High-throughput cis-regulatory element dissection [Systems Biology]

High-throughput cis-regulatory element dissection [Systems Biology]: 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...

Convergent Evolution within an Adaptive Radiation of Cichlid Fishes

Convergent Evolution within an Adaptive Radiation of Cichlid Fishes: Moritz Muschick, Adrian Indermaur, Walter Salzburger. The recurrent evolution of convergent forms is a widespread phenomenon in adaptive radiations (e.g., [1–9]). For example, similar ecotypes of anoles lizards have evolved on different islands of th....

Controls of Nucleosome Positioning in the Human Genome

Controls of Nucleosome Positioning in the Human Genome:
by Daniel J. Gaffney, Graham McVicker, Athma A. Pai, Yvonne N. Fondufe-Mittendorf, Noah Lewellen, Katelyn Michelini, Jonathan Widom, Yoav Gilad, Jonathan K. Pritchard

Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase–seq) from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase–seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7%) of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned—in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.

Genomic Variation and Its Impact on Gene Expression in Drosophila melanogaster

Genomic Variation and Its Impact on Gene Expression in Drosophila melanogaster:
by Andreas Massouras, Sebastian M. Waszak, Monica Albarca-Aguilera, Korneel Hens, Wiebke Holcombe, Julien F. Ayroles, Emmanouil T. Dermitzakis, Eric A. Stone, Jeffrey D. Jensen, Trudy F. C. Mackay, Bart Deplancke

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.

Recombination Modulates How Selection Affects Linked Sites in Drosophila

Recombination Modulates How Selection Affects Linked Sites in Drosophila:
by Suzanne E. McGaugh, Caiti S. S. Heil, Brenda Manzano-Winkler, Laurence Loewe, Steve Goldstein, Tiffany L. Himmel, Mohamed A. F. Noor

One of the most influential observations in molecular evolution has been a strong association between local recombination rate and nucleotide polymorphisms across the genome. This is interpreted as evidence for ubiquitous natural selection. The alternative explanation, that recombination is mutagenic, has been rejected by the absence of a similar association between local recombination rate and nucleotide divergence between species. However, many recent studies show that recombination rates are often very different even in closely related species, questioning whether an association between recombination rate and divergence between species has been tested satisfactorily. To circumvent this problem, we directly surveyed recombination across approximately 43% of the D. pseudoobscura physical genome in two separate recombination maps and 31% of the D. miranda physical genome, and we identified both global and local differences in recombination rate between these two closely related species. Using only regions with conserved recombination rates between and within species and accounting for multiple covariates, our data support the conclusion that recombination is positively related to diversity because recombination modulates Hill–Robertson effects in the genome and not because recombination is predominately mutagenic. Finally, we find evidence for dips in diversity around nonsynonymous substitutions. We infer that at least some of this reduction in diversity resulted from selective sweeps and examine these dips in the context of recombination rate.

Mechanistic Differences in the Transcriptional Interpretation of Local and Long-Range Shh Morphogen Signaling

Mechanistic Differences in the Transcriptional Interpretation of Local and Long-Range Shh Morphogen Signaling: Tony Oosterveen, Sanja Kurdija, Zhanna Alekseenko, Christopher W. Uhde, Maria Bergsland, Magnus Sandberg, Elisabet Andersson, José M. Dias, Jonas Muhr, Johan Ericson. Morphogens orchestrate tissue patterning in a concentration-dependent fashion during vertebrate embryogenesis, yet little is known of how positional information provided by such signals is transla....

Drift and the evolution of mutation rates [Evolution]

Drift and the evolution of mutation rates [Evolution]: Mutation dictates the tempo and mode of evolution, and like all traits, the mutation rate is subject to evolutionary modification. Here, we report refined estimates of the mutation rate for a prokaryote with an exceptionally small genome and for a unicellular eukaryote with a large genome. Combined with prior results,...

Boolean modeling of a GRN [Developmental Biology]

Boolean modeling of a GRN [Developmental Biology]: Conducting research on sea urchins at the Naples Zoological Station, 19th century developmental biologist Hans Driesch demonstrated the totipotent nature of early embryonic cells, contributing significantly to the then-nascent field of “developmental mechanics.” Driesch discussed the possibility of understanding the clocklike development of this organism in physical/mathematical terms, but ultimately...

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome:
by Xiaochun Ni, Yong E. Zhang, Nicolas Nègre, Sidi Chen, Manyuan Long, Kevin P. White

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.

