Identification of Genomic Alterations Through Multilevel DNA Structural Analysis.

Current methods to identify genomic alterations using whole-genome sequencing (WGS) data are often limited to single nucleotide polymorphisms and insertions and deletions that are less than 10 bp in length. These limitations are largely due to challenges in accurately mapping short sequencing reads that significantly diverge from the reference genome. Newer sequencing-based methods have been developed to define and characterize larger DNA structural elements. This is achieved by enriching for and sequencing regions of the genome that contain a specific element, followed by identifying genomic regions with high densities of mapped short reads that designate the location of these elements. This process essentially aggregates short read data into larger structural units for further characterization. Here, we describe protocols for identifying various types of genomic alterations using differential analysis of these structural units. We focus on changes in DNA accessibility, protein-DNA interactions, and chromosomal contacts as measured by ATAC-Seq, ChIP-Seq, and Hi-C respectively. As many protocols have been published describing the generation and processing of these data, we focus on simple methods that can be used to identify mutations in these data, and can be executed by someone with limited computational expertise.

Agnostic detection of genomic alterations by holistic DNA structural interrogation.

There is an established relationship between primary DNA sequence, secondary and tertiary chromatin structure, and transcriptional activity, suggesting that observed differences in one of these properties may reflect changes in the others. Here, we exploit these relationships to show that variations in DNA structure can be used to identify a wide range of genomic alterations in mammalian samples. In this proof-of-concept study we characterized and compared genome-wide histone occupancy by ChIP-Seq, DNA accessibility by ATAC-Seq, and chromosomal conformation by Hi-C for five CRISPR/Cas9-modified mammalian cell lines and their unmodified parent strains, as well as in one modified tissue sample and its parent strain. The results showed that the impact of genomic alterations on each of the levels of DNA organization varied depending on mutation type (insertion or deletion), size, and genomic location. The largest genomic alterations we identified included chromosomal rearrangements and deletions (greater than 200 Kb) in four of the modified cell lines, which can be difficult to identify by standard whole genome sequencing analysis. This multi-level DNA organizational analysis provides a sensitive approach for identifying a wide range of genomic and epigenomic perturbations that can be utilized for biomedical and biosecurity applications.

Redox crisis underlies conditional light-dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA.

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light-dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day.

The essential gene set of a photosynthetic organism.

Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼ 250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism's 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA(Leu), which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism's physiology and defines the essential gene set required for the growth of a photosynthetic organism.

Giving Time Purpose: The Synechococcus elongatus Clock in a Broader Network Context.

Early research on the cyanobacterial clock focused on characterizing the genes needed to keep, entrain, and convey time within the cell. As the scope of assays used in molecular genetics has expanded to capture systems-level properties (e.g., RNA-seq, ChIP-seq, metabolomics, high-throughput screening of genetic variants), so has our understanding of how the clock fits within and influences a broader cellular context. Here we review the work that has established a global perspective of the clock, with a focus on (a) an emerging network-centric view of clock architecture, (b) mechanistic insights into how temporal and environmental cues are transmitted and integrated within this network,

High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

Single mutations in sasA enable a simpler ΔcikA gene network architecture with equivalent circadian properties.

The circadian input kinase of the cyanobacterium Synechococcus elongatus PCC 7942 (CikA) is important both for synchronizing circadian rhythms with external environmental cycles and for transferring temporal information between the oscillator and the global transcriptional regulator RpaA (regulator of phycobilisome-associated A). KOs of cikA result in one of the most severely altered but still rhythmic circadian phenotypes observed. We chemically mutagenized a cikA-null S. elongatus strain and screened for second-site suppressor mutations that could restore normal circadian rhythms. We identified two independent mutations in the Synechococcus adaptive sensor A (sasA) gene that produce nearly WT rhythms of gene expression, likely because they compensate for the loss of CikA on the temporal phosphorylation of RpaA. Additionally, these mutations restore the ability to reset the clock after a short dark pulse through an output-independent pathway, suggesting that SasA can influence entrainment through direct interactions with KaiC, a property previously unattributed to it. These experiments question the evolutionary advantage of integrating CikA into the cyanobacterial clock, challenge the conventional construct of separable input and output pathways, and show how easily the cell can adapt to restore phenotype in a severely compromised genetic network.

Probing the informational and regulatory plasticity of a transcription factor DNA-binding domain.

Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinity that appropriately modulates the rate of transcription. Since both are determined by the biophysical properties of the DNA-binding domain, selection on one will ultimately affect the other. We were interested in understanding how plastic the informational and regulatory properties of a transcription factor are and how transcription factors evolve to balance these constraints. To study this, we developed an in vivo selection system in Escherichia coli to identify variants of the helix-turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selections were done within the context of the initiation complex, selecting for both specific binding within the genome and for a physiologically significant strength of interaction to maintain function of the factor. Using MITOMI, quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities could be accessed with a few mutations, suggesting that transcriptional regulatory networks are highly adaptable and expandable.

Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay.

RNA levels are determined by the rates of both transcription and decay, and a mechanistic understanding of the complex networks regulating gene expression requires methods that allow dynamic measurements of transcription and decay in living cells with minimal perturbation. Here, we describe a metabolic pulse-chase labeling protocol using 4-thiouracil combined with large-scale RNA sequencing to determine decay rates of all mRNAs in Saccharomyces cerevisiae. Profiling in various growth and stress conditions reveals that mRNA turnover is highly regulated both for specific groups of transcripts and at the system-wide level. For example, acute glucose starvation induces global mRNA stabilization but increases the degradation of all 132 detected ribosomal protein mRNAs. This effect is transient and can be mimicked by inhibiting the target-of-rapamycin kinase. Half-lives of mRNAs critical for galactose (GAL) metabolism are also highly sensitive to changes in carbon source. The fast reduction of GAL transcripts in glucose requires their dramatically enhanced turnover, highlighting the importance of mRNA decay in the control of gene expression. The approach described here provides a general platform for the global analysis of mRNA turnover and transcription and can be applied to dissect gene expression programs in a wide range of organisms and conditions.

