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1.  Regulation of cellular chromatin state 
Organogenesis  2010;6(1):37-47.
The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation.
PMCID: PMC2861742  PMID: 20592864
hox; polycomb; trithorax; quiescence; myogenesis; satellite cells; regeneration; chromatin state
2.  Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb 
Nucleic Acids Research  2011;39(11):4664-4679.
To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb’s ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation.
doi:10.1093/nar/gkr024
PMCID: PMC3113568  PMID: 21317192
3.  The GATA1-HS2 Enhancer Allows Persistent and Position-Independent Expression of a β-globin Transgene 
PLoS ONE  2011;6(12):e27955.
Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.
doi:10.1371/journal.pone.0027955
PMCID: PMC3229501  PMID: 22164220
4.  ChIP'ing the mammalian genome: technical advances and insights into functional elements 
Genome Medicine  2009;1(9):89.
Characterization of the functional components in mammalian genomes depends on our ability to completely elucidate the genetic and epigenetic regulatory networks of chromatin states and nuclear architecture. Such endeavors demand the availability of robust and effective approaches to characterizing protein-DNA associations in their native chromatin environments. Consider able progress has been made through the applica tion of chromatin immunoprecipitation (ChIP) to study chromatin biology in cells. Coupled with genome-wide analyses, ChIP-based assays enable us to take a global, unbiased and comprehensive view of transcriptional control, epigenetic regulation and chromatin structures, with high precision and versatility. The integrated knowledge derived from these studies is used to decipher gene regulatory networks and define genome organization. In this review, we discuss this powerful approach and its current advances. We also explore the possible future developments of ChIP-based approaches to interrogating long-range chromatin interactions and their impact on the mechanisms regulating gene expression.
doi:10.1186/gm89
PMCID: PMC2768996  PMID: 19804607
5.  Chromatin state maps: new technologies, new insights 
Recent years have seen unprecedented characterization of mammalian chromatin thanks to advances in chromatin assays, antibody development and genomics. Genome-wide maps of chromatin state can now be readily acquired using microarrays or next-generation sequencing technologies. These datasets reveal local and long-range chromatin patterns that offer insight into the locations and functions of underlying regulatory elements and genes. These patterns are dynamic across developmental stages and lineages. Global studies of chromatin in embryonic stem cells have led to intriguing hypotheses regarding Polycomb/trithorax and RNA polymerase roles in ‘poising’ transcription. Chromatin state maps thus provide a rich resource for understanding chromatin at a ‘systems level’, and a starting point for mechanistic studies aimed at defining epigenetic controls that underlie development.
doi:10.1016/j.gde.2008.01.010
PMCID: PMC2486450  PMID: 18339538
6.  Chromatin structure of pluripotent stem cells and induced pluripotent stem cells 
Pluripotent embryonic stem (ES) cells are specialized cells with a dynamic chromatin structure, which is intimately connected with their pluripotency and physiology. In recent years somatic cells have been reprogrammed to a pluripotent state through over-expression of a defined set of transcription factors. These cells, known as induced pluripotent stem (iPS) cells, recapitulate ES cell properties and can be differentiated to apparently all cell lineages, making iPS cells a suitable replacement for ES cells in future regenerative medicine. Chromatin modifiers play a key function in establishing and maintaining pluripotency, therefore, elucidating the mechanisms controlling chromatin structure in both ES and iPS cells is of utmost importance to understanding their properties and harnessing their therapeutic potential. In this review, we discuss recent studies that provide a genome-wide view of the chromatin structure signature in ES cells and iPS cells and that highlight the central role of histone modifiers and chromatin remodelers in pluripotency maintenance and induction.
doi:10.1093/bfgp/elq038
PMCID: PMC3080763  PMID: 21325400
embryonic stem cells; induced pluripotent stem cells; reprogramming; epigenetics; chromatin structure; differentiation
7.  Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells 
Nature immunology  2010;12(2):129-136.
The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non–DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex–binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.
doi:10.1038/ni.1978
PMCID: PMC3766981  PMID: 21186366
8.  Tracking the mechanical dynamics of human embryonic stem cell chromatin 
Background
A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells.
Results
We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs) are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation.
Conclusions
We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.
doi:10.1186/1756-8935-5-20
PMCID: PMC3570407  PMID: 23259580
9.  Genomewide Chromatin Maps Derived from Limited Numbers of Hematopoietic Progenitors 
Nature methods  2010;7(8):615-618.
Current methods for whole genome mapping of protein-DNA interactions, performed by coupling chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq), require large amounts of starting materials which precludes their application to rare cell types. Here, we combine a high-sensitivity ChIP assay with a novel library preparation procedure to map histone modifications in as few as 10,000 cells. We apply the technique to characterize murine hematopoietic progenitors and thereby gain insight into their developmental program.
doi:10.1038/nmeth.1478
PMCID: PMC2924612  PMID: 20622861
10.  Chromatin: the interface between extrinsic cues and the epigenetic regulation of muscle regeneration 
Trends in cell biology  2009;19(6):286-294.
Muscle regeneration provides a paradigm by which to study how extrinsic signals coordinate gene expression in somatic stem cells (satellite cells) by directing the genome distribution of chromatin-modifying complexes. Understanding the signal-dependent control of the epigenetic events underlying the transition of muscle stem cells through sequential regeneration stages holds the promise to reveal new targets for selective interventions toward repairing diseased muscles. This review describes the latest findings on how regeneration cues are integrated at the chromatin level to build the transcription network that regulates progression of endogenous muscle progenitors throughout the myogenic program. In particular, we describe how specific epigenetic signatures can confer responsiveness to extrinsic cues on discrete regions of the muscle stem cell genome.
doi:10.1016/j.tcb.2009.03.002
PMCID: PMC2807132  PMID: 19394225
11.  Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays 
PLoS ONE  2010;5(12):e15651.
Background
Uncovering epigenetic states by chromatin immunoprecipitation and microarray hybridization (ChIP-chip) has significantly contributed to the understanding of gene regulation at the genome-scale level. Many studies have been carried out in mice and humans; however limited high-resolution information exists to date for non-mammalian vertebrate species.
Principal Findings
We report a 2.1-million feature high-resolution Nimblegen tiling microarray for ChIP-chip interrogations of epigenetic states in zebrafish (Danio rerio). The array covers 251 megabases of the genome at 92 base-pair resolution. It includes ∼15 kb of upstream regulatory sequences encompassing all RefSeq promoters, and over 5 kb in the 5′ end of coding regions. We identify with high reproducibility, in a fibroblast cell line, promoters enriched in H3K4me3, H3K27me3 or co-enriched in both modifications. ChIP-qPCR and sequential ChIP experiments validate the ChIP-chip data and support the co-enrichment of trimethylated H3K4 and H3K27 on a subset of genes. H3K4me3- and/or H3K27me3-enriched genes are associated with distinct transcriptional status and are linked to distinct functional categories.
Conclusions
We have designed and validated for the scientific community a comprehensive high-resolution tiling microarray for investigations of epigenetic states in zebrafish, a widely used developmental and disease model organism.
doi:10.1371/journal.pone.0015651
PMCID: PMC3004928  PMID: 21187971
12.  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.
Availability: http://home.gwu.edu/∼wpeng/Software.htm
Contact: wpeng@gwu.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btp340
PMCID: PMC2732366  PMID: 19505939
13.  Epigenetics of induced pluripotency, the seven-headed dragon 
Induction of pluripotency from somatic cells by exogenous transcription factors is made possible by a variety of epigenetic changes that take place during the reprogramming process. The derivation of fully reprogrammed induced pluripotent stem (iPS) cells is achieved through establishment of embryonic stem cell (ESC)-like epigenetic architecture permitting the reactivation of key endogenous pluripotency-related genes, establishment of appropriate bivalent chromatin domains and DNA hypomethylation of genomic heterochromatic regions. Restructuring of the epigenetic landscape, however, is a very inefficient process and the vast majority of the induced cells fail to complete the reprogramming process. Optimal ESC-like epigenetic reorganization is necessary for all reliable downstream uses of iPS cells, including in vitro modeling of disease and clinical applications. Here, we discuss the key advancements in the understanding of dynamic epigenetic changes taking place over the course of the reprogramming process and how aberrant epigenetic remodeling may impact downstream applications of iPS cell technology.
