transcription; Genomics; Transcription Factors
20.  Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3 
Genome Biology  2012;13(9):R52.
The TCF7L2 transcription factor is linked to a variety of human diseases, including type 2 diabetes and cancer. One mechanism by which TCF7L2 could influence expression of genes involved in diverse diseases is by binding to distinct regulatory regions in different tissues. To test this hypothesis, we performed ChIP-seq for TCF7L2 in six human cell lines.
We identified 116,000 non-redundant TCF7L2 binding sites, with only 1,864 sites common to the six cell lines. Using ChIP-seq, we showed that many genomic regions that are marked by both H3K4me1 and H3K27Ac are also bound by TCF7L2, suggesting that TCF7L2 plays a critical role in enhancer activity. Bioinformatic analysis of the cell type-specific TCF7L2 binding sites revealed enrichment for multiple transcription factors, including HNF4alpha and FOXA2 motifs in HepG2 cells and the GATA3 motif in MCF7 cells. ChIP-seq analysis revealed that TCF7L2 co-localizes with HNF4alpha and FOXA2 in HepG2 cells and with GATA3 in MCF7 cells. Interestingly, in MCF7 cells the TCF7L2 motif is enriched in most TCF7L2 sites but is not enriched in the sites bound by both GATA3 and TCF7L2. This analysis suggested that GATA3 might tether TCF7L2 to the genome at these sites. To test this hypothesis, we depleted GATA3 in MCF7 cells and showed that TCF7L2 binding was lost at a subset of sites. RNA-seq analysis suggested that TCF7L2 represses transcription when tethered to the genome via GATA3.
Our studies demonstrate a novel relationship between GATA3 and TCF7L2, and reveal important insights into TCF7L2-mediated gene regulation.
PMCID: PMC3491396  PMID: 22951069
21.  Autophagy Driven by a Master Regulator of Hematopoiesis 
Molecular and Cellular Biology  2012;32(1):226-239.
Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy.
PMCID: PMC3255705  PMID: 22025678
22.  Integration of Hi-C and ChIP-seq data reveals distinct types of chromatin linkages 
Nucleic Acids Research  2012;40(16):7690-7704.
We have analyzed publicly available K562 Hi-C data, which enable genome-wide unbiased capturing of chromatin interactions, using a Mixture Poisson Regression Model and a power-law decay background to define a highly specific set of interacting genomic regions. We integrated multiple ENCODE Consortium resources with the Hi-C data, using DNase-seq data and ChIP-seq data for 45 transcription factors and 9 histone modifications. We classified 12 different sets (clusters) of interacting loci that can be distinguished by their chromatin modifications and which can be categorized into two types of chromatin linkages. The different clusters of loci display very different relationships with transcription factor-binding sites. As expected, many of the transcription factors show binding patterns specific to clusters composed of interacting loci that encompass promoters or enhancers. However, cluster 9, which is distinguished by marks of open chromatin but not by active enhancer or promoter marks, was not bound by most transcription factors but was highly enriched for three transcription factors (GATA1, GATA2 and c-Jun) and three chromatin modifiers (BRG1, INI1 and SIRT6). To investigate the impact of chromatin organization on gene regulation, we performed ribonucleicacid-seq analyses before and after knockdown of GATA1 or GATA2. We found that knockdown of the GATA factors not only alters the expression of genes having a nearby bound GATA but also affects expression of genes in interacting loci. Our work, in combination with previous studies linking regulation by GATA factors with c-Jun and BRG1, provides genome-wide evidence that Hi-C data identify sets of biologically relevant interacting loci.
