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Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Here, we explored DNA methylation in bacteria of the genus Ensifer, including its potential role in regulating terminal differentiation during nitrogen-fixing symbiosis with legumes. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cell cultures, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent of methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. Based on these observations, we suggest that CcrM activity is dysregulated and constitutive during terminal differentiation, which we hypothesize is a driving factor for endoreduplication of terminally differentiated bacteroids. IMPORTANCE Nitrogen fixation by rhizobia in symbiosis with legumes is economically and ecologically important. The symbiosis can involve a complex bacterial transformation—terminal differentiation—that includes major shifts in the transcriptome and cell cycle. Epigenetic regulation is an important regulatory mechanism in diverse bacteria; however, the roles of DNA methylation in rhizobia and symbiotic nitrogen fixation have been poorly investigated. We show that aside from cell cycle regulation, DNA methyltransferases are unlikely to have conserved roles in the biology of bacteria of the genus Ensifer. However, we present evidence consistent with an interpretation that the cell cycle methyltransferase CcrM is dysregulated during symbiosis, which we hypothesize may be a key factor driving the cell cycle switch in terminal differentiation required for effective symbioses.

Here, we report the restriction modification system of Nostoc punctiforme ATCC 29133, along with its methylated genome sequence, under contrasting nitrate availability. Generated methylation profiles revealed increased methylation for key enzymes of assimilatory nitrate reduction, suggesting that Nostoc punctiforme employs DNA methylation to regulate its nitrogen metabolism.

Significance Understanding the molecular response of plants to drought is critical to efforts to improve agricultural yields under increasingly frequent droughts. We grew 2 cultivars of the naturally drought-tolerant food crop sorghum in the field under drought stress. We sequenced the mRNA from weekly samples of these plants, resulting in a molecular profile of drought response over the growing season. We find molecular differences in the 2 cultivars that help explain their differing tolerances to drought and evidence of a disruption in the plant’s symbiosis with arbuscular mycorrhizal fungi. Our findings are of practical importance for agricultural breeding programs, while the resulting data are a resource for the plant and microbial communities for studying the dynamics of drought response., Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants’ roots.

Author(s): Rajderkar, Sudha; Barozzi, Iros; Zhu, Yiwen; Hu, Rong; Zhang, Yanxiao; Li, Bin; Fukuda-Yuzawa, Yoko; Kelman, Guy; Akeza, Adyam; Blow, Matthew; Pham, Quan; Harrington, Anne; Godoy, Janeth; Meky, Eman; von Maydell, Kianna; Novak, Catherine; Plajzer-Frick, Ingrid; Afzal, Veena; Tran, Stella; Talkowski, Michael; Kent Lloyd, KC; Ren, Bing; Dickel, Diane; Visel, Axel; Pennacchio, Len | Abstract: Summary Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype 1–3 , but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo . We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

Significance Microorganisms have evolved signaling networks to identify and prioritize utilization of carbon sources. For fungi that degrade plant biomass, such as Neurospora crassa, signaling networks dictate the metabolic response to carbon sources present in plant cell walls, resulting in optimal utilization of nutrient sources. However, within a fungal colony, regulatory hierarchies associated with activation of transcription factors and temporal and spatial production of proteins for plant biomass utilization are unclear. Here, we perform expression profiling of N. crassa on simple sugars to complex carbohydrates to identify regulatory factors and direct targets of regulatory transcription factors using DNA affinity purification sequencing (DAP-seq). These findings will enable more precise tailoring of metabolic networks in filamentous fungi for the production of second-generation biofuels., Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

Sharing of bioinformatics data within research communities holds the promise of facilitating more rapid discovery, yet the volume of data is growing at a pace exponentially greater than what traditional biocuration can support. We present here an approach that we have used to empower data producing researchers to curate high quality shared data that is ready for reuse and re-analysis.

Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems., Microorganisms that release plant-available phosphate from natural soil phosphate stores may serve as biological alternatives to costly and environmentally damaging phosphate fertilizers. To explore this possibility, we engineered a collection of root bacteria to release plant-available orthophosphate from phytate, an abundant phosphate source in many soils. We identified 82 phylogenetically diverse phytase genes, refactored their sequences for optimal expression in Proteobacteria, and then synthesized and engineered them into the genomes of three root-colonizing bacteria. Liquid culture assays revealed 41 engineered strains with high levels of phytate hydrolysis. Among these, we identified 12 strains across three bacterial hosts that confer a growth advantage on the model plant Arabidopsis thaliana when phytate is the sole phosphate source. These data demonstrate that DNA synthesis approaches can be used to generate plant-associated strains with novel phosphate-solubilizing capabilities. IMPORTANCE Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems.

In previous work (D. R. Harris et al., J Bacteriol 191:5240–5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans. After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans. Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident. IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus. Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.

Author(s): Spurrell, Cailyn; Barozzi, Iros; Mannion, Brandon; Blow, Matthew; Fukuda-Yuzawa, Yoko; Afzal, Sarah; Akiyama, Jennifer; Afzal, Veena; Tran, Stella; Plajzer-Frick, Ingrid; Novak, Catherine; Kato, Momoe; Lee, Elizabeth; Garvin, Tyler; Pham, Quan; Harrington, Anne; Lisgo, Steven; Bristow, James; Cappola, Thomas; Morley, Michael; Margulies, Kenneth; Pennacchio, Len; Dickel, Diane; Visel, Axel | Abstract: Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the genome-wide regulatory landscape in heart disease has remained obscure. Here we show that pervasive epigenomic changes occur in heart disease, with thousands of regulatory sequences reacquiring fetal-like chromatin signatures. We used RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from 18 healthy controls and 18 individuals with idiopathic dilated cardiomyopathy (DCM). Healthy individuals had a highly reproducible epigenomic landscape, consisting of more than 31,000 predicted heart enhancers. In contrast, we observed reproducible disease-associated gains or losses of activity at more than 7,500 predicted heart enhancers. Next, we profiled human fetal heart tissue by ChIP-seq and RNA-seq. Comparison with adult tissues revealed that the heart disease epigenome and transcrip-tome both shift toward a fetal-like state, with 3,400 individual enhancers sharing fetal regulatory properties. Our results demonstrate widespread epigenomic changes in DCM, and we provide a comprehensive data resource ( http://heart.lbl.gov ) for the mechanistic exploration of heart disease etiology.

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.

Background Switchgrass (Panicum virgatum L.) is an important bioenergy crop widely used for lignocellulosic research. While extensive transcriptomic analyses have been conducted on this species using short read-based sequencing techniques, very little has been reliably derived regarding alternatively spliced (AS) transcripts. Results We present an analysis of transcriptomes of six switchgrass tissue types pooled together, sequenced using Pacific Biosciences (PacBio) single-molecular long-read technology. Our analysis identified 105,419 unique transcripts covering 43,570 known genes and 8795 previously unknown genes. 45,168 are novel transcripts of known genes. A total of 60,096 AS transcripts are identified, 45,628 being novel. We have also predicted 1549 transcripts of genes involved in cell wall construction and remodeling, 639 being novel transcripts of known cell wall genes. Most of the predicted transcripts are validated against Illumina-based short reads. Specifically, 96% of the splice junction sites in all the unique transcripts are validated by at least five Illumina reads. Comparisons between genes derived from our identified transcripts and the current genome annotation revealed that among the gene set predicted by both analyses, 16,640 have different exon–intron structures. Conclusions Overall, substantial amount of new information is derived from the PacBio RNA data regarding both the transcriptome and the genome of switchgrass. Electronic supplementary material The online version of this article (10.1186/s13068-018-1167-z) contains supplementary material, which is available to authorized users.

© 2017, The Author(s). Cyanobacteria have the potential to produce bulk and fine chemicals and members belonging to Nostoc sp. have received particular attention due to their relatively fast growth rate and the relative ease with which they can be harvested. Nostoc punctiforme is an aerobic, motile, Gram-negative, filamentous cyanobacterium that has been studied intensively to enhance our understanding of microbial carbon and nitrogen fixation. The genome of the type strain N. punctiforme ATCC 29133 was sequenced in 2001 and the scientific community has used these genome data extensively since then. Advances in bioinformatics tools for sequence annotation and the importance of this organism prompted us to resequence and reanalyze its genome and to make both, the initial and improved annotation, available to the scientific community. The new draft genome has a total size of 9.1 Mbp and consists of 65 contiguous pieces of DNA with a GC content of 41.38% and 7664 protein-coding genes. Furthermore, the resequenced genome is slightly (5152 bp) larger and contains 987 more genes with functional prediction when compared to the previously published version. We deposited the annotation of both genomes in the Department of Energy’s IMG database to facilitate easy genome exploration by the scientific community without the need of in-depth bioinformatics skills. We expect that an facilitated access and ability to search the N. punctiforme ATCC 29133 for genes of interest will significantly facilitate metabolic engineering and genome prospecting efforts and ultimately the synthesis of biofuels and natural products from this keystone organism and closely related cyanobacteria.