doi:10.1186/scrt3
PMCID: PMC2873696  PMID: 20504284
14.  Temporal and epigenetic regulation of neurodevelopmental plasticity 
The anticipated therapeutic uses of neural stem cells depend on their ability to retain a certain level of developmental plasticity. In particular, cells must respond to developmental manipulations designed to specify precise neural fates. Studies in vivo and in vitro have shown that the developmental potential of neural progenitor cells changes and becomes progressively restricted with time. For in vitro cultured neural progenitors, it is those derived from embryonic stem cells that exhibit the greatest developmental potential. It is clear that both extrinsic and intrinsic mechanisms determine the developmental potential of neural progenitors and that epigenetic, or chromatin structural, changes regulate and coordinate hierarchical changes in fate-determining gene expression. Here, we review the temporal changes in developmental plasticity of neural progenitor cells and discuss the epigenetic mechanisms that underpin these changes. We propose that understanding the processes of epigenetic programming within the neural lineage is likely to lead to the development of more rationale strategies for cell reprogramming that may be used to expand the developmental potential of otherwise restricted progenitor populations.
doi:10.1098/rstb.2006.2010
PMCID: PMC2605484  PMID: 17311782
neural stem cell; progenitor; development; plasticity; differentiation; epigenetic
15.  Characterization of Chromatin Structure-associated Histone Modifications in Breast Cancer Cells 
Genomics & Informatics  2012;10(3):145-152.
Chromatin structure and dynamics that are influenced by epigenetic marks, such as histone modification and DNA methylation, play a crucial role in modulating gene transcription. To understand the relationship between histone modifications and regulatory elements in breast cancer cells, we compared our chromatin immunoprecipitation sequencing (ChIP-Seq) histone modification patterns for histone H3K4me1, H3K4me3, H3K9/16ac, and H3K27me3 in MCF-7 cells with publicly available formaldehyde-assisted isolation of regulatory elements (FAIRE)-chip signals in human chromosomes 8, 11, and 12, identified by a method called FAIRE. Active regulatory elements defined by FAIRE were highly associated with active histone modifications, like H3K4me3 and H3K9/16ac, especially near transcription start sites. The H3K9/16ac-enriched genes that overlapped with FAIRE signals (FAIRE-H3K9/14ac) were moderately correlated with gene expression levels. We also identified functional sequence motifs at H3K4me1-enriched FAIRE sites upstream of putative promoters, suggesting that regulatory elements could be associated with H3K4me1 to be regarded as distal regulatory elements. Our results might provide an insight into epigenetic regulatory mechanisms explaining the association of histone modifications with open chromatin structure in breast cancer cells.
doi:10.5808/GI.2012.10.3.145
PMCID: PMC3492650  PMID: 23166525
breast neoplasms; ChIP-Seq; epigenetic regulation; formaldehyde-assisted isolation of regulatory elements (FAIRE); histone modification
16.  Global Chromatin Architecture Reflects Pluripotency and Lineage Commitment in the Early Mouse Embryo 
PLoS ONE  2010;5(5):e10531.
An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.
doi:10.1371/journal.pone.0010531
PMCID: PMC2866533  PMID: 20479880
17.  The Epigenomics of Embryonic Stem Cell Differentiation 
Embryonic stem cells (ESCs) possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency. The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues. Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes.
Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.
doi:10.7150/ijbs.7998
PMCID: PMC3858586  PMID: 24339734
Epigenomics; chromatin; embryonic stem cells; differentiation
18.  ChIP-less analysis of chromatin states 
Background
Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools.
Results
Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA.