PMCID: PMC3439894  PMID: 22675074
23.  The Transcription Factor Encyclopedia 
Yusuf, Dimas | Butland, Stefanie L | Swanson, Magdalena I | Bolotin, Eugene | Ticoll, Amy | Cheung, Warren A | Cindy Zhang, Xiao Yu | Dickman, Christopher TD | Fulton, Debra L | Lim, Jonathan S | Schnabl, Jake M | Ramos, Oscar HP | Vasseur-Cognet, Mireille | de Leeuw, Charles N | Simpson, Elizabeth M | Ryffel, Gerhart U | Lam, Eric W-F | Kist, Ralf | Wilson, Miranda SC | Marco-Ferreres, Raquel | Brosens, Jan J | Beccari, Leonardo L | Bovolenta, Paola | Benayoun, Bérénice A | Monteiro, Lara J | Schwenen, Helma DC | Grontved, Lars | Wederell, Elizabeth | Mandrup, Susanne | Veitia, Reiner A | Chakravarthy, Harini | Hoodless, Pamela A | Mancarelli, M Michela | Torbett, Bruce E | Banham, Alison H | Reddy, Sekhar P | Cullum, Rebecca L | Liedtke, Michaela | Tschan, Mario P | Vaz, Michelle | Rizzino, Angie | Zannini, Mariastella | Frietze, Seth | Farnham, Peggy J | Eijkelenboom, Astrid | Brown, Philip J | Laperrière, David | Leprince, Dominique | de Cristofaro, Tiziana | Prince, Kelly L | Putker, Marrit | del Peso, Luis | Camenisch, Gieri | Wenger, Roland H | Mikula, Michal | Rozendaal, Marieke | Mader, Sylvie | Ostrowski, Jerzy | Rhodes, Simon J | Van Rechem, Capucine | Boulay, Gaylor | Olechnowicz, Sam WZ | Breslin, Mary B | Lan, Michael S | Nanan, Kyster K | Wegner, Michael | Hou, Juan | Mullen, Rachel D | Colvin, Stephanie C | Noy, Peter John | Webb, Carol F | Witek, Matthew E | Ferrell, Scott | Daniel, Juliet M | Park, Jason | Waldman, Scott A | Peet, Daniel J | Taggart, Michael | Jayaraman, Padma-Sheela | Karrich, Julien J | Blom, Bianca | Vesuna, Farhad | O'Geen, Henriette | Sun, Yunfu | Gronostajski, Richard M | Woodcroft, Mark W | Hough, Margaret R | Chen, Edwin | Europe-Finner, G Nicholas | Karolczak-Bayatti, Magdalena | Bailey, Jarrod | Hankinson, Oliver | Raman, Venu | LeBrun, David P | Biswal, Shyam | Harvey, Christopher J | DeBruyne, Jason P | Hogenesch, John B | Hevner, Robert F | Héligon, Christophe | Luo, Xin M | Blank, Marissa Cathleen | Millen, Kathleen Joyce | Sharlin, David S | Forrest, Douglas | Dahlman-Wright, Karin | Zhao, Chunyan | Mishima, Yuriko | Sinha, Satrajit | Chakrabarti, Rumela | Portales-Casamar, Elodie | Sladek, Frances M | Bradley, Philip H | Wasserman, Wyeth W
Genome Biology  2012;13(3):R24.
Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at
PMCID: PMC3439975  PMID: 22458515
24.  Epigenetic Modulation of miR-122 Facilitates Human Embryonic Stem Cell Self-Renewal and Hepatocellular Carcinoma Proliferation 
PLoS ONE  2011;6(11):e27740.
The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn “on” this miRNA is observed in uncontrollably proliferating HCCs.
PMCID: PMC3225380  PMID: 22140464
25.  L3MBTL2 protein acts in concert with PcG protein mediated monoubiquitination of H2A to establish a repressive chromatin structure 
Molecular cell  2011;42(4):438-450.
We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb Repressive Complex 1 (PRC1)-like 4 (PRC1L4) given the co-presence of PcG proteins RING1, RING2 and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/ HPlγ known to function in transcriptional repression. PRCIL4-mediated repression necessitated L3MBTL2 that compacted chromatin in a histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT-domain containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genes that exhibited a significant reduction in H2A lysine 119 monoubiquitination. These findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks.
PMCID: PMC3142354  PMID: 21596310

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