© 2014 . The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue specific noncoding RNAs yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis we identified and characterized for the first time a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA sequencing demonstrates that many of these transcripts are polyadenylated multi exonic long noncoding RNAs. Moreover knockdown of two enhancer associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly the reactivation of the fetal gene program a hallmark of the stress response in the adult heart is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer derived lncRNAs. •Fetal cardiac enhancers are transcribed generating enhancer derived lncRNAs.

SummaryThe mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

SummaryThe function of nearly half of all protein-coding genes identified in bacterial genomes remains unknown. To systematically explore the functions of these proteins, we generated saturated transposon mutant libraries from 25 diverse bacteria and we assayed mutant phenotypes across hundreds of distinct conditions. From 3,903 genome-wide mutant fitness assays, we obtained 14.9 million gene phenotype measurements and we identified a mutant phenotype for 8,487 proteins with previously unknown functions. The majority of these hypothetical proteins (57%) had phenotypes that were either specific to a few conditions or were similar to that of another gene, thus enabling us to make informed predictions of protein function. For 1,914 of these hypothetical proteins, the functional associations are conserved across related proteins from different bacteria, which confirms that these associations are genuine. This comprehensive catalogue of experimentally-annotated protein functions also enables the targeted exploration of specific biological processes. For example, sensitivity to a DNA-damaging agent revealed 28 known families of DNA repair proteins and 11 putative novel families. Across all sequenced bacteria, 14% of proteins that lack detailed annotations have an ortholog with a functional association in our data set. Our study demonstrates the utility and scalability of high-throughput genetics for large-scale annotation of bacterial proteins and provides a vast compendium of experimentally-determined protein functions across diverse bacteria.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems., Author Summary DNA methylation is a chemical modification of DNA present in many prokaryotic genomes. The best-known role of DNA methylation is as a component of restriction-modification systems. In these systems, restriction enzymes target foreign DNA for cleavage, while DNA methylation protects the host genome from destruction. Studies in a handful of organisms show that DNA methylation may also act independently of restriction systems and function in genome regulation. However, a lack of technologies has limited the study of DNA methylation to a small number of organisms, and the broader patterns and functions of DNA methylation remain unknown. Here we use SMRT-sequencing to determine the genome wide DNA methylation patterns of more than 200 diverse bacteria and archaea. We show that DNA methylation is pervasive and present in more than 90% of studied organisms. Analysis of this data enabled annotation of the specific DNA binding sites of more than 600 restriction systems, revealing their extraordinary diversity. Strikingly, we observed widespread DNA methylation in the absence of restriction systems. Analyses of these patterns reveal that they are conserved through evolution, and likely function in genome regulation. Thus DNA methylation may play a far wider function in prokaryotic genome biology than was previously supposed.

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here we present the results of chromatin immunoprecipitation with the enhancer-associated protein p300 followed by massively parallel sequencing, and map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases demonstrated reproducible enhancer activity in the tissues that were predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities, and suggest that such data sets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.

The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters ( rsaF a and rsaF b ), a stress-responsive transcription factor ( cztR ), or a ppGpp synthetase/hydrolase ( spoT ) exhibited a significantly lower survival rate under U stress. RsaF a and RsaF b , which are homologues of TolC in Escherichia coli , have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaF a and rsaF b by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaF a mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaF a and RsaF b in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus . IMPORTANCE Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus . The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus . In particular, we show that outer membrane transporters RsaF a and RsaF b , previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling., IMPORTANCE A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.

