High-throughput functional testing of ENCODE segmentation predictions

Abstract

The histone modification state of genomic regions is hypothesized to reflect the regulatory activity of the underlying genomic DNA. Based on this hypothesis, the ENCODE Project Consortium measured the status of multiple histone modifications across the genome in several cell types and used these data to segment the genome into regions with different predicted regulatory activities. We measured the cis-regulatory activity of more than 2000 of these predictions in the K562 leukemia cell line. We tested genomic segments predicted to be Enhancers, Weak Enhancers, or Repressed elements in K562 cells, along with other sequences predicted to be Enhancers specific to the H1 human embryonic stem cell line (H1-hESC). Both Enhancer and Weak Enhancer sequences in K562 cells were more active than negative controls, although surprisingly, Weak Enhancer segmentations drove expression higher than did Enhancer segmentations. Lower levels of the covalent histone modifications H3K36me3 and H3K27ac, thought to mark active enhancers and transcribed gene bodies, associate with higher expression and partly explain the higher activity of Weak Enhancers over Enhancer predictions. While DNase I hypersensitivity (HS) is a good predictor of active sequences in our assay, transcription factor (TF) binding models need to be included in order to accurately identify highly expressed sequences. Overall, our results show that a significant fraction (~26%) of the ENCODE enhancer predictions have regulatory activity, suggesting that histone modification states can reflect the cis-regulatory activity of sequences in the genome, but that specific sequence preferences, such as TF-binding sites, are the causal determinants of cis-regulatory activity.[Supplemental material is available for this article.]It is widely reported that specific combinations of covalent histone modifications reflect the regulatory function of underlying genomic DNA sequence (Strahl and Allis 2000). As part of the ENCODE Project, the genomic locations of a variety of covalent histone modifications were determined by chromatin immunoprecipitation sequencing (ChIP-seq) in a number of cell types and cell lines. Two studies used these data to train computational models that predict different functional regions of the human genome. These unsupervised learning algorithms, Segway (Hoffman et al. 2012) and ChromHMM Kellis 2010, 2012), take functional genomics data as input (DNase-seq; FAIRE-seq; and ChIP-seq of histone modifications, RNA polymerase II large subunit [POLR2A], and CTCF) and return segmentation classes, which are then assigned a hypothesized function using current knowledge of histone modification function. As part of the ENCODE Project, these two sets of predictions were consolidated to create a unified annotation of the entire human genome with seven functional classes in multiple cell types. These segmentations include Transcription Start Site, Promoter Flanking, Transcribed, CTCF-bound, Enhancer, Weak Enhancer, and Repressed or Inactive segments (The ENCODE Project ...

Promoter architectures and developmental gene regulation

“…High-resolution mapping of numerous histone modifications in multiple cell types contributed to detection of most common combinations and associated functional genomic elements [62][63][64] and allowed segmentation of the genome into distinct domains based on the levels of various modifications [62,63,65]. Although specific histone modification combinations generally reflect the identity of the underlying DNA element, recent study has shown that actual levels of modification do not necessarily reflect the predicted regulatory activity [66].…”

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

“…Recently, Kwasnieski et al (2014) used a massively parallel reporter gene assay (MPRA) to compare the enhancer activity of genomic regions in different chromatin states in K562 cells. We adopted a similar approach to compare H2BK20ac-only and H3K27ac-only enhancers.…”

Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay

“…This assay is based upon the well-established reporter gene assay, in which a vector containing a reporter gene (e.g. luciferase or green fluorescent protein [GFP]), a promoter and a potential regulatory sequence is inserted into a plasmid, which is transfected into a cell; sequences that regulate gene expression then alter the amount of luciferase/GFP expressed (Arnold et al, 2013;Melnikov et al, 2012;Ow et al, 1986;Patwardhan et al, 2012;Vockley et al, 2015;Kwasnieski et al, 2014). Through the use of unique barcodes in the 3′ untranslated region (UTR) of the reporter to differentiate expression of individual oligos, MPRA can test many different sequences simultaneously, and it has been shown to reproducibly detect segments of the genome that change expression levels (Kheradpour et al, 2013;Mogno et al, 2013).…”