In September 2012, 30 research papers, including 6 in Nature, were published online (http://www.nature.com/encode/) about genomescale data from a decade-long project, the Encyclopedia of DNA Elements (ENCODE) [1]. Aiming to delineate all functional elements encoded in the human genome [1-4], the ENCODE project examined 1% of the human genome in its pilot phase and scaled up to the whole genome in its second phase. It released data from more than 1,600 sets of experiments from 147 types of tissues [4]. These data include a catalog of human protein-coding and noncoding RNAs as well as protein-DNA interactions, chromatin and DNA accessibility, histone modifications, DNA methylation, and long-range chromosomal interactions. The ENCODE consortium reported that 80.4% of the human genome serves some type of known biochemical function [4]. The unprecedented volume and span of this study will make it an excellent resource for biological analyses. On the other hand, it can be overwhelming for a researcher to access and interpret the data. ENCODE also developed novel (or refined existing) techniques for data generation and computational analyses. These techniques are by no means restricted to ENCODE and can be employed in a wide array of applications. Because of the vast span of ENCODE, we will limit our scope to the human transcriptome and its association with chromatin modifications in this review. We aim to introduce the data that ENCODE generated and the techniques (both experimental and computational) that were used to generate them. We summarize the observations that ENCODE found. We also provide a detailed discussion of a machine learning method that was used to predict gene expression from chromatin modifications with higher accuracy than its predecessors.

DNA sequences are wrapped around octamers of histone proteins to form nucleosomes, the unit of chromatin. The nucleosome core is composed of two copies each of four histone proteins. Each histone has an N-terminal tail that faces outward from the nucleosome and can be chemically modified to influence the accessibility of the chromatin and interactions with other chromatin-binding proteins. These histone modifications are associated with the activation or repression of gene transcription [5] and many other activities of the genome, such as enhancer activities [6] and splicing regulation [7, 8]. There has been great interest in dissecting the interactions between histones and other chromatin modifications and transcriptional regulation in recent years [9]. One of ENCODE's major goals [4] is to use these chromatin modifications to define regulatory elements of the human genome in multiple cell lines and to investigate their interactions with RNA transcription.

Chromatin immunoprecipitation (ChIP) is an experimental technique used to investigate protein-DNA interactions in the cell. ChIP, followed by microarray (ChIP-chip) or high-throughput sequencing (ChIP-seq), is widely used to determine genomewide binding locations of proteins, including transcription factors, covalent modifications of histones, and other chromatin regulatory proteins. ChIP-chip was the chosen technology for the pilot phase of ENCODE to dissect the regulatory regions in the 1% selected portion of the human genome [2, 10, 11]. As next-generation sequencing technology advanced [12], it soon became clear that ChIP-seq was a superior approach [13, 14].

ChIP-seq was the chosen technology for the second phase of the ENCODE project [3, 4, 15]. Although ChIP-seq shows clear advantages over ChIP-chip, it is by no means a perfect technology. In an evaluation study performed by Chen et al. [16], multiple factors were found to influence the fidelity of ChIP-seq data. For example, ChIP-seq data were found to be biased toward open chromatin regions, leading to false positives if not corrected; comparison of different algorithms also showed notable variation in sensitivity and specificity. The ENCODE consortium has established a set of guidelines to ensure the quality of ChIP-seq experiments [15]. Because antibodies play a predominant role in the success of ChIP experiments, a significant effort has been made by ENCODE to characterize the specificity and efficiency of a large number of antibodies. The list of the antibodies used and validated by ENCODE can be found at: http://genome.ucsc.edu/ENCODE/antibodies.html. A large-scale assessment of histone modification antibodies (>200) was performed by multiple groups [17] of the ENCODE consortium, with the most up-to-date information available at: http://compbio.med.harvard.edu/antibodies/. In addition, multiple quality metrics have been developed and used by the ENCODE project [15], as listed in Table 1. Having employed these quality metrics, the ENCODE consortium mapped 11 histone modifications plus one histone variant across 46 human cell types, including a complete matrix of the 12 marks across two groups of cell lines, designated as tier 1 and tier 2.

Enabled by the unprecedented volume of data generated by the ENCODE project, Dong et al. [57] performed a very interesting study that attempted to predict gene expression from chromatin features using machine learning techniques. As usually practiced, a machine learning study is composed of a series of procedures that typically involve data collection, data representation, model building, and testing. Using machine learning terminology, the response variables here are gene expression patterns that are predicted/modeled, while the predictors or features are various chromatin measures. Chromatin data were collected from the ENCODE project with 11 chromatin modifications, one histone variant, and DNase I hypersensitivity, all mapped in seven cell lines (Table 3). Gene expression data were from different cellular compartments, using two different RNA isolation approaches and sequenced with different technologies (Table 3). With such diversity of RNA sources, Dong et al. [57] answered not only the general question of whether gene expression can be predicted with satisfactory accuracy but also the questions of whether different RNA sources that are sequenced by different technologies can be predicted differently using the same chromatin features.

