Peter J. Park

ChIP-seq: advantages and challenges of a maturing technology

Chromatin immunoprecipitation followed by sequencing (ChIP–seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. Owing to the tremendous progress in next-generation sequencing technology, ChIP–seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP–chip. With the decreasing cost of sequencing, ChIP–seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this Review, I describe the benefits and challenges in harnessing this technique with an emphasis on issues related to experimental design and data analysis. ChIP–seq experiments generate large quantities of data, and effective computational analysis will be crucial for uncovering biological mechanisms.

Identification of functional elements and regulatory circuits by Drosophila modENCODE

From Genome to Regulatory Networks For biologists, having a genome in hand is only the beginning—much more investigation is still needed to characterize how the genome is used to help to produce a functional organism (see the Perspective by Blaxter). In this vein, Gerstein et al. (p. 1775) summarize for the Caenorhabditis elegans genome, and The modENCODE Consortium (p. 1787) summarize for the Drosophila melanogaster genome, full transcriptome analyses over developmental stages, genome-wide identification of transcription factor binding sites, and high-resolution maps of chromatin organization. Both studies identified regions of the nematode and fly genomes that show highly occupied targets (or HOT) regions where DNA was bound by more than 15 of the transcription factors analyzed and the expression of related genes were characterized. Overall, the studies provide insights into the organization, structure, and function of the two genomes and provide basic information needed to guide and correlate both focused and genome-wide studies. The Drosophila modENCODE project demonstrates the functional regulatory network of flies. To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

Human Decidual Natural Killer Cells Are a Unique NK Cell Subset with Immunomodulatory Potential

Natural killer cells constitute 50–90% of lymphocytes in human uterine decidua in early pregnancy. Here, CD56bright uterine decidual NK (dNK) cells were compared with the CD56bright and CD56dim peripheral NK cell subsets by microarray analysis, with verification of results by flow cytometry and RT-PCR. Among the ∼10,000 genes studied, 278 genes showed at least a threefold change with P ≤ 0.001 when comparing the dNK and peripheral NK cell subsets, most displaying increased expression in dNK cells. The largest number of these encoded surface proteins, including the unusual lectinlike receptors NKG2E and Ly-49L, several killer cell Ig-like receptors, the integrin subunits αD, αX, β1, and β5, and multiple tetraspanins (CD9, CD151, CD53, CD63, and TSPAN-5). Additionally, two secreted proteins, galectin-1 and progestagen-associated protein 14, known to have immunomodulatory functions, were selectively expressed in dNK cells.

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Design and analysis of ChIP-seq experiments for DNA-binding proteins

Recent progress in massively parallel sequencing platforms has enabled genome-wide characterization of DNA-associated proteins using the combination of chromatin immunoprecipitation and sequencing (ChIP-seq). Although a variety of methods exist for analysis of the established alternative ChIP microarray (ChIP-chip), few approaches have been described for processing ChIP-seq data. To fill this gap, we propose an analysis pipeline specifically designed to detect protein-binding positions with high accuracy. Using previously reported data sets for three transcription factors, we illustrate methods for improving tag alignment and correcting for background signals. We compare the sensitivity and spatial precision of three peak detection algorithms with published methods, demonstrating gains in spatial precision when an asymmetric distribution of tags on positive and negative strands is considered. We also analyze the relationship between the depth of sequencing and characteristics of the detected binding positions, and provide a method for estimating the sequencing depth necessary for a desired coverage of protein binding sites.

Landscape of somatic retrotransposition in human cancers.

Movement in the Cancer Genome Transposable elements are genetic sequences that can replicate and move within the genome. The factors that make an element mobile are unknown but are generally considered rare in mammals. Lee et al. (p. 967, published online 28 June) analyzed five cancer types occurring among several individuals and found that three types of epithelial tumors exhibited high rates of element movement relative to brain and blood cancers. Furthermore, these somatically acquired, tumor-specific elements targeted genes in colorectal cancer that, when disrupted, impact gene expression and thus may be a factor in the progression of the cancers. Whole-genome sequencing provides evidence for somatic insertions in colorectal, prostate, and ovarian cancers. Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.

Interferon-α/β–mediated innate immune mechanisms in dermatomyositis

Dermatomyositis has been modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B and T helper cell muscle infiltration, antibody and complement‐mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. To further understand the pathophysiology of dermatomyositis, we used microarrays, computational methods, immunohistochemistry and electron microscopy to study muscle specimens from 67 patients, 54 with inflammatory myopathies, 14 with dermatomyositis. In dermatomyositis, genes induced by interferon‐α/β were highly overexpressed, and immunohistochemistry for the interferon‐α/β inducible protein MxA showed dense staining of perifascicular, and, sometimes all myofibers in 8/14 patients and on capillaries in 13/14 patients. Of 36 patients with other inflammatory myopathies, 1 patient had faint MxA staining of myofibers and 3 of capillaries. Plasmacytoid dendritic cells, potent CD4+ cellular sources of interferon‐α, are present in substantial numbers in dermatomyositis and may account for most of the cells previously identified as T helper cells. In addition to an adaptive immune response, an innate immune response characterized by plasmacytoid dendritic cell infiltration and interferon‐α/β inducible gene and protein expression may be an important part of the pathogenesis of dermatomyositis, as it appears to be in systemic lupus erythematosus. Ann Neurol 2005;57:664–678

Comparative analysis of algorithms for identifying amplifications and deletions in array CGH data

MOTIVATION Array Comparative Genomic Hybridization (CGH) can reveal chromosomal aberrations in the genomic DNA. These amplifications and deletions at the DNA level are important in the pathogenesis of cancer and other diseases. While a large number of approaches have been proposed for analyzing the large array CGH datasets, the relative merits of these methods in practice are not clear. RESULTS We compare 11 different algorithms for analyzing array CGH data. These include both segment detection methods and smoothing methods, based on diverse techniques such as mixture models, Hidden Markov Models, maximum likelihood, regression, wavelets and genetic algorithms. We compute the Receiver Operating Characteristic (ROC) curves using simulated data to quantify sensitivity and specificity for various levels of signal-to-noise ratio and different sizes of abnormalities. We also characterize their performance on chromosomal regions of interest in a real dataset obtained from patients with Glioblastoma Multiforme. While comparisons of this type are difficult due to possibly sub-optimal choice of parameters in the methods, they nevertheless reveal general characteristics that are helpful to the biological investigator.

Postmeiotic Sex Chromatin in the Male Germline of Mice

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.