Why Transcription Factor Binding Sites Are Ten Nucleotides Long [Population and Evolutionary Genetics]

Why Transcription Factor Binding Sites Are Ten Nucleotides Long [Population and Evolutionary Genetics]:
Gene expression is controlled primarily by transcription factors, whose DNA binding sites are typically 10 nt long. We develop a population-genetic model to understand how the length and information content of such binding sites evolve. Our analysis is based on an inherent trade-off between specificity, which is greater in long binding sites, and robustness to mutation, which is greater in short binding sites. The evolutionary stable distribution of binding site lengths predicted by the model agrees with the empirical distribution (5–31 nt, with mean 9.9 nt for eukaryotes), and it is remarkably robust to variation in the underlying parameters of population size, mutation rate, number of transcription factor targets, and strength of selection for proper binding and selection against improper binding. In a systematic data set of eukaryotic and prokaryotic transcription factors we also uncover strong relationships between the length of a binding site and its information content per nucleotide, as well as between the number of targets a transcription factor regulates and the information content in its binding sites. Our analysis explains these features as well as the remarkable conservation of binding site characteristics across diverse taxa.

HDAC4 Governs a Transcriptional Program Essential for Synaptic Plasticity and Memory

HDAC4 Governs a Transcriptional Program Essential for Synaptic Plasticity and Memory: Richard Sando, Natalia Gounko, Simon Pieraut, Lujian Liao, John Yates, Anton Maximov. Neuronal activity influences genes involved in circuit development and information processing. However, the molecular basis of this process remains poorly understood. We found that HDAC4, a histon....

Nucleosomal Elements that Control the Topography of the Barrier to Transcription

Nucleosomal Elements that Control the Topography of the Barrier to Transcription: Lacramioara Bintu, Toyotaka Ishibashi, Manchuta Dangkulwanich, Yueh-Yi Wu, Lucyna Lubkowska, Mikhail Kashlev, Carlos Bustamante. The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component—the histone tails, the specif....

Nuclear Aggregation of Olfactory Receptor Genes Governs Their Monogenic Expression

Nuclear Aggregation of Olfactory Receptor Genes Governs Their Monogenic Expression: E. Josephine Clowney, Mark A. LeGros, Colleen P. Mosley, Fiona G. Clowney, Eirene C. Markenskoff-Papadimitriou, Markko Myllys, Gilad Barnea, Carolyn A. Larabell, Stavros Lomvardas. Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from differen....

Precision of Hunchback Expression in the Drosophila Embryo

Precision of Hunchback Expression in the Drosophila Embryo: Michael W. Perry, Jacques P. Bothma, Ryan D. Luu, Michael Levine. Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to u....

[Report] Gene Loops Enhance Transcriptional Directionality

[Report] Gene Loops Enhance Transcriptional Directionality: A protein constrains double-helical DNA physically, thereby pointing RNA polymerases in the right direction.

Authors: Sue Mei Tan-Wong, Judith B. Zaugg, Jurgi Camblong, Zhenyu Xu, David W. Zhang, Hannah E. Mischo, Aseem Z. Ansari, Nicholas M. Luscombe, Lars M. Steinmetz, Nick J. Proudfoot

The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila species [RESEARCH]

The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila species [RESEARCH]:
Understanding the relationship between genome organization and expression is central to understanding genome function. Closely apposed genes in a head-to-head orientation share the same upstream region and are likely to be coregulated. Here we identify the Drosophila BEAF-32 insulator as a cis regulatory element separating close head-to-head genes with different transcription regulation modes. We then compare the binding landscapes of the BEAF-32 insulator protein in four different Drosophila genomes and highlight the evolutionarily conserved presence of this protein between close adjacent genes. We find that changes in binding of BEAF-32 to sites in the genome of different Drosophila species correlate with alterations in genome organization caused by DNA rearrangements or genome size expansion. The cross-talk between BEAF-32 genomic distribution and genome organization contributes to new gene-expression profiles, which in turn translate into specific and distinct phenotypes. The results suggest a mechanism for the establishment of differences in transcription patterns during evolution.

Nature and function of insulator protein binding sites in the Drosophila genome [RESEARCH]

Nature and function of insulator protein binding sites in the Drosophila genome [RESEARCH]:

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains [RESEARCH]

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains [RESEARCH]:
Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

Cohesin regulates tissue-specific expression by stabilizing highly occupied cis-regulatory modules [RESEARCH]

Cohesin regulates tissue-specific expression by stabilizing highly occupied cis-regulatory modules [RESEARCH]:

The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wild-type cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein–DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels.