Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Unique Cell Subpopulations and Disease Progression Markers in Canines with Atopic Dermatitis.

Atopic dermatitis (AD) is a common pruritic inflammatory skin disease with unclear molecular and cellular contributions behind the complex etiology. To unravel these differences between healthy control and AD skin we employed single-cell transcriptomics, utilizing the canine AD model for its resemblance to human clinical and molecular phenotypes. In this study, we show that there are overall increases in keratinocytes and T cells and decreases in fibroblast populations in AD dogs. Within immune cell types, we identified an enriched γδ T cell population in AD, which may contribute to cutaneous inflammation. A prominent IL26-positive fibroblast subpopulation in AD was detected, which may activate neighboring cells in the dermal-epidermal niche. Lastly, by comparing dogs with different disease severities, we found genes that follow disease progression and may serve as potential biomarkers. In this study, we characterized key AD cell types and cellular processes that can be further leveraged in diagnosis and treatment.

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

CRISPR/Cas9 guided genome and epigenome engineering and its therapeutic applications in immune mediated diseases.

Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.

Integrative analysis of 111 reference human epigenomes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Reactivation of γ-globin in adult ß-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Disorders involving ß-globin gene mutations, primarily ß-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human ß-globin locus-transgenic (ß-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from ß-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into ß-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human ß- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors.

Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

Comprehensive identification and analysis of human accelerated regulatory DNA.

It has long been hypothesized that changes in gene regulation have played an important role in human evolution, but regulatory DNA has been much more difficult to study compared with protein-coding regions. Recent large-scale studies have created genome-scale catalogs of DNase I hypersensitive sites (DHSs), which demark potentially functional regulatory DNA. To better define regulatory DNA that has been subject to human-specific adaptive evolution, we performed comprehensive evolutionary and population genetics analyses on over 18 million DHSs discovered in 130 cell types. We identified 524 DHSs that are conserved in nonhuman primates but accelerated in the human lineage (haDHS), and estimate that 70% of substitutions in haDHSs are attributable to positive selection. Through extensive computational and experimental analyses, we demonstrate that haDHSs are often active in brain or neuronal cell types; play an important role in regulating the expression of developmentally important genes, including many transcription factors such as SOX6, POU3F2, and HOX genes; and identify striking examples of adaptive regulatory evolution that may have contributed to human-specific phenotypes. More generally, our results reveal new insights into conserved and adaptive regulatory DNA in humans and refine the set of genomic substrates that distinguish humans from their closest living primate relatives.

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Next-generation genomics: an integrative approach.

Integrating results from diverse experiments is an essential process in our effort to understand the logic of complex systems, such as development, homeostasis and responses to the environment. With the advent of high-throughput methods--including genome-wide association (GWA) studies, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequencing (RNA-seq)--acquisition of genome-scale data has never been easier. Epigenomics, transcriptomics, proteomics and genomics each provide an insightful, and yet one-dimensional, view of genome function; integrative analysis promises a unified, global view. However, the large amount of information and diverse technology platforms pose multiple challenges for data access and processing. This Review discusses emerging issues and strategies related to data integration in the era of next-generation genomics.

Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.

Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome.

cChIP-seq: a robust small-scale method for investigation of histone modifications.

BACKGROUND: ChIP-seq is highly utilized for mapping histone modifications that are informative about gene regulation and genome annotations. For example, applying ChIP-seq to histone modifications such as H3K4me1 has facilitated generating epigenomic maps of putative enhancers. This powerful technology, however, is limited in its application by the large number of cells required. ChIP-seq involves extensive manipulation of sample material and multiple reactions with limited quality control at each step, therefore, scaling down the number of cells required has proven challenging. Recently, several methods have been proposed to overcome this limit but most of these methods require extensive optimization to tailor the protocol to the specific antibody used or number of cells being profiled. RESULTS: Here we describe a robust, yet facile method, which we named carrier ChIP-seq (cChIP-seq), for use on limited cell amounts. cChIP-seq employs a DNA-free histone carrier in order to maintain the working ChIP reaction scale, removing the need to tailor reactions to specific amounts of cells or histone modifications to be assayed. We have applied our method to three different histone modifications, H3K4me3, H3K4me1 and H3K27me3 in the K562 cell line, and H3K4me1 in H1 hESCs. We successfully obtained epigenomic maps for these histone modifications starting with as few as 10,000 cells. We compared cChIP-seq data to data generated as part of the ENCODE project. ENCODE data are the reference standard in the field and have been generated starting from tens of million of cells. Our results show that cChIP-seq successfully recapitulates bulk data. Furthermore, we showed that the differences observed between small-scale ChIP-seq data and ENCODE data are largely to be due to lab-to-lab variability rather than operating on a reduced scale. CONCLUSIONS: Data generated using cChIP-seq are equivalent to reference epigenomic maps from three orders of magnitude more cells. Our method offers a robust and straightforward approach to scale down ChIP-seq to as low as 10,000 cells. The underlying principle of our strategy makes it suitable for being applied to a vast range of chromatin modifications without requiring expensive optimization. Furthermore, our strategy of a DNA-free carrier can be adapted to most ChIP-seq protocols.

Methods for identifying higher-order chromatin structure.

Eukaryotic genomic DNA is combined with histones, nonhistone proteins, and RNA to form chromatin, which is extensively packaged hierarchically to fit inside a cell's nucleus. The nucleosome-comprising a histone octamer with 147 base pairs of DNA wrapped around it-is the initial level and the repeating unit of chromatin packaging, which electron microscopy first made visible to the human eye as "beads on a string" nearly four decades ago. The mechanism and nature of chromatin packaging are still under intense research. Recently, classic methods like chromatin immunoprecipitation and digestion with deoxyribonuclease and micrococcal nuclease have been combined with high-throughput sequencing to provide detailed nucleosome occupancy maps, and chromosome conformation capture and its variants have revealed that higher-order chromatin structure involves long-range loop formation between distant genomic elements. This review discusses the methods for identifying higher-order chromatin structure and the information they have provided on this important topic.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Epigenomics of autoimmune diseases.

Autoimmune diseases are complex disorders of largely unknown etiology. Genetic studies have identified a limited number of causal genes from a marginal number of individuals, and demonstrated a high degree of discordance in monozygotic twins. Studies have begun to reveal epigenetic contributions to these diseases, primarily through the study of DNA methylation, but chromatin and non-coding RNA changes are also emerging. Moving forward an integrative analysis of genomic, transcriptomic and epigenomic data, with the latter two coming from specific cell types, will provide an understanding that has been missed from genetics alone. We provide an overview of the current state of the field and vision for deriving the epigenomics of autoimmunity.

Human DNA methylomes at base resolution show widespread epigenomic differences.

DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA-protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

Histone modifications at human enhancers reflect global cell-type-specific gene expression.

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression.