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 ...

Cis-regulatory elements (CREs) control gene expression by recruiting transcription factors (TFs) and other DNA binding proteins. We aim to understand how individual nucleotides contribute to the function of CREs. Here we introduce CRE analysis by sequencing (CRE-seq), a high-throughput method for producing and testing large numbers of reporter genes in mammalian cells. We used CRE-seq to assay >1,000 single and double nucleotide mutations in a 52-bp CRE in the Rhodopsin promoter that drives strong and specific expression in mammalian photoreceptors. We find that this particular CRE is remarkably complex. The majority (86%) of single nucleotide substitutions in this sequence exert significant effects on regulatory activity. Although changes in the affinity of known TF binding sites explain some of these expression changes, we present evidence for complex phenomena, including binding site turnover and TF competition. Analysis of double mutants revealed complex, nucleotide-specific interactions between residues in different TF binding sites. We conclude that some mammalian CREs are finely tuned by evolution and function through complex, nonadditive interactions between bound TFs. CRE-seq will be an important tool to uncover the rules that govern these interactions.utations in cis-regulatory elements (CREs) often have unexpected effects on gene regulation. We lack models with the predictive power to accurately interpret the functional consequences of noncoding polymorphisms. More generally, we do not understand the nucleotide-level architecture that distinguishes true CREs from nonfunctional groupings of transcription factor (TF) binding sites (TFBS). Although consortium-driven efforts continue to predict that large numbers of mammalian sequences are CREs (1, 2), we lack a corresponding high-throughput method for functionally analyzing the consequences of variants in these elements. Addressing these problems requires fine structure mutational analysis of mammalian CREs on a large scale-experiments that are difficult to perform using traditional assays. To facilitate such experiments, we developed CRE analysis by sequencing (CREseq), a high-throughput reporter gene assay for mammalian cells.CRE-seq leverages recent advances in oligonucleotide (oligo) synthesis (3) and high-throughput sequencing (4). Using arraybased oligo synthesis, we construct large numbers of reporter genes with unique sequence barcodes in their 3′ UTRs. These libraries of barcoded reporter genes are then transfected, en masse, into mammalian cells and quantified by performing RNA sequencing (RNA-Seq) (5) on the sequence barcodes. Here we present a study using CRE-seq to dissect a CRE in mouse Rhodopsin (Rho), a gene that is expressed strongly and specifically in the mammalian retina.Tight control of Rho expression is critical for the function of mammalian retinas (6, 7). Rho expression is regulated in mice by multiple CREs located at varying distances from the transcription start site (TSS) (8, 9). These elements are occupied in vivo by CRX, a re...

Because maturing oocytes and early embryos lack appreciable transcription, posttranscriptional regulatory processes control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly regulate translation until gastrulation, when this coupling disappears. During egg activation, relative changes in poly(A)-tail length, and thus translational efficiency, were largely retained in the absence of cytoplasmic polyadenylation, which indicated that selective poly(A)-tail shortening primarily specifies these changes. Many translational changes depended on PAN GU and Smaug, and these changes were largely attributable to tail-length changes. Our results also revealed the presence of tail-length–independent mechanisms that maintained translation despite tail-length shortening during oocyte maturation, and prevented essentially all translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies.DOI: http://dx.doi.org/10.7554/eLife.16955.001