Conclusions
Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.
doi:10.1186/1756-8935-7-7
PMCID: PMC4022240  PMID: 24872844
Affinity enrichment; Antibody-free; Chromatin; Histone; Peptide microarray; PTM; Reader domain
19.  Chromatin higher-order structures and gene regulation 
Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin to fit inside the nucleus. The dynamics of higher-order chromatin compaction play a critical role in transcription and other biological processes inherent to DNA. Many factors, including histone variants, histone modifications, DNA methylation and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. In this review, we focus on the recent progress in elucidating the structure of the 30-nm chromatin fiber. We also discuss the structural plasticity/dynamics and epigenetic inheritance of higher-order chromatin and the roles of chromatin higher-order organization in eukaryotic gene regulation.
doi:10.1016/j.gde.2011.01.022
PMCID: PMC3124554  PMID: 21342762
20.  Biased, Non-equivalent Gene-Proximal and -Distal Binding Motifs of Orphan Nuclear Receptor TR4 in Primary Human Erythroid Cells 
PLoS Genetics  2014;10(5):e1004339.
We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34+ hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.
Author Summary
Sequential genome-wide binding studies investigated by deep sequencing (ChIP-seq) represent a powerful tool for investigating the temporal sequence of gene activation and repression events that take place as cells differentiate. Here, we report the binding of an “orphan” nuclear receptor (one for which no ligand has been identified) to its cognate genomic regulatory sites and perform the functional analysis to validate its downstream targets as precursor cells differentiate from very early human hematopoietic progenitors into red blood cells. We discovered that when this receptor is bound at gene proximal promoters, it recognizes a different DNA sequence than when it binds to more distant regulatory sites (enhancers and silencers). Since this receptor can either activate or repress specific target genes, the data suggest the intriguing possibility that the two different modes of DNA recognition may reflect association of the receptor with different partner molecules when regulating gene expression from proximal or distal sequences.
doi:10.1371/journal.pgen.1004339
PMCID: PMC4014424  PMID: 24811540
21.  An Overview of Epigenetic Assays 
Molecular biotechnology  2007;38(2):179-183.
A significant portion of ongoing epigenetic research involves the investigation of DNA methylation and chromatin modification patterns seen throughout many biological processes. Over the last few years, epigenetic research has undergone a gradual shift and recent studies have been directed toward a genome-wide assessment. DNA methylation and chromatin modifications are essential components of the regulation of gene activity. DNA methylation effectively down-regulates gene activity by addition of a methyl group to the five-carbon of a cytosine base. Less specifically, modification of the chromatin structure can be carried out by multiple mechanisms leading to either the upregulation or down-regulation of the associated gene. Of the many assays used to assess the effects of epigenetic modifications, chromatin immunoprecipitation (ChIP), which serves to monitor changes in chromatin structure, and bisulfite modification, which tracks changes in DNA methylation, are the two most commonly used techniques.
doi:10.1007/s12033-007-9010-y
PMCID: PMC2423347  PMID: 17943463
Epigenetics; DNA methylation; DNMTs; CpG islands; HDACs; HMTs; Chromatin immunoprecipitation; Bisulfite modification
22.  Genomic Characterization of the Mouse Ribosomal DNA Locus 
G3: Genes|Genomes|Genetics  2013;4(2):243-254.
The transcription of rRNA is critical to all living cells and is tightly controlled at the level of chromatin structure. Although the widespread adoption of genomic technologies including chromatin immunoprecipitation with massively parallel short-read sequencing (ChIP-seq) has allowed for the interrogation of chromatin structure on a genome-wide scale, until recently rDNA has not been analyzed by this technique. We extended genomic analysis of rDNA to mouse (Mus musculus), in which rDNA is similar in structure but highly divergent in sequence compared with human rDNA. Comparison of rDNA histone marks between mouse embryonic stem cells (mESCs) and more differentiated mouse cell types revealed differences between pluripotent and differentiated states. We also observed substantial divergence in rDNA histone modification patterns between mESCs and human embryonic stem cells (hESCs). Surprisingly, we found that the pluripotency factor OCT4 was bound to rDNA in similar patterns in mESCs and hESCs. Extending this analysis, we found that an additional 17 pluripotency-associated factors were bound to rDNA in mESCs, suggesting novel modes of rDNA regulation in pluripotent cells. Taken together, our results provide a detailed view of rDNA chromatin structure in an important model system and enable high-resolution comparison of rDNA regulation between mouse and human.
doi:10.1534/g3.113.009290
PMCID: PMC3931559  PMID: 24347625
rDNA; rRNA; ChIP-seq; Oct4; polycomb
23.  Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis 
DNA methylation and histone modifications have essential roles in remodeling chromatin structure of genes necessary for multi-lineage differentiation of mammary stem/progenitor cells. The role of this well-defined epigenetic programming is to heritably maintain transcriptional plasticity of these loci over multiple cell divisions in the differentiated progeny. Epigenetic events can be deregulated in progenitor cells chronically exposed to xenoestrogen or inflammatory microenvironment. In addition, epigenetically mediated silencing of genes associated with tumor suppression can take place, resulting in clonal proliferation of undifferentiated or semidifferentiated cells. Alternatively, microRNAs that negatively regulate the expression of their protein-coding targets may become epigenetically repressed, leading to oncogenic expression of these genes. Here we further discuss interactions between DNA methylation and histone modifications that have significant contributions to the differentiation of mammary stem/progenitor cells and to tumor initiation and progression.
Mammary gland stem cells and their progeny display characteristic DNA methylation and histone modification patterns. Carcinogens and inflammation can disrupt such epigenetic programs, leading to tumorigenesis.
doi:10.1101/cshperspect.a004515
PMCID: PMC2926750  PMID: 20610549
24.  Integrative genome-wide approaches in embryonic stem cell research 
Integrative Biology  2010;2(10):510-516.
Embryonic stem (ES) cells are derived from blastocysts. They can differentiate into the three embryonic germ layers and essentially any type of somatic cells. Therefore, they hold great potentials in tissue regeneration therapy. The ethical issues associated with the use of human embryonic stem cells are resolved by the technical break-through of generating induced pluripotent stem (iPS) cells from various types of somatic cells. However, how ES and iPS cells self-renew and maintain their pluripotency is still largely unknown in spite of the great progresses that have been made in the last two decades. Integrative genome-wide approaches, such as gene expression microarray, chromatin immunoprecipitation based microarray (ChIP-chip) and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) offer unprecedented opportunities to elucidate the mechanism of the pluripotency, reprogramming and DNA damage response of ES and iPS cells. This review summarized the fundamental biological questions about ES and iPS cells and reviewed the recent advances in ES and iPS cell research using genome-wide technologies. In the end, we offered our perspectives on the future of genome-wide studies on stem cells.
doi:10.1039/c0ib00068j
PMCID: PMC3400334  PMID: 20852801
25.  Transcriptional targeting by microRNA-Polycomb complexes 
Cell Cycle  2012;11(19):3543-3549.
Advances in the understanding of the epigenetic events underlying the regulation of developmental genes expression and cell lineage commitment are revealing novel regulatory networks. These also involve distinct components of the epigenetic pathways, including chromatin histone modification, DNA methylation, repression by polycomb complexes and microRNAs. Changes in chromatin structure, DNA methylation status and microRNA expression levels represent flexible, reversible and heritable mechanisms for the maintenance of stem cell states and cell fate decisions. We recently provided novel evidence showing that microRNAs, besides determining the post-transcriptional gene silencing of their targets, also bind to evolutionarily conserved complementary genomic seed-matches present on target gene promoters. At these sites, microRNAs can function as a critical interface between chromatin remodeling complexes and the genome for transcriptional gene silencing. Here, we discuss our novel findings supporting a role of the transcriptional chromatin targeting by polycomb-microRNA complexes in lineage fate determination of human hematopoietic cells.
doi:10.4161/cc.21468
PMCID: PMC3478304  PMID: 22895111
microRNA; chromatin remodeling; Polycomb complexes; microRNA-DNA interaction; transcriptional gene silencing

Results 1-25 (1089402)