cis-regulatory evolution: July 2013

Monday, July 29, 2013

Frequency of TERT promoter mutations in human cancers

by João Vinagre

Article

Reactivation of telomerase has been implicated in human tumorigenesis. Here, somatic mutations in the TERT promoter are reported in cancers of the central nervous system, bladder, follicular cell-derived thyroid and melanoma, thus demonstrating that TERT promoter mutations are a frequent event in human cancer.

Nature Communications doi: 10.1038/ncomms3185

Authors: João Vinagre, Ana Almeida, Helena Pópulo, Rui Batista, Joana Lyra, Vasco Pinto, Ricardo Coelho, Ricardo Celestino, Hugo Prazeres, Luis Lima, Miguel Melo, Adriana Gaspar da Rocha, Ana Preto, Patrícia Castro, Ligia Castro, Fernando Pardal, José Manuel Lopes, Lúcio Lara Santos, Rui Manuel Reis, José Cameselle-Teijeiro, Manuel Sobrinho-Simões, Jorge Lima, Valdemar Máximo, Paula Soares

Friday, July 26, 2013

Local features determine cis-regulatory function [Genetics]

by White, M. A., Myers, C. A., Corbo, J. C., Cohen, B. A.

Transcription factors (TFs) recognize short sequence motifs that are present in millions of copies in large eukaryotic genomes. TFsmust distinguish their target binding sites from a vast genomic excess of spurious motif occurrences; however, it is unclear whether functional sites are distinguished from nonfunctional motifs by local primary sequence features...

DNA-binding site changes in forkhead evolution [Evolution]

by Nakagawa, S., Gisselbrecht, S. S., Rogers, J. M., Hartl, D. L., Bulyk, M. L.

The evolution of transcriptional regulatory networks entails the expansion and diversification of transcription factor (TF) families. The forkhead family of TFs, defined by a highly conserved winged helix DNA-binding domain (DBD), has diverged into dozens of subfamilies in animals, fungi, and related protists. We have used a combination of maximum-likelihood...

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

by Robin Smith

Background: Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. Results: We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. Conclusions: This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

An Epigenetic Trap Stabilizes Singular Olfactory Receptor Expression

David B. Lyons, William E. Allen, Tracie Goh, Lulu Tsai, Gilad Barnea, Stavros Lomvardas. The molecular mechanisms regulating olfactory receptor (OR) expression in the mammalian nose are not yet understood. Here, we identify the transient expression of histone demethylase LSD1 and the ....

SET1 and p300 Act Synergistically, through Coupled Histone Modifications, in Transcriptional Activation by p53

Zhanyun Tang, Wei-Yi Chen, Miho Shimada, Uyen T.T. Nguyen, Jaehoon Kim, Xiao-Jian Sun, Toru Sengoku, Robert K. McGinty, Joseph P. Fernandez, Tom W. Muir, Robert G. Roeder. The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with....

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Publication date: 15 August 2013
Source:Developmental Biology, Volume 380, Issue 2
Author(s): Heather Marlow , David Q. Matus , Mark Q. Martindale
The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral–aboral (O–Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O–Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3β inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O–Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal–vegetal patterning at earlier stages of anthozoan development.

Deciphering the onychophoran ‘segmentation gene cascade’: Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network

Publication date: Available online 20 July 2013
Source:Developmental Biology
Author(s): Ralf Janssen , Graham E. Budd
The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilizing the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods.

Differential and redundant functions of gooseberry and gooseberry neuro in the central nervous system and segmentation of the Drosophila embryo