The fitness landscapes of cis-acting binding sites in different promoter and environmental contexts.

The biophysical nature of the interaction between a transcription factor and its target sequences in vitro is sufficiently well understood to allow for the effects of DNA sequence alterations on affinity to be predicted. But even in relatively simple in vivo systems, the complexities of promoter organization and activity have made it difficult to predict how altering specific interactions between a transcription factor and DNA will affect promoter output. To better understand this, we measured the relative fitness of nearly all Escherichia coli sigma(70) -35 binding sites in different promoter and environmental contexts by competing four randomized -35 promoter libraries controlling the expression of the tetracycline resistance gene (tet)against each other in increasing concentrations of drug. We sequenced populations after competition to determine the relative enrichment of each -35 sequence. We observed a consistent relationship between the frequency of recovery of each -35 binding site and its predicted affinity for sigma(70) that varied depending on the sequence context of the promoter and drug concentration. Overall the relative fitness of each promoter could be predicted by a simple thermodynamic model of transcriptional regulation, in which the rate of transcriptional initiation (and hence fitness) is dependent upon the overall stability of the initiation complex, which in turn is dependent upon the energetic contributions of all sites within the complex. As implied by this model, a decrease in the free energy of association at one site could be compensated for by an increase in the binding energy at another to produce a similar output. Furthermore, these data show that a large and continuous range of transcriptional outputs can be accessed by merely changing the -35, suggesting that evolved or engineered mutations at this site could allow for subtle and precise control over gene expression.

Determining physical constraints in transcriptional initiation complexes using DNA sequence analysis.

Eukaryotic gene expression is often under the control of cooperatively acting transcription factors whose binding is limited by structural constraints. By determining these structural constraints, we can understand the "rules" that define functional cooperativity. Conversely, by understanding the rules of binding, we can infer structural characteristics. We have developed an information theory based method for approximating the physical limitations of cooperative interactions by comparing sequence analysis to microarray expression data. When applied to the coordinated binding of the sulfur amino acid regulatory protein Met4 by Cbf1 and Met31, we were able to create a combinatorial model that can correctly identify Met4 regulated genes. Interestingly, we found that the major determinant of Met4 regulation was the sum of the strength of the Cbf1 and Met31 binding sites and that the energetic costs associated with spacing appeared to be minimal.

Discovery of Fur binding site clusters in Escherichia coli by information theory models.

Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints ( approximately 83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur-DNA recognition mechanism may be conserved for even distantly related bacteria.

Correlation between binding rate constants and individual information of E. coli Fis binding sites.

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.

Anatomy of Escherichia coli sigma70 promoters.

Information theory was used to build a promoter model that accounts for the -10, the -35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base -7, rather than -11, of the -10 may be flipping to initiate transcription. We found that the sequence conservation of sigma70 binding sites is 6.5 +/- 0.1 bits. Some promoters lack a -35 region, but have a 6.7 +/- 0.2 bit extended -10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended -10 binding and the -35 suggest that the flexible bipartite sigma factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5' mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the -35, so bending allows distal activators to reach the polymerase.

Flexible promoter architecture requirements for coactivator recruitment.

BACKGROUND: The spatial organization of transcription factor binding sites in regulatory DNA, and the composition of intersite sequences, influences the assembly of the multiprotein complexes that regulate RNA polymerase recruitment and thereby affects transcription. We have developed a genetic approach to investigate how reporter gene transcription is affected by varying the spacing between transcription factor binding sites. We characterized the components of promoter architecture that govern the yeast transcription factors Cbf1 and Met31/32, which bind independently, but collaboratively recruit the coactivator Met4. RESULTS: A Cbf1 binding site was required upstream of a Met31/32 binding site for full reporter gene expression. Distance constraints on coactivator recruitment were more flexible than those for cooperatively binding transcription factors. Distances from 18 to 50 bp between binding sites support efficient recruitment of Met4, with only slight modulation by helical phasing. Intriguingly, we found that certain sequences located between the binding sites abolished gene expression. CONCLUSION: These results yield insight to the influence of both binding site architecture and local DNA flexibility on gene expression, and can be used to refine computational predictions of gene expression from promoter sequences. In addition, our approach can be applied to survey promoter architecture requirements for arbitrary combinations of transcription factor binding sites.

The C-terminal domain of DNA gyrase A adopts a DNA-bending beta-pinwheel fold.

DNA gyrase is unique among enzymes for its ability to actively introduce negative supercoils into DNA. This function is mediated in part by the C-terminal domain of its A subunit (GyrA CTD). Here, we report the crystal structure of this approximately 35-kDa domain determined to 1.75-A resolution. The GyrA CTD unexpectedly adopts an unusual fold, which we term a beta-pinwheel, that is globally reminiscent of a beta-propeller but is built of blades with a previously unobserved topology. A large, conserved basic patch on the outer edge of this domain suggests a likely site for binding and bending DNA; fluorescence resonance energy transfer-based assays show that the GyrA CTD is capable of bending DNA by > or =180 degrees over a 40-bp region. Surprisingly, we find that the CTD of the topoisomerase IV A subunit, which shares limited sequence homology with the GyrA CTD, also bends DNA. Together, these data provide a physical explanation for the ability of DNA gyrase to constrain a positive superhelical DNA wrap, and also suggest that the particular substrate preferences of topoisomerase IV might be dictated in part by the function of this domain.