The gene expression data are relatively easy to represent. They were separated into two classes: transcriptional start site (TSS)-based and transcript-based (Tx-based). TSS-based expression data are read counts within a 101-bp window centered on the TSS, which measures transcriptional initiation. Tx-based expression data are summarized read counts from the whole transcript, which measures transcriptional elongation. However, the representation of chromatin data seems to be tricky and requires further research. Dong et al. [57] used a strategy called "bestbin," which considers the chromatin signals across the entire gene body, including 2-kb flanking regions. It basically segregates each genic region into equal bins of 100 bp and summarizes the chromatin signals within each. A training dataset was used to identify the bin that correlates most with gene expression, and the learned parameter values were applied to testing data. Other strategies [58, 59] are possible, but the "bestbin" strategy was found to be superior [57].

RNA sequencing data are known to contain very little or no background noise, with a large portion of the genes having 0 read counts. Therefore, the response variables become a mixture of a 0 component and a positive counting component. Neither a classifier nor regression method seems to be able to capture the variability of both. To deal with the challenge, a two-step approach was used by [57], so that a classifier first categorized genes as "expressed" or "unexpressed," Then, a regression method was used to predict the expression levels of the expressed genes. The final prediction is the product of the classifier and the regression method.

To test the performance of their approach, each dataset was separated into a training set and a testing set. On the training set, the "best bin" and a few other parameters were determined. After that, a 10-fold crossvalidation was performed on the testing set to evaluate the model. AUC [60] was used to represent the accuracy of the classifier. Two criteria were used to represent the accuracy of the regression method. Pearson correlation coefficient (PCC) was used to measure the similarity between the predicted value and experimental value. Root mean square error was used to measure the disparity between the predicted value and experimental value.

Overall, the two-step model achieved very satisfactory performance, with a PCC > 0.9 for a number of datasets and a PCC > 0.8 for 71% of the whole data. Looking at the two steps separately, the AUC can be as high as 0.95 for the classifier, and the PCC can be as high as 0.77 for the regression method when predicting CAGE-measured polyA+ cytosolic RNA expression in K562 cells. Similar performance was achieved in other datasets. It was also found that H3K9ac and H3K4me3 are the most important predictors for the classifier, strengthening their roles as activation marks at TSSs. In contrast, H3K79me2 and H3K36me3 are the most important predictors for the regression methods, strengthening their roles as elongation marks at gene bodies. These findings show that the two-step model not only improved the accuracy of prediction but also enabled the identification of the chromatin features that are associated with different transcriptional roles.

During the past decade, the ENCODE project has evolved into a genomewide scale, and the dataset it generated has expanded in quantity as well as in scope. The ENCODE project has provided a global view about the human transcriptome and most noticeably found that the transcribed region of the human genome is more abundant than we previously thought. This finding significantly reduced the so-called intergenic regions, as defined in the traditional sense. The quantitative measurement of RNA species in several cellular compartments as well as their polyadenylation provided a comprehensive view of RNA generation. In this review, we have revisited the characteristics of both coding and noncoding transcripts in association with their structures and locations in cells.

Besides the human transcriptome and the associated chromatin modification data that we discussed, the ENCODE consortium also mapped transcription factor binding sites and their associated DNA motifs, as well as DNA methylation and long-range chromosomal interactions [4]. In parallel, the Roadmap Epigenomics Project (http://www.roadmapepigenomics.org) and International Human Epigenome Consortium (http://www.ihec-epigenomes.org) have been accumulating data of a similar scale to understand the human genome in other tissues and conditions. It is remarkable that the ENCODE data altogether have associated more than 80% of the human genome with some type of biochemical function so far, and the coverage will continue to increase as we map additional protein-DNA interactions in the near future. It has now become very clear that so-called "junk DNA" is not evolutionarily vestigial but has specific structural or biochemical functions.

While data generation has been a major goal of ENCODE, the need to integrate the current datasets is becoming more and more important. Computational approaches have been developed to exploit the ENCODE data at to a fuller potential. For instance, chromatin features were used to model gene expression [57, 61]; integrative methods were developed to annotate genomes [62-64]; visualization tools were developed to investigate epigenomic regulation at a global scale (also see ngs.plot at https://code.google.com/p/ngsplot/) [65, 66]; and large regulatory networks were reconstructed, based on TFs and DNaseI footprinting [67, 68]. The network-based approach as well as the chromosomal interactions [69] provided novel angles in studying gene regulation at higher levels. New approaches to integrate the large amount of data to provide new biological insights are on the horizon.