Publication date: Available online 22 July 2013
Source:Developmental Biology
Author(s): Haihuai He , Markus Noll
The gooseberry locus of Drosophila consists of two homologous Pax genes, gooseberry neuro (gsbn) and gooseberry (gsb). Originally characterized by genetics as a single segment-polarity gene, its role in segmentation has been enigmatic, as only deficiencies uncovering both genes showed a strong segmentation phenotype while mutants of gsb did not. To solve this conundrum and assay for differential roles of gsbn and gsb, we have obtained by homologous recombination for the first time null mutants of either gene as well as a deficiency inactivating only gsbn and gsb. Our analysis shows that (i) gsbn null mutants are subviable while all surviving males and most females are sterile; (ii) gsb and gsbn share overlapping functions in segmentation and the CNS, in which gsbn largely, but not completely depends on the transcriptional activation by the product of gsb; (iii) as a consequence, in the absence of gsbn, gsb becomes haploinsufficient for its function in the CNS, and gsbn−/−gsb−/+ mutants die as larvae. Such mutants display defects in the proper specification of the SNa branch of the segmental nerve, which appears intact in gsbn−/− mutants. Lineage analysis in the embryonic CNS showed that gsbn is expressed in the entire lineage derived from NB5-4, which generates 4 or 5 motoneurons whose axons are part of the SNa branch and all of which except one also express BarH1. Analysis of gsbn−/−gsb−/+ clones originating from NB5-4 further suggests that gsb and gsbn specify the SNa fate and concomitantly repress the SNc fate in this lineage and that their products activate BarH1 transcription. Specification of the SNa fate by Gsb and Gsbn occurs mainly at the NB and GMC stage. However, the SNa mutant phenotype can be rescued by providing Gsbn as late as at the postmitotic stage. The hierarchical relationship between gsb and gsbn, the haploinsufficiency of gsb in gsbn mutants, and their redundant roles in the epidermis and CNS are discussed. A model is proposed how selection for both genes occurred after their duplication during evolution.

Friday, July 12, 2013

Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

by Adriana D. Briscoe et al.

by Adriana D. Briscoe, Aide Macias-Muñoz, Krzysztof M. Kozak, James R. Walters, Furong Yuan, Gabriel A. Jamie, Simon H. Martin, Kanchon K. Dasmahapatra, Laura C. Ferguson, James Mallet, Emmanuelle Jacquin-Joly, Chris D. Jiggins

Secondary plant compounds are strong deterrents of insect oviposition and feeding, but may also be attractants for specialist herbivores. These insect-plant interactions are mediated by insect gustatory receptors (Grs) and olfactory receptors (Ors). An analysis of the reference genome of the butterfly Heliconius melpomene, which feeds on passion-flower vines (Passiflora spp.), together with whole-genome sequencing within the species and across the Heliconius phylogeny has permitted an unprecedented opportunity to study the patterns of gene duplication and copy-number variation (CNV) among these key sensory genes. We report in silico gene predictions of 73 Gr genes in the H. melpomene reference genome, including putative CO2, sugar, sugar alcohol, fructose, and bitter receptors. The majority of these Grs are the result of gene duplications since Heliconius shared a common ancestor with the monarch butterfly or the silkmoth. Among Grs but not Ors, CNVs are more common within species in those gene lineages that have also duplicated over this evolutionary time-scale, suggesting ongoing rapid gene family evolution. Deep sequencing (∼1 billion reads) of transcriptomes from proboscis and labial palps, antennae, and legs of adult H. melpomene males and females indicates that 67 of the predicted 73 Gr genes and 67 of the 70 predicted Or genes are expressed in these three tissues. Intriguingly, we find that one-third of all Grs show female-biased gene expression (n = 26) and nearly all of these (n = 21) are Heliconius-specific Grs. In fact, a significant excess of Grs that are expressed in female legs but not male legs are the result of recent gene duplication. This difference in Gr gene expression diversity between the sexes is accompanied by a striking sexual dimorphism in the abundance of gustatory sensilla on the forelegs of H. melpomene, suggesting that female oviposition behaviour drives the evolution of new gustatory receptors in butterfly genomes.

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

Luke A. Gilbert, Matthew H. Larson, Leonardo Morsut, Zairan Liu, Gloria A. Brar, Sandra E. Torres, Noam Stern-Ginossar, Onn Brandman, Evan H. Whitehead, Jennifer A. Doudna, Wendell A. Lim, Jonathan S. Weissman, Lei S. Qi.

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 pro....