The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development. ispartof: GENOME RESEARCH vol:24 issue:6 pages:920-929 ispartof: location:United States status: published

We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli . Furthermore, comparative analyses suggest that many Gammaproteobacteria , including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.

Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers. To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ∼6,200 candidate enhancer sequences directly from fetal and adult human heart tissue. Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease. To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart. These results support the discovery of a genome-wide set of non-coding sequences highly enriched in human heart enhancers which is likely to facilitate down-stream studies of the role of enhancers in development and pathological conditions of the heart.

Accurate control of tissue-specific gene expression plays a pivotal role in heart development, but few cardiac transcriptional enhancers have thus far been identified. Extreme noncoding-sequence conservation has successfully predicted enhancers that are active in many tissues but has failed to identify substantial numbers of heart-specific enhancers. Here, we used ChIP-Seq with the enhancer-associated protein p300 from mouse embryonic day 11.5 heart tissue to identify over 3,000 candidate heart enhancers genome wide. Compared to enhancers active in other tissues we studied at this time point, most candidate heart enhancers were less deeply conserved in vertebrate evolution. Nevertheless, transgenic mouse assays of 130 candidate regions revealed that most function reproducibly as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for a large population of poorly conserved heart enhancers and suggest that the evolutionary conservation of embryonic enhancers can vary depending on tissue type.

Sequence polymorphisms in a 58-kilobase (kb) interval on chromosome 9p21 confer a markedly increased risk of coronary artery disease (CAD), the leading cause of death worldwide. The variants have a substantial effect on the epidemiology of CAD and other life-threatening vascular conditions because nearly one-quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein-coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70-kb non-coding interval on mouse chromosome 4 affects cardiac expression of neighbouring genes, as well as proliferation properties of vascular cells. Chr4(Delta70kb/Delta70kb) mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the non-coding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4(Delta70kb/Delta70kb) mice, indicating that distant-acting gene regulatory functions are located in the non-coding CAD risk interval. Allele-specific expression of Cdkn2b transcripts in heterozygous mice showed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4(Delta70kb/Delta70kb) aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval has a pivotal role in regulation of cardiac Cdkn2a/b expression, and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation. ispartof: NATURE vol:464 issue:7287 pages:409-U103 ispartof: location:England status: published

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates—a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

A survey of RNA editing of miRNAs from ten human tissues indicates that RNA editing increases the diversity of miRNAs and their targets., Background MicroRNAs (miRNAs) are short RNAs of around 22 nucleotides that regulate gene expression. The primary transcripts of miRNAs contain double-stranded RNA and are therefore potential substrates for adenosine to inosine (A-to-I) RNA editing. Results We have conducted a survey of RNA editing of miRNAs from ten human tissues by sequence comparison of PCR products derived from matched genomic DNA and total cDNA from the same individual. Six out of 99 (6%) miRNA transcripts from which data were obtained were subject to A-to-I editing in at least one tissue. Four out of seven edited adenosines were in the mature miRNA and were predicted to change the target sites in 3' untranslated regions. For a further six miRNAs, we identified A-to-I editing of transcripts derived from the opposite strand of the genome to the annotated miRNA. These miRNAs may have been annotated to the wrong genomic strand. Conclusion Our results indicate that RNA editing increases the diversity of miRNAs and their targets, and hence may modulate miRNA function.

We have conducted a survey of RNA editing in human brain by comparing sequences of clones from a human brain cDNA library to the reference human genome sequence and to genomic DNA from the same individual. In the RNA sample from which the library was constructed, ∼1:2000 nucleotides were edited out of >3 Mb surveyed. All edits were adenosine to inosine (A→I) and were predominantly in intronic and in intergenic RNAs. No edits were found in translated exons and few in untranslated exons. Most edits were in high-copy-number repeats, usually Alus. Analysis of the genome in the vicinity of edited sequences strongly supports the idea that formation of intramolecular double-stranded RNA with an inverted copy underlies most A→I editing. The likelihood of editing is increased by the presence of two inverted copies of a sequence within the same intron, proximity of the two sequences to each other (preferably within 2 kb), and by a high density of inverted copies in the vicinity. Editing exhibits sequence preferences and is less likely at an adenosine 3′ to a guanosine and more likely at an adenosine 5′ to a guanosine. Simulation by BLAST alignment of the double-stranded RNA molecules that underlie known edits indicates that there is a greater likelihood of A→I editing at A:C mismatches than editing at other mismatches or at A:U matches. However, because A:U matches in double-stranded RNA are more common than all mismatches, overall the likely effect of editing is to increase the number of mismatches in double-stranded RNA.

Introduction The shape of the face is one of the most distinctive features among humans, and differences in facial morphology have substantial implications in areas such as social interaction, psychology, forensics, and clinical genetics. Craniofacial shape is highly heritable, including the normal spectrum of morphological variation as well as susceptibility to major craniofacial birth defects. In this study, we explored the role of transcriptional enhancers in the development of the craniofacial complex. Our study is based on the rationale that such enhancers, which can be hundreds of kilobases away from their target genes, regulate the spatial patterns, levels, and timing of gene expression in normal development. Methods To identify distant-acting enhancers active during craniofacial development, we used chromatin immunoprecipitation on embryonic mouse face tissue followed by sequencing to identify noncoding genome regions bound by the enhancer-associated p300 protein. We used LacZ reporter assays in transgenic mice and optical projection tomography (OPT) to determine three-dimensional expression patterns of a subset of these candidate enhancers. Last, we deleted three of the craniofacial enhancers from the mouse genome to assess their effect on gene expression and craniofacial morphology during development. Results We identified more than 4000 candidate enhancer sequences predicted to be active in the developing craniofacial complex. The majority of these sequences are at least partially conserved between humans and mice, and many are located in chromosomal regions associated with normal facial morphology or craniofacial birth defects. Characterization of more than 200 candidate enhancer sequences in transgenic mice revealed a remarkable spatial complexity of in vivo expression patterns. Targeted deletions of three craniofacial enhancers near genes with known roles in craniofacial development resulted in changes of expression of those genes as well as quantitatively subtle but definable alterations of craniofacial shape. Discussion Our analysis identifies enhancers that fine tune expression of genes during craniofacial development in mice. These results support that variation in the sequence or copy number of craniofacial enhancers may contribute to the spectrum of facial variation we find in human populations. Because many craniofacial enhancers are located in genome regions associated with craniofacial birth defects, such as clefts of the lip and palate, our results also offer a starting point for exploring the contribution of noncoding sequences to these disorders.

Background: MicroRNAs (miRNAs) are short RNAs of around 22 nucleotides that regulate gene expression. The primary transcripts of miRNAs contain double-stranded RNA and are therefore potential substrates for adenosine to inosine (A-to-I) RNA editing. Results: We have conducted a survey of RNA editing of miRNAs from ten human tissues by sequence comparison of PCR products derived from matched genomic DNA and total cDNA from the same individual. Six out of 99 (6%) miRNA transcripts from which data were obtained were subject to A-to-I editing in at least one tissue. Four out of seven edited adenosines were in the mature miRNA and were predicted to change the target sites in 3' untranslated regions. For a further six miRNAs, we identified A-to-I editing of transcripts derived from the opposite strand of the genome to the annotated miRNA. These miRNAs may have been annotated to the wrong genomic strand. Conclusion: Our results indicate that RNA editing increases the diversity of miRNAs and their targets, and hence may modulate miRNA function.

Background Comprehensive annotation and quantification of transcriptomes are outstanding problems in functional genomics. While high throughput mRNA sequencing (RNA-Seq) has emerged as a powerful tool for addressing these problems, its success is dependent upon the availability and quality of reference genome sequences, thus limiting the organisms to which it can be applied. Results Here, we describe Rnnotator, an automated software pipeline that generates transcript models by de novo assembly of RNA-Seq data without the need for a reference genome. We have applied the Rnnotator assembly pipeline to two yeast transcriptomes and compared the results to the reference gene catalogs of these organisms. The contigs produced by Rnnotator are highly accurate (95%) and reconstruct full-length genes for the majority of the existing gene models (54.3%). Furthermore, our analyses revealed many novel transcribed regions that are absent from well annotated genomes, suggesting Rnnotator serves as a complementary approach to analysis based on a reference genome for comprehensive transcriptomics. Conclusions These results demonstrate that the Rnnotator pipeline is able to reconstruct full-length transcripts in the absence of a complete reference genome.