Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output

Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output: "

by Barbara Celona, Assaf Weiner, Francesca Di Felice, Francesco M. Mancuso, Elisa Cesarini, Riccardo L. Rossi, Lorna Gregory, Dilair Baban, Grazisa Rossetti, Paolo Grianti, Massimiliano Pagani, Tiziana Bonaldi, Jiannis Ragoussis, Nir Friedman, Giorgio Camilloni, Marco E. Bianchi, Alessandra Agresti

The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation."

Reshuffling genomic landscapes to study the regulatory evolution of Hox gene clusters [Genetics]

Reshuffling genomic landscapes to study the regulatory evolution of Hox gene clusters [Genetics]: "The emergence of Vertebrata was accompanied by two rounds of whole-genome duplications. This enabled paralogous genes to acquire novel functions with high evolutionary potential, a process suggested to occur mostly by changes in gene regulation, rather than in protein sequences. In the case of Hox gene clusters, such duplications favored the appearance of distinct global regulations. To assess the impact of such “regulatory evolution” upon neo-functionalization, we developed PANTHERE (PAN-genomic Translocation for Heterologous Enhancer RE-shuffling) to bring the entire megabase-scale HoxD regulatory landscape in front of the HoxC gene cluster via a targeted translocation in vivo. At this chimeric locus, Hoxc genes could both interpret this foreign regulation and functionally substitute for their Hoxd counterparts. Our results emphasize the importance of evolving regulatory modules rather than their target genes in the process of neo-functionalization and offer a genetic tool to study the complexity of the vertebrate regulatory genome."

p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation [Biochemistry]

p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation [Biochemistry]: "It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5′) and proximal (3′) p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal RE within the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites."

Single-tube linear DNA amplification (LinDA) for robust ChIP-seq

Single-tube linear DNA amplification (LinDA) for robust ChIP-seq: "

Single-tube linear DNA amplification (LinDA) for robust ChIP-seq

Nature Methods 8, 565 (2011).
doi:10.1038/nmeth.1626

Authors: Pattabhiraman Shankaranarayanan, Marco-Antonio Mendoza-Parra, Mannu Walia, Li Wang, Ning Li, Luisa M Trindade & Hinrich Gronemeyer

Genome-wide profiling of transcription factors based on massive parallel sequencing of immunoprecipitated chromatin (ChIP-seq) requires nanogram amounts of DNA. Here we describe a high-fidelity, single-tube linear DNA amplification method (LinDA) for ChIP-seq and reChIP-seq with picogram DNA amounts obtained from a few thousand cells. This amplification technology will facilitate global analyses of transcription-factor binding and chromatin with very small cell populations, such as stem or cancer-initiating cells.

"

A gene regulatory network controlling the embryonic specification ofendoderm

A gene regulatory network controlling the embryonic specification of<br>endoderm: "

A gene regulatory network controlling the embryonic specification of
endoderm

Nature 474, 7353 (2011). doi:10.1038/nature10100

Authors: Isabelle S. Peter & Eric H. Davidson

Specification of endoderm is the prerequisite for gut formation in the
embryogenesis of bilaterian organisms. Modern lineage labelling studies have shown that in the sea urchin embryo model system, descendants
of the veg1 and veg2 cell lineages produce the endoderm, and that the veg2
lineage also gives rise to mesodermal cell types. It is known that Wnt/β-catenin
signalling is required for endoderm specification and
Delta/Notch signalling is required for mesoderm specification.
Some direct cis-regulatory targets of these signals have been found and various phenomenological patterns of gene expression have
been observed in the pre-gastrular endomesoderm. However, no comprehensive,
causal explanation of endoderm specification has been conceived for sea urchins,
nor for any other deuterostome. Here we propose a model, on the basis of the
underlying genomic control system, that provides such an explanation, built
at several levels of biological organization. The hardwired core of the control
system consists of the cis-regulatory apparatus of endodermal regulatory
genes, which determine the relationship between the inputs to which these
genes are exposed and their outputs. The architecture of the network circuitry
controlling the dynamic process of endoderm specification then explains, at
the system level, a sequence of developmental logic operations, which generate
the biological process. The control system initiates non-interacting endodermal
and mesodermal gene regulatory networks in veg2-derived cells and extinguishes
the endodermal gene regulatory network in mesodermal precursors. It also generates
a cross-regulatory network that specifies future anterior endoderm in veg2
descendants and institutes a distinct network specifying posterior endoderm
in veg1-derived cells. The network model provides an explanatory framework
that relates endoderm specification to the genomic regulatory code.

"

Determinants of nucleosome organization in primary human cells

Determinants of nucleosome organization in primary human cells: "

Determinants of nucleosome organization in primary human cells

Nature 474, 7352 (2011). doi:10.1038/nature10002

Authors: Anton Valouev, Steven M. Johnson, Scott D. Boyd, Cheryl L. Smith, Andrew Z. Fire & Arend Sidow

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.

"

Non-adaptive origins of interactome complexity

Non-adaptive origins of interactome complexity: "

Non-adaptive origins of interactome complexity

Nature 474, 7352 (2011). doi:10.1038/nature09992

Authors: Ariel Fernández & Michael Lynch

The boundaries between prokaryotes, unicellular eukaryotes and multicellular eukaryotes are accompanied by orders-of-magnitude reductions in effective population size, with concurrent amplifications of the effects of random genetic drift and mutation. The resultant decline in the efficiency of selection seems to be sufficient to influence a wide range of attributes at the genomic level in a non-adaptive manner. A key remaining question concerns the extent to which variation in the power of random genetic drift is capable of influencing phylogenetic diversity at the subcellular and cellular levels. Should this be the case, population size would have to be considered as a potential determinant of the mechanistic pathways underlying long-term phenotypic evolution. Here we demonstrate a phylogenetically broad inverse relation between the power of drift and the structural integrity of protein subunits. This leads to the hypothesis that the accumulation of mildly deleterious mutations in populations of small size induces secondary selection for protein–protein interactions that stabilize key gene functions. By this means, the complex protein architectures and interactions essential to the genesis of phenotypic diversity may initially emerge by non-adaptive mechanisms.

"

Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences [Evolution]

Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences [Evolution]: "Spatiotemporal changes in gene expression underlie many evolutionary novelties in nature. However, the evolutionary origins of novel expression patterns, and the transcriptional control elements (“enhancers”) that govern them, remain unclear. Here, we sought to explore the molecular genetic mechanisms by which new enhancers arise. We undertook a survey of closely related Drosophila species to identify recently evolved novel gene expression patterns and traced their evolutionary history. Analyses of gene expression in a variety of developing tissues of the Drosophila melanogaster species subgroup revealed high rates of expression pattern divergence, including numerous evolutionary losses, heterochronic shifts, and expansions or contractions of expression domains. However, gains of novel expression patterns were much less frequent. One gain was observed for the Neprilysin-1 (Nep1) gene, which has evolved a unique expression pattern in optic lobe neuroblasts of Drosophila santomea. Dissection of the Nep1 cis-regulatory region localized a newly derived optic lobe enhancer activity to a region of an intron that has accumulated a small number of mutations. The Nep1 optic lobe enhancer overlaps with other enhancer activities, from which the novel activity was co-opted. We suggest that the novel optic lobe enhancer evolved by exploiting the cryptic activity of extant regulatory sequences, and this may reflect a general mechanism whereby new enhancers evolve."

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation [RESEARCH ARTICLES]

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation [RESEARCH ARTICLES]: "Mark D. Schroeder, Christina Greer, and Ulrike Gaul

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The ‘primary’ pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas ‘secondary’ pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, we have conducted a comprehensive systems-level examination of the regulatory architecture underlying pair-rule stripe formation. We find that runt (run), fushi tarazu (ftz) and odd skipped (odd) establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, we observe long-range cis-regulation across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, our study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo.

"

Minimal regulatory spaces in yeast genomes

Minimal regulatory spaces in yeast genomes: "Background:
The regulatory information encoded in the DNA of promoter regions usually enforces a minimal, non-zero distance between the coding regions of neighboring genes. However, the size of this minimal regulatory space is not generally known. In particular, it is unclear if minimal promoter size differs between species and between uni- and bi-directionally acting regulatory regions.
Results:
Analyzing the genomes of 11 yeasts, we show that the lower size limit on promoter-containing regions is species-specific within a relatively narrow range (80-255bp). This size limit applies equally to regions that initiate transcription on one or both strands, indicating that bi-directional promoters and uni-directional promoters are constrained similarly. We further find that young, species-specific regions are on average much longer than older regions, suggesting either a bias towards deletions or selection for genome compactness in yeasts. While the length evolution of promoter-less intergenic regions is well described by a simplistic, purely neutral model, regions containing promoters typically show an excess of unusually long regions. Regions flanked by divergently transcribed genes have a bi-modal length distribution, with short lengths found preferentially among older regions. These old, short regions likely harbor evolutionarily conserved bi-directionally active promoters. Surprisingly, some of the evolutionarily youngest regions in two of the eleven species (S. cerevisiae and K. waltii) are shorter than the lower limit observed in older regions.
Conclusions:
The minimal chromosomal space required for transcriptional regulation appears to be relatively similar across yeast species, and is the same for uni-directional and bi-directional promoters. New intergenic regions created by genome rearrangements tend to evolve towards the more narrow size distribution found among older regions."

Beyond DNA: integrating inclusive inheritance into an extended theory of evolution

Beyond DNA: integrating inclusive inheritance into an extended theory of evolution: "

Beyond DNA: integrating inclusive inheritance into an extended theory of evolution

Nature Reviews Genetics 12, 475 (2011).
doi:10.1038/nrg3028

Authors: Étienne Danchin, Anne Charmantier, Frances A. Champagne, Alex Mesoudi, Benoit Pujol & Simon Blanchet

Many biologists are calling for an 'extended evolutionary synthesis' that would 'modernize the modern synthesis' of evolution. Biological information is typically considered as being transmitted across generations by the DNA sequence alone, but accumulating evidence indicates that both genetic and non-genetic inheritance, and the interactions

"

Trait Variation in Yeast Is Defined by Population History

Trait Variation in Yeast Is Defined by Population History: "

by Jonas Warringer, Enikö Zörgö, Francisco A. Cubillos, Amin Zia, Arne Gjuvsland, Jared T. Simpson, Annabelle Forsmark, Richard Durbin, Stig W. Omholt, Edward J. Louis, Gianni Liti, Alan Moses, Anders Blomberg

A fundamental goal in biology is to achieve a mechanistic understanding of how and to what extent ecological variation imposes selection for distinct traits and favors the fixation of specific genetic variants. Key to such an understanding is the detailed mapping of the natural genomic and phenomic space and a bridging of the gap that separates these worlds. Here we chart a high-resolution map of natural trait variation in one of the most important genetic model organisms, the budding yeast Saccharomyces cerevisiae, and its closest wild relatives and trace the genetic basis and timing of major phenotype changing events in its recent history. We show that natural trait variation in S. cerevisiae exceeds that of its relatives, despite limited genetic variation, and follows the population history rather than the source environment. In particular, the West African population is phenotypically unique, with an extreme abundance of low-performance alleles, notably a premature translational termination signal in GAL3 that cause inability to utilize galactose. Our observations suggest that many S. cerevisiae traits may be the consequence of genetic drift rather than selection, in line with the assumption that natural yeast lineages are remnants of recent population bottlenecks. Disconcertingly, the universal type strain S288C was found to be highly atypical, highlighting the danger of extrapolating gene-trait connections obtained in mosaic, lab-domesticated lineages to the species as a whole. Overall, this study represents a step towards an in-depth understanding of the causal relationship between co-variation in ecology, selection pressure, natural traits, molecular mechanism, and alleles in a key model organism."

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor: "

by Sean Michael Carroll, Eric A. Ortlund, Joseph W. Thornton

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution."

The Role of Response Elements Organization in Transcription Factor Selectivity: The IFN-β Enhanceosome Example

The Role of Response Elements Organization in Transcription Factor Selectivity: The IFN-β Enhanceosome Example: "

by Yongping Pan, Ruth Nussinov

What is the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA? The IFN-β enhanceosome provides a good model system: it is small; its components' crystal structures are available; and there are biochemical and cellular data. In the IFN-β enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization."

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA: "

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA

Nature 474, 7351 (2011). doi:10.1038/nature10006

Authors: Dong Wang, Ivan Garcia-Bassets, Chris Benner, Wenbo Li, Xue Su, Yiming Zhou, Jinsong Qiu, Wen Liu, Minna U. Kaikkonen, Kenneth A. Ohgi, Christopher K. Glass, Michael G. Rosenfeld & Xiang-Dong Fu

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer–promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

"

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins [Cell Biology]

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins [Cell Biology]: "Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations."

Clusters of Nucleotide Substitutions and Insertion/Deletion Mutations Are Associated with Repeat Sequences

Clusters of Nucleotide Substitutions and Insertion/Deletion Mutations Are Associated with Repeat Sequences: "
by Michael J. McDonald, Wei-Chi Wang, Hsien-Da Huang, Jun-Yi Leu


The genome-sequencing gold rush has facilitated the use of comparative genomics to uncover patterns of genome evolution, although their causal mechanisms remain elusive. One such trend, ubiquitous to prokarya and eukarya, is the association of insertion/deletion mutations (indels) with increases in the nucleotide substitution rate extending over hundreds of base pairs. The prevailing hypothesis is that indels are themselves mutagenic agents. Here, we employ population genomics data from Escherichia coli, Saccharomyces paradoxus, and Drosophila to provide evidence suggesting that it is not the indels per se but the sequence in which indels occur that causes the accumulation of nucleotide substitutions. We found that about two-thirds of indels are closely associated with repeat sequences and that repeat sequence abundance could be used to identify regions of elevated sequence diversity, independently of indels. Moreover, the mutational signature of indel-proximal nucleotide substitutions matches that of error-prone DNA polymerases. We propose that repeat sequences promote an increased probability of replication fork arrest, causing the persistent recruitment of error-prone DNA polymerases to specific sequence regions over evolutionary time scales. Experimental measures of the mutation rates of engineered DNA sequences and analyses of experimentally obtained collections of spontaneous mutations provide molecular evidence supporting our hypothesis. This study uncovers a new role for repeat sequences in genome evolution and provides an explanation of how fine-scale sequence contextual effects influence mutation rates and thereby evolution."

Gene Regulation by MAPK Substrate Competition

Gene Regulation by MAPK Substrate Competition: "Yoosik Kim, María José Andreu, Bomyi Lim, Kwanghun Chung, Mark Terayama, Gerardo Jiménez, Celeste A. Berg, Hang Lu, Stanislav Y. Shvartsman. Developing tissues are patterned by coordinated activities of signaling systems, which can be integrated by a regulatory region of a gene that binds multiple transcription factors or by a transcri...."

A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

A method to sequence and quantify DNA integration for monitoring outcome in gene therapy: "

Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

"

Towards an Evolutionary Model of Transcription Networks

Towards an Evolutionary Model of Transcription Networks: "
by Dan Xie, Chieh-Chun Chen, Xin He, Xiaoyi Cao, Sheng Zhong


DNA evolution models made invaluable contributions to comparative genomics, although it seemed formidable to include non-genomic features into these models. In order to build an evolutionary model of transcription networks (TNs), we had to forfeit the substitution model used in DNA evolution and to start from modeling the evolution of the regulatory relationships. We present a quantitative evolutionary model of TNs, subjecting the phylogenetic distance and the evolutionary changes of cis-regulatory sequence, gene expression and network structure to one probabilistic framework. Using the genome sequences and gene expression data from multiple species, this model can predict regulatory relationships between a transcription factor (TF) and its target genes in all species, and thus identify TN re-wiring events. Applying this model to analyze the pre-implantation development of three mammalian species, we identified the conserved and re-wired components of the TNs downstream to a set of TFs including Oct4, Gata3/4/6, cMyc and nMyc. Evolutionary events on the DNA sequence that led to turnover of TF binding sites were identified, including a birth of an Oct4 binding site by a 2nt deletion. In contrast to recent reports of large interspecies differences of TF binding sites and gene expression patterns, the interspecies difference in TF-target relationship is much smaller. The data showed increasing conservation levels from genomic sequences to TF-DNA interaction, gene expression, TN, and finally to morphology, suggesting that evolutionary changes are larger at molecular levels and smaller at functional levels. The data also showed that evolutionarily older TFs are more likely to have conserved target genes, whereas younger TFs tend to have larger re-wiring rates."

Interlocked Feedforward Loops Control Cell-Type-Specific Rhodopsin Expression in the Drosophila Eye

Interlocked Feedforward Loops Control Cell-Type-Specific Rhodopsin Expression in the Drosophila Eye: "Robert J. Johnston, Yoshiaki Otake, Pranidhi Sood, Nina Vogt, Rudy Behnia, Daniel Vasiliauskas, Elizabeth McDonald, Baotong Xie, Sebastian Koenig, Reinhard Wolf, Tiffany Cook, Brian Gebelein, Edo Kussell, Hideki Nakagoshi, Claude Desplan. How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of...."

The Relationship Between Relative Solvent Accessibility and Evolutionary Rate in Protein Evolution [Genome and systems biology]

The Relationship Between Relative Solvent Accessibility and Evolutionary Rate in Protein Evolution [Genome and systems biology]: "

Recent work with Saccharomyces cerevisiae shows a linear relationship between the evolutionary rate of sites and the relative solvent accessibility (RSA) of the corresponding residues in the folded protein. Here, we aim to develop a mathematical model that can reproduce this linear relationship. We first demonstrate that two models that both seem reasonable choices (a simple model in which selection strength correlates with RSA and a more complex model based on RSA-dependent amino acid distributions) fail to reproduce the observed relationship. We then develop a model on the basis of observed site-specific amino acid distributions and show that this model behaves appropriately. We conclude that evolutionary rates are directly linked to the distribution of amino acids at individual sites. Because of this link, any future insight into the biophysical mechanisms that determine amino acid distributions will improve our understanding of evolutionary rates.

"

Circumventing Heterozygosity: Sequencing the Amplified Genome of a Single Haploid Drosophila melanogaster Embryo [Methods, technology, and resources]

Circumventing Heterozygosity: Sequencing the Amplified Genome of a Single Haploid Drosophila melanogaster Embryo [Methods, technology, and resources]: "

Heterozygosity is a major challenge to efficient, high-quality genomic assembly and to the full genomic survey of polymorphism and divergence. In Drosophila melanogaster lines derived from equatorial populations are particularly resistant to inbreeding, thus imposing a major barrier to the determination and analyses of genomic variation in natural populations of this model organism. Here we present a simple genome sequencing protocol based on the whole-genome amplification of the gynogenetically derived haploid genome of a progeny of females mated to males homozygous for the recessive male sterile mutation, ms(3)K81. A single 'lane' of paired-end sequences (2 x 76 bp) provides a good syntenic assembly with >95% high-quality coverage (more than five reads). The amplification of the genomic DNA moderately inflates the variation in coverage across the euchromatic portion of the genome. It also increases the frequency of chimeric clones. But the low frequency and random genomic distribution of the chimeric clones limits their impact on the final assemblies. This method provides a solid path forward for population genomic sequencing and offers applications to many other systems in which small amounts of genomic DNA have unique experimental relevance.

"

Patterns and Mechanisms of Ancestral Histone Protein Inheritance in Budding Yeast

Patterns and Mechanisms of Ancestral Histone Protein Inheritance in<br> Budding Yeast: "
by Marta Radman-Livaja, Kitty F. Verzijlbergen, Assaf Weiner, Tibor van Welsem, Nir Friedman, Oliver J. Rando, Fred van Leeuwen


Replicating chromatin involves disruption of histone-DNA contacts and subsequent
reassembly of maternal histones on the new daughter genomes. In bulk, maternal
histones are randomly segregated to the two daughters, but little is known about
the fine details of this process: do maternal histones re-assemble at preferred
locations or close to their original loci? Here, we use a recently developed
method for swapping epitope tags to measure the disposition of ancestral histone
H3 across the yeast genome over six generations. We find that ancestral H3 is
preferentially retained at the 5′ ends of most genes, with strongest
retention at long, poorly transcribed genes. We recapitulate these observations
with a quantitative model in which the majority of maternal histones are
reincorporated within 400 bp of their pre-replication locus during replication,
with replication-independent replacement and transcription-related retrograde
nucleosome movement shaping the resulting distributions of ancestral histones.
We find a key role for Topoisomerase I in retrograde histone movement during
transcription, and we find that loss of Chromatin Assembly Factor-1 affects
replication-independent turnover. Together, these results show that specific
loci are enriched for histone proteins first synthesized several generations
beforehand, and that maternal histones re-associate close to their original
locations on daughter genomes after replication. Our findings further suggest
that accumulation of ancestral histones could play a role in shaping histone
modification patterns."

The Mismeasure of Science: Stephen Jay Gould versus Samuel George Morton on Skulls and Bias

The Mismeasure of Science: Stephen Jay Gould versus Samuel George Morton on Skulls and Bias: "
by Jason E. Lewis, David DeGusta, Marc R. Meyer, Janet M. Monge, Alan E. Mann, Ralph L. Holloway
"

Convergent transcription confers a bistable switch in Enterococcus faecalis conjugation [Systems Biology]

Convergent transcription confers a bistable switch in Enterococcus faecalis conjugation [Systems Biology]: "Convergent gene pairs with head-to-head configurations are widespread in both eukaryotic and prokaryotic genomes and are speculated to be involved in gene regulation. Here we present a unique mechanism of gene regulation due to convergent transcription from the antagonistic prgX/prgQ operon in Enterococcus faecalis controlling conjugative transfer of the antibiotic resistance plasmid pCF10 from donor cells to recipient cells. Using mathematical modeling and experimentation, we demonstrate that convergent transcription in the prgX/prgQ operon endows the system with the properties of a robust genetic switch through premature termination of elongating transcripts due to collisions between RNA polymerases (RNAPs) transcribing from opposite directions and antisense regulation between complementary counter-transcripts. Evidence is provided for the presence of truncated RNAs resulting from convergent transcription from both the promoters that are capable of sense–antisense interactions. A mathematical model predicts that both RNAP collision and antisense regulation are essential for a robust bistable switch behavior in the control of conjugation initiation by prgX/prgQ operons. Moreover, given that convergent transcription is conserved across species, the mechanism of coupling RNAP collision and antisense interaction is likely to have a significant regulatory role in gene expression."

Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development [RESEARCH ARTICLES]

Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development [RESEARCH ARTICLES]: "Shengzhan D. Luo, Guang W. Shi, and Bruce S. Baker

Uncovering the direct regulatory targets of doublesex (dsx) and fruitless (fru) is crucial for an understanding of how they regulate sexual development, morphogenesis, differentiation and adult functions (including behavior) in Drosophila melanogaster. Using a modified DamID approach, we identified 650 DSX-binding regions in the genome from which we then extracted an optimal palindromic 13 bp DSX-binding sequence. This sequence is functional in vivo, and the base identity at each position is important for DSX binding in vitro. In addition, this sequence is enriched in the genomes of D. melanogaster (58 copies versus approximately the three expected from random) and in the 11 other sequenced Drosophila species, as well as in some other Dipterans. Twenty-three genes are associated with both an in vivo peak in DSX binding and an optimal DSX-binding sequence, and thus are almost certainly direct DSX targets. The association of these 23 genes with optimum DSX binding sites was used to examine the evolutionary changes occurring in DSX and its targets in insects.

"

Scaling of the Bicoid morphogen gradient by a volume-dependent production rate [RESEARCH ARTICLES]

Scaling of the Bicoid morphogen gradient by a volume-dependent production rate [RESEARCH ARTICLES]: "David Cheung, Cecelia Miles, Martin Kreitman, and Jun Ma

An important feature of development is the formation of patterns that are proportional to the overall size of the embryo. But how such proportionality, or scaling, is achieved mechanistically remains poorly understood. Furthermore, it is currently unclear whether organisms utilize similar or distinct mechanisms to achieve scaling within a species and between species. Here we investigate within-species scaling mechanisms for anterior-posterior (A-P) patterning in Drosophila melanogaster, focusing specifically on the properties of the Bicoid (Bcd) morphogen gradient. Using embryos from lines artificially selected for large and small egg volume, we show that large embryos have higher nuclear Bcd concentrations in the anterior than small embryos. This anterior difference leads to scaling properties of the Bcd gradient profiles: in broad regions of the large and small embryos along the A-P axis, normalizing their positions to embryo length reduces the differences in both the nuclear Bcd concentrations and Bcd-encoded positional information. We further trace the origin of Bcd gradient scaling by showing directly that large embryos have more maternally deposited bcd mRNA than small embryos. Our results suggest a simple model for how within-species Bcd gradient scaling can be achieved. In this model, the Bcd production rate, which is dependent on the total number of bcd mRNA molecules in the anterior, is scaled with embryo volume.

"

Distinct Functional Constraints Partition Sequence Conservation in a cis-Regulatory Element

Distinct Functional Constraints Partition Sequence Conservation in a cis-Regulatory Element: "

by Antoine Barrière, Kacy L. Gordon, Ilya Ruvinsky

Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter."

HIGH-DIMENSIONAL VARIANCE PARTITIONING REVEALS THE MODULAR GENETIC BASIS OF ADAPTIVE DIVERGENCE IN GENE EXPRESSION DURING REPRODUCTIVE CHARACTER DISPLACEMENT

HIGH-DIMENSIONAL VARIANCE PARTITIONING REVEALS THE MODULAR GENETIC BASIS OF ADAPTIVE DIVERGENCE IN GENE EXPRESSION DURING REPRODUCTIVE CHARACTER DISPLACEMENT: "

ABSTRACT

Although adaptive change is usually associated with complex changes in phenotype, few genetic investigations have been conducted of adaptations that involve sets of high dimensional traits. Microarrays have supplied high-dimensional descriptions of gene expression, and phenotypic change resulting from adaptation often results in large-scale changes in gene expression. We demonstrate how genetic analysis of large-scale changes in gene expression generated during adaptation can be accomplished by determining by high-dimensional variance partitioning within classical genetic experimental designs. A microarray experiment conducted on a panel of recombinant inbred lines (RILs) generated from two populations of Drosophila serrata that have diverged in response to natural selection, revealed genetic divergence in 10.6% of 3762 gene products examined. Over 97% of the genetic divergence in transcript abundance was explained by only 12 genetic modules. The two most important modules, explaining 50% of the genetic variance in transcript abundance, were genetically correlated with the morphological traits that are known to be under selection. The expression of three candidate genes from these two important genetic modules was assessed in an independent experiment using qRT-PCR on 430 individuals from the panel of RILs, and confirmed the genetic association between transcript abundance and morphological traits under selection.

"

Cryptic genetic variation promotes rapid evolutionary adaptation in anRNA enzyme

Cryptic genetic variation promotes rapid evolutionary adaptation in an<br>RNA enzyme: "

Cryptic genetic variation promotes rapid evolutionary adaptation in an
RNA enzyme

Nature 474, 7349 (2011). doi:10.1038/nature10083

Authors: Eric J. Hayden, Evandro Ferrada & Andreas Wagner

Cryptic variation is caused by the robustness of phenotypes to mutations. Cryptic variation has no effect on phenotypes in a given genetic
or environmental background, but it can have effects after mutations or environmental
change. Because evolutionary adaptation by natural
selection requires phenotypic variation, phenotypically revealed cryptic genetic
variation may facilitate evolutionary adaptation. This
is possible if the cryptic variation happens to be pre-adapted, or “exapted”, to a new environment, and is thus advantageous once revealed.
However, this facilitating role for cryptic variation has not been proven,
partly because most pertinent work focuses on complex phenotypes of whole
organisms whose genetic basis is incompletely understood. Here we show that
populations of RNA enzymes with accumulated cryptic variation adapt more rapidly
to a new substrate than a population without cryptic variation. A detailed
analysis of our evolving RNA populations in genotype space shows that cryptic
variation allows a population to explore new genotypes that become adaptive
only in a new environment. Our observations show that cryptic variation contains
new genotypes pre-adapted to a changed environment. Our results highlight
the positive role that robustness and epistasis can have in adaptive evolution.

"

High sensitivity to aligner and high rate of false positives in the estimates of positive selection in the 12 Drosophila genomes [RESEARCH]

High sensitivity to aligner and high rate of false positives in the estimates of positive selection in the 12 Drosophila genomes [RESEARCH]: "

We investigate the effect of aligner choice on inferences of positive selection using site-specific models of molecular evolution. We find that independently of the choice of aligner, the rate of false positives is unacceptably high. Our study is a whole-genome analysis of all protein-coding genes in 12 Drosophila genomes annotated in either all 12 species (~6690 genes) or in the six melanogaster group species. We compare six popular aligners: PRANK, T-Coffee, ClustalW, ProbCons, AMAP, and MUSCLE, and find that the aligner choice strongly influences the estimates of positive selection. Differences persist when we use (1) different stringency cutoffs, (2) different selection inference models, (3) alignments with or without gaps, and/or additional masking, (4) per-site versus per-gene statistics, (5) closely related melanogaster group species versus more distant 12 Drosophila genomes. Furthermore, we find that these differences are consequential for downstream analyses such as determination of over/under-represented GO terms associated with positive selection. Visual analysis indicates that most sites inferred as positively selected are, in fact, misaligned at the codon level, resulting in false positive rates of 48%–82%. PRANK, which has been reported to outperform other aligners in simulations, performed best in our empirical study as well. Unfortunately, PRANK still had a high, and unacceptable for most applications, false positives rate of 50%–55%. We identify misannotations and indels, many of which appear to be located in disordered protein regions, as primary culprits for the high misalignment-related error levels and discuss possible workaround approaches to this apparently pervasive problem in genome-wide evolutionary analyses.

"

Natural genetic variation caused by small insertions and deletions in the human genome [RESEARCH]

Natural genetic variation caused by small insertions and deletions in the human genome [RESEARCH]: "

Human genetic variation is expected to play a central role in personalized medicine. Yet only a fraction of the natural genetic variation that is harbored by humans has been discovered to date. Here we report almost 2 million small insertions and deletions (INDELs) that range from 1 bp to 10,000 bp in length in the genomes of 79 diverse humans. These variants include 819,363 small INDELs that map to human genes. Small INDELs frequently were found in the coding exons of these genes, and several lines of evidence indicate that such variation is a major determinant of human biological diversity. Microarray-based genotyping experiments revealed several interesting observations regarding the population genetics of small INDEL variation. For example, we found that many of our INDELs had high levels of linkage disequilibrium (LD) with both HapMap SNPs and with high-scoring SNPs from genome-wide association studies. Overall, our study indicates that small INDEL variation is likely to be a key factor underlying inherited traits and diseases in humans.

"

Genetic basis of wing morphogenesis in Drosophila: sexual dimorphism and non-allometric effects of shape variation

Genetic basis of wing morphogenesis in Drosophila: sexual dimorphism and non-allometric effects of shape variation: "Background:
The Drosophila wing represents a particularly appropriate model to investigate the developmental control of phenotypic variation. Previous studies which aimed to identify candidate genes for wing morphology demonstrated that the genetic basis of wing shape variation in D. melanogaster is composed of numerous genetic factors causing small, additive effects. In this study, we analyzed wing shape in males and females from 191 lines of D. melanogaster, homozygous for a single P-element insertion, using geometric morphometrics techniques. The analysis allowed us to identify known and novel candidate genes that may contribute to the expression of wing shape in each sex separately and to compare them to candidate genes affecting wing size which have been identified previously using the same lines.
Results:
Our results indicate that more than 63% of induced mutations affected wing shape in one or both sexes, although only 33% showed significant differences in both males and females. The joint analysis of wing size and shape revealed that only 19% of the P-element insertions caused coincident effects on both components of wing form in one or both sexes. Further morphometrical analyses revealed that the intersection between veins showed the smallest displacements in the proximal region of the wing. Finally, we observed that mutations causing general deformations were more common than expected in both sexes whereas the opposite occurred with those generating local changes. For most of the 94 candidate genes identified, this seems to be the first record relating them with wing shape variation.
Conclusions:
Our results support the idea that the genetic architecture of wing shape is complex with many different genes contributing to the trait in a sexually dimorphic manner. This polygenic basis, which is relatively independent from that of wing size, is composed of genes generally involved in development and/or metabolic functions, especially related to the regulation of different cellular processes such as motility, adhesion, communication and signal transduction. This study suggests that understanding the genetic basis of wing shape requires merging the regulation of vein patterning by signalling pathways with processes that occur during wing development at the cellular level."