Monday, July 8, 2013

Evolution of the pair rule gene network: Insights from a centipede

Publication date: Available online 26 June 2013
Source:Developmental Biology
Author(s): Jack Green , Michael Akam
Comparative studies have examined the expression and function of homologues of the Drosophila melanogaster pair rule and segment polarity genes in a range of arthropods. The segment polarity gene homologues have a conserved role in the specification of the parasegment boundary, but the degree of conservation of the upstream patterning genes has proved more variable. Using genomic resources we identify a complete set of pair rule gene homologues from the centipede Strigamia maritima, and document a detailed time series of expression during trunk segmentation. We find supportive evidence for a conserved hierarchical organisation of the pair rule genes, with a division into early- and late-activated genes which parallels the functional division into primary and secondary pair rule genes described in insects. We confirm that the relative expression of sloppy-paired and paired with respect to wingless and engrailed at the parasegment boundary is conserved between myriapods and insects; suggesting that functional interactions between these genes might be an ancient feature of arthropod segment patterning. However, we find that the relative expression of a number of the primary pair rule genes is divergent between myriapods and insects. This corroborates suggestions that the evolution of upper tiers in the segmentation gene network is more flexible. Finally, we find that the expression of the Strigamia pair rule genes in periodic patterns is restricted to the ectoderm. This suggests that any direct role of these genes in segmentation is restricted to this germ layer, and that mesoderm segmentation is either dependent on the ectoderm, or occurs through an independent mechanism.

Size-dependent regulation of dorsal–ventral patterning in the early Drosophila embryo

Publication date: Available online 22 June 2013
Source:Developmental Biology
Author(s): Mayra Garcia , Marcos Nahmad , Gregory T. Reeves , Angelike Stathopoulos

How natural variation in embryo size affects patterning of the Drosophila embryo dorsal–ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates in this system and suggest that individual targets respond to changes in embryo size in a gene-specific manner.

Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system

Publication date: 15 August 2013
Source:Developmental Biology, Volume 380, Issue 2
Author(s): Joung-Woo Hong , Kye Won Park , Michael S. Levine
Differentiation of a specific organ or tissue requires sequential activation of regulatory genes. However, little is known about how serial gene expression is temporally regulated. Here, we present evidence that differential expression of single-minded (sim) target genes can be attributed, in part, to the number of Sim and Tango (Tgo) heterodimer binding sites within their enhancer regions. The Sim, termed a master regulator, directs ventral midline differentiation of Drosophila central nervous system (CNS). According to data on the onset timing of ventral midline gene expression, sim target genes are classified into at least 2 groups (early and late). The sim and rhomboid (rho) genes are activated during early midline differentiation whereas orthodenticle (otd), CG10249, and slit (sli) genes undergo activation during later stages of midline differentiation. Germline transformation and in situ hybridization with transgenic embryos demonstrate that enhancers activating sim and rho expression contain 4 Sim–Tgo binding sites whereas only 1 Sim–Tgo binding site is found in an enhancer of sli. A mutagenized version of the rho enhancer lacking either 1, 2, or 3 Sim–Tgo binding sites mediated progressively more delayed expression of a lacZ reporter gene in the ventral midline. In contrast, a modified sli enhancer displayed progressively earlier onset of lacZ expression when 1, 2, or 3 more Sim–Tgo binding sites were added. Taken together, these results suggest that the number of Sim–Tgo-binding sites is decisive in determining the timing of gene expression in the developing ventral midline. We also discuss a combinatorial model accounting for the sequential expression of sim target genes.

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Variation and genetic control of protein abundance in humans

by Linfeng Wu

Variation and genetic control of protein abundance in humans

Nature 499, 7456 (2013). doi:10.1038/nature12223

Authors: Linfeng Wu, Sophie I. Candille, Yoonha Choi, Dan Xie, Lihua Jiang, Jennifer Li-Pook-Than, Hua Tang & Michael Snyder

Gene expression differs among individuals and populations and is thought to be a major determinant of phenotypic variation. Although variation and genetic loci responsible for RNA expression levels have been analysed extensively in human populations, our knowledge is limited regarding the differences in human protein abundance and the genetic basis for this difference. Variation in messenger RNA expression is not a perfect surrogate for protein expression because the latter is influenced by an array of post-transcriptional regulatory mechanisms, and, empirically, the correlation between protein and mRNA levels is generally modest. Here we used isobaric tag-based quantitative mass spectrometry to determine relative protein levels of 5,953 genes in lymphoblastoid cell lines from 95 diverse individuals genotyped in the HapMap Project. We found that protein levels are heritable molecular phenotypes that exhibit considerable variation between individuals, populations and sexes. Levels of specific sets of proteins involved in the same biological process covary among individuals, indicating that these processes are tightly regulated at the protein level. We identified cis-pQTLs (protein quantitative trait loci), including variants not detected by previous transcriptome studies. This study demonstrates the feasibility of high-throughput human proteome quantification that, when integrated with DNA variation and transcriptome information, adds a new dimension to the characterization of gene expression regulation.

A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans

A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans [Research Papers]

by Doitsidou, M., Flames, N., Topalidou, I., Abe, N., Felton, T., Remesal, L., Popovitchenko, T., Mann, R., Chalfie, M., Hobert, O.

Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis eleganshermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.

Understanding the regulatory and transcriptional complexity of the genome through structure

Understanding the regulatory and transcriptional complexity of the genome through structure [PERSPECTIVE]

by Mercer, T. R., Mattick, J. S.

An expansive functionality and complexity has been ascribed to the majority of the human genome that was unanticipated at the outset of the draft sequence and assembly a decade ago. We are now faced with the challenge of integrating and interpreting this complexity in order to achieve a coherent view of genome biology. We argue that the linear representation of the genome exacerbates this complexity and an understanding of its three-dimensional structure is central to interpreting the regulatory and transcriptional architecture of the genome. Chromatin conformation capture techniques and high-resolution microscopy have afforded an emergent global view of genome structure within the nucleus. Chromosomes fold into complex, territorialized three-dimensional domains in concert with specialized subnuclear bodies that harbor concentrations of transcription and splicing machinery. The signature of these folds is retained within the layered regulatory landscapes annotated by chromatin immunoprecipitation, and we propose that genome contacts are reflected in the organization and expression of interweaved networks of overlapping coding and noncoding transcripts. This pervasive impact of genome structure favors a preeminent role for the nucleoskeleton and RNA in regulating gene expression by organizing these folds and contacts. Accordingly, we propose that the local and global three-dimensional structure of the genome provides a consistent, integrated, and intuitive framework for interpreting and understanding the regulatory and transcriptional complexity of the human genome.

Gene expression drives local adaptation in humans

Gene expression drives local adaptation in humans [RESEARCH]

by Fraser, H. B.

The molecular basis of adaptation—and, in particular, the relative roles of protein-coding versus gene expression changes—has long been the subject of speculation and debate. Recently, the genotyping of diverse human populations has led to the identification of many putative "local adaptations" that differ between populations. Here I show that these local adaptations are over 10-fold more likely to affect gene expression than amino acid sequence. In addition, a novel framework for identifying polygenic local adaptations detects recent positive selection on the expression levels of genes involved in UV radiation response, immune cell proliferation, and diabetes-related pathways. These results provide the first examples of polygenic gene expression adaptation in humans, as well as the first genome-scale support for the hypothesis that changes in gene expression have driven human adaptation.

Maps of open chromatin highlight cell type-restricted patterns of regulatory sequence variation at hematological trait loci [RESEARCH]

Maps of open chromatin highlight cell type-restricted patterns of regulatory sequence variation at hematological trait loci

by Paul, D. S., Albers, C. A., Rendon, A., Voss, K., Stephens, J., HaemGen Consortium, van der Harst, P., Chambers, J. C., Soranzo

, N., Ouwehand, W. H., Deloukas, P.

Nearly three-quarters of the 143 genetic signals associated with platelet and erythrocyte phenotypes identified by meta-analyses of genome-wide association (GWA) studies are located at non-protein-coding regions. Here, we assessed the role of candidate regulatory variants associated with cell type–restricted, closely related hematological quantitative traits in biologically relevant hematopoietic cell types. We used formaldehyde-assisted isolation of regulatory elements followed by next-generation sequencing (FAIRE-seq) to map regions of open chromatin in three primary human blood cells of the myeloid lineage. In the precursors of platelets and erythrocytes, as well as in monocytes, we found that open chromatin signatures reflect the corresponding hematopoietic lineages of the studied cell types and associate with the cell type–specific gene expression patterns. Dependent on their signal strength, open chromatin regions showed correlation with promoter and enhancer histone marks, distance to the transcription start site, and ontology classes of nearby genes. Cell type–restricted regions of open chromatin were enriched in sequence variants associated with hematological indices. The majority (63.6%) of such candidate functional variants at platelet quantitative trait loci (QTLs) coincided with binding sites of five transcription factors key in regulating megakaryopoiesis. We experimentally tested 13 candidate regulatory variants at 10 platelet QTLs and found that 10 (76.9%) affected protein binding, suggesting that this is a frequent mechanism by which regulatory variants influence quantitative trait levels. Our findings demonstrate that combining large-scale GWA data with open chromatin profiles of relevant cell types can be a powerful means of dissecting the genetic architecture of closely related quantitative traits.

Sunday, July 7, 2013

Understanding the regulatory and transcriptional complexity of the genome through structure

Understanding the regulatory and transcriptional complexity of the genome through structure [PERSPECTIVE]

by Mercer, T. R., Mattick, J. S.

DNase-seq predicts regions of rotational nucleosome stability across diverse human cell types

DNase-seq predicts regions of rotational nucleosome stability across diverse human cell types [RESEARCH]

by Winter, D. R., Song, L., Mukherjee, S., Furey, T. S., Crawford, G. E.

DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, ~40 yr ago it was demonstrated that DNase I also digests with a ~10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation.

Maps of open chromatin highlight cell type-restricted patterns of regulatory sequence variation at hematological trait loci

Maps of open chromatin highlight cell type-restricted patterns of regulatory sequence variation at hematological trait loci [RESEARCH]

by Paul, D. S., Albers, C. A., Rendon, A., Voss, K., Stephens, J., HaemGen Consortium, van der Harst, P., Chambers, J. C., Soranzo, N., Ouwehand, W. H., Deloukas, P.

Interplay between chromatin state, regulator binding, and regulatory motifs in six human cell types

Interplay between chromatin state, regulator binding, and regulatory motifs in six human cell types [RESEARCH]

by Ernst, J., Kellis, M.

The regions bound by sequence-specific transcription factors can be highly variable across different cell types despite the static nature of the underlying genome sequence. This has been partly attributed to changes in chromatin accessibility, but a systematic picture has been hindered by the lack of large-scale data sets. Here, we use 456 binding experiments for 119 regulators and 84 chromatin maps generated by the ENCODE in six human cell types, and relate those to a global map of regulatory motif instances for these factors. We find specific and robust chromatin state preferences for each regulator beyond the previously reported open-chromatin association, suggesting a much richer chromatin landscape beyond simple accessibility. The preferentially bound chromatin states of regulators were enriched for sequence motifs of regulators relative to all states, suggesting that these preferences are at least partly encoded by the genomic sequence. Relative to all regions bound by a regulator, however, regulatory motifs were surprisingly depleted in the regulator's preferentially bound states, suggesting additional non-sequence-specific binding beyond the level predicted by the regulatory motifs. Such permissive binding was largely restricted to open-chromatin regions showing histone modification marks characteristic of active enhancer and promoter regions, whereas open-chromatin regions lacking such marks did not show permissive binding. Lastly, the vast majority of cobinding of regulator pairs is predicted by the chromatin state preferences of individual regulators. Overall, our results suggest a joint role of sequence motifs and specific chromatin states beyond mere accessibility in mediating regulator binding dynamics across different cell types.

A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans

A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans [Research Papers]

by Doitsidou, M., Flames, N., Topalidou, I., Abe, N., Felton, T., Remesal, L., Popovitchenko, T., Mann, R., Chalfie, M., Hobert, O.

Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.

Wednesday, July 3, 2013

TALE-mediated modulation of transcriptional enhancers in vivo

Justin Crocker

& David L Stern

http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2543.html

We tested whether transcription activator-like effectors (TALES) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERS) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expresion. TALEs thus provide a novel tool for the detection and functional modulation of transcriptional enhancers in their native genomic context.