Transcriptional enhancers direct precise on-off patterns of gene expression during development. To explore the basis for this precision, we conducted a high-throughput analysis of the Otx-a enhancer, which mediates expression in the neural plate of Ciona embryos in response to fibroblast growth factor (FGF) signaling and a localized GATA determinant. We provide evidence that enhancer specificity depends on submaximal recognition motifs having reduced binding affinities (“suboptimization”). Native GATA and ETS (FGF) binding sites contain imperfect matches to consensus motifs. Perfect matches mediate robust but ectopic patterns of gene expression. The native sites are not arranged at optimal intervals, and subtle changes in their spacing alter enhancer activity. Multiple tiers of enhancer suboptimization produce specific, but weak, patterns of expression, and we suggest that clusters of weak enhancers, including certain “superenhancers,” circumvent this trade-off in specificity and activity.

Summary In this study, we used whole genome sequencing and gene expression profiling of 215 human induced pluripotent stem cell (iPSC) lines from different donors to identify genetic variants associated with RNA expression for 5,746 genes. We were able to predict causal variants for these expression quantitative trait loci (eQTLs) that disrupt transcription factor binding and validated a subset of them experimentally. We also identified copy number variant (CNV) eQTLs, including some that appear to affect gene expression by altering the copy number of intergenic regulatory regions. In addition, we were able to identify effects on gene expression of rare genic CNVs and regulatory single nucleotide variants, and found that reactivation of gene expression on the X chromosome depends on gene chromosomal position. Our work highlights the value of iPSCs for genetic association analyses and provides a unique resource for investigating the genetic regulation of gene expression in pluripotent cells.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.enhancer | gene regulation | transcription | enhancer grammar | regulatory principles P revious studies have highlighted the importance of sequence constraints within developmental enhancers for tissue-specific patterns of gene expression in both Drosophila and Ciona embryos (1-6). For example, the 69-bp orthodenticle homeobox (Otx)-a enhancer mediates restricted expression in the Ciona neural plate in response to pleiotropic fibroblast growth factor (FGF) signaling (7-9). Specificity depends on a series of low-affinity binding sites for the transcription factors ETS (FGF signaling) and GATA (ectoderm determinant) (2). Modification of these sites to improve their binding affinities resulted in augmented levels of gene expression in the neural plate, as well as ectopic expression in additional tissues that respond to FGF signaling (2).These observations prompted the suggestion that the evolution of developmental enhancers depends on the selection of submaximal binding sites. This "suboptimization" might also apply to the organization of enhancers, as changing the spacing of neighboring GATA and ETS binding sites resulted in a significant increase in enhancer activity (2). Modified Otx-a enhancers containing both optimal binding sites and optimal spacing of neighboring sites mediated intense expression in a variety of tissues responding to FGF, including the neural plate, notochord, an...

Although cancer genomes are replete with noncoding mutations, the effects of these mutations remain poorly characterized. Here we perform an integrative analysis of 930 tumor whole genomes and matched transcriptomes, identifying a network of 193 noncoding loci in which mutations disrupt target gene expression. These “somatic eQTLs” (expression Quantitative Trait Loci) are frequently mutated in specific cancer tissues, and the majority can be validated in an independent cohort of 3,382 tumors. Among these, we find that the effects of noncoding mutations on DAAM1, MTG2 and HYI transcription are recapitulated in multiple cancer cell lines, and that increasing DAAM1 expression leads to invasive cell migration. Collectively the noncoding loci converge on a set of core pathways, permitting a classification of tumors into pathway-based subtypes. The somatic eQTL network is disrupted in 88% of tumors, suggesting widespread impact of noncoding mutations in cancer.

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Understanding the control of cell-fate choices during embryonic stem cell (ESC) differentiation is crucial for harnessing strategies for efficient production of desired cell types for pharmaceutical drug screening and cell transplantation. Here we report the identification of the zinc finger-like doublesex and mab-3-related transcription factor 5 (Dmrt5) as a marker for mammalian ventralmedial mesencephalic neuroepithelium that give rise to dopamine neurons. Gain-and loss-of-function studies in ESC demonstrate that Dmrt5 is critically involved in the specification of ventralmedial neural progenitor cell fate and the subsequent generation of dopamine neurons expressing essential midbrain characteristics. Genome-wide analysis of Dmrt5-mediated transcriptome changes and expression profiling of ventral-medial and ventral-lateral mesencephalic neuroepithelium revealed suppressive and inductive regulatory roles for Dmrt5 in the transcription program associated with the ventral-medial neural progenitor fates. Together, these data identify Dmrt5 as an important player in ventral mesencephalic neural fate specification.A major goal of embryonic stem cell (ESC) research is to direct the cells' differentiation toward specific cell types, especially those targeted by devastating degenerative diseases. The advent of induced pluripotent stem cell technology, with its promise for disease modeling, drug screening, and cell therapy, places further demand on a better understanding on the control of lineage/cell-fate specification from pluripotent stem cells. One neuronal cell type in particular, the midbrain dopaminergic (mDA) neuron, is a prime target in applied stem cell research because of its association with Parkinson's disease.The mDA neurons are generated in the floor plate (FP) region of the ventral midbrain and are uniquely identified by their coexpression of tyrosine hydroxylase (TH) with the mDA-specific homeobox protein Pitx3 (1, 2). During development, local inductive signals-Shh, FGF8, and Wnt1-induce distinct cell-fate potentials through initiation of transcriptional cascades that govern the subsequent differentiation, migration, and maturation of the ventral-most progenitors into mDA neurons (3-5). The distinct cell-fate potentials of ventral midbrain progenitors are defined by domain-identifiable expression of transcription factors. For example, the Lmx1a + Foxa2 + FP exclusively gives rise to mDA neurons, whereas the ventral-lateral domains marked by Meis2, Mab21l2, Helt, and Lhx1 produce glutamatergic or GABAergic neurons (6-9). Perturbation of such a transcription "code" seen in genetic studies often led to misspecification of progenitor identity and subsequently to neural transmitter phenotypes (10-12). These studies demonstrate the mechanism of cell-fate determination to be a balance of the activation of "specification" programs and the repression of alternative fates, as observed in the spinal cord (13). However, how transcription factors coordinate distinct fate choice in the ventral midbrain remains poorl...

Transcriptional enhancers are short segments of genomic DNA (50 bp to 1 kb in length) that can work over long distances (≥1 Mb) to regulate gene expression in specific cells and tissues. Genomic assays have identified on the order of 400,000 to one million putative enhancers in the human genome (e.g., ENCODE Consortium). This suggests that a typical gene is regulated by tens of enhancers, ensuring stringent regulation of gene expression in response to a variety of intrinsic and external signals. Despite the discovery of the first transcriptional enhancer more than 30 years ago, we know surprisingly little about how enhancers regulate gene expression. In particular, the relationship between primary DNA sequence and enhancer specificity remains obscure. Here we summarize recent high-throughput studies in whole embryos aimed at the systematic identification of the sequence and organizational constraints underlying enhancer function and specificity.