Integrated omics analyses of retrograde signaling mutant delineate interrelated stress-response strata.

To maintain homeostasis in the face of intrinsic and extrinsic insults, cells have evolved elaborate quality control networks to resolve damage at multiple levels. Interorganellar communication is a key requirement for this maintenance, however the underlying mechanisms of this communication have remained an enigma. Here we integrate the outcome of transcriptomic, proteomic, and metabolomics analyses of genotypes including ceh1, a mutant with constitutively elevated levels of both the stress-specific plastidial retrograde signaling metabolite methyl-erythritol cyclodiphosphate (MEcPP) and the defense hormone salicylic acid (SA), as well as the high MEcPP but SA deficient genotype ceh1/eds16, along with corresponding controls. Integration of multi-omic analyses enabled us to delineate the function of MEcPP from SA, and expose the compartmentalized role of this retrograde signaling metabolite in induction of distinct but interdependent signaling cascades instrumental in adaptive responses. Specifically, here we identify strata of MEcPP-sensitive stress-response cascades, among which we focus on selected pathways including organelle-specific regulation of jasmonate biosynthesis; simultaneous induction of synthesis and breakdown of SA; and MEcPP-mediated alteration of cellular redox status in particular glutathione redox balance. Collectively, these integrated multi-omic analyses provided a vehicle to gain an in-depth knowledge of genome-metabolism interactions, and to further probe the extent of these interactions and delineate their functional contributions. Through this approach we were able to pinpoint stress-mediated transcriptional and metabolic signatures and identify the downstream processes modulated by the independent or overlapping functions of MEcPP and SA in adaptive responses.

SIST: stress-induced structural transitions in superhelical DNA.

SUMMARY: Supercoiling imposes stress on a DNA molecule that can drive susceptible sequences into alternative non-B form structures. This phenomenon occurs frequently in vivo and has been implicated in biological processes, such as replication, transcription, recombination and translocation. SIST is a software package that analyzes sequence-dependent structural transitions in kilobase length superhelical DNA molecules. The numerical algorithms in SIST are based on a statistical mechanical model that calculates the equilibrium probability of transition for each base pair in the domain. They are extensions of the original stress-induced duplex destabilization (SIDD) method, which analyzes stress-driven DNA strand separation. SIST also includes algorithms to analyze B-Z transitions and cruciform extrusion. The SIST pipeline has an option to use the DZCBtrans algorithm, which analyzes the competition among these three transitions within a superhelical domain. AVAILABILITY AND IMPLEMENTATION: The package and additional documentation are freely available at https://bitbucket.org/benhamlab/sist_codes. CONTACT: dzhabinskaya@ucdavis.edu.

Potential non-B DNA regions in the human genome are associated with higher rates of nucleotide mutation and expression variation.

While individual non-B DNA structures have been shown to impact gene expression, their broad regulatory role remains elusive. We utilized genomic variants and expression quantitative trait loci (eQTL) data to analyze genome-wide variation propensities of potential non-B DNA regions and their relation to gene expression. Independent of genomic location, these regions were enriched in nucleotide variants. Our results are consistent with previously observed mutagenic properties of these regions and counter a previous study concluding that G-quadruplex regions have a reduced frequency of variants. While such mutagenicity might undermine functionality of these elements, we identified in potential non-B DNA regions a signature of negative selection. Yet, we found a depletion of eQTL-associated variants in potential non-B DNA regions, opposite to what might be expected from their proposed regulatory role. However, we also observed that genes downstream of potential non-B DNA regions showed higher expression variation between individuals. This coupling between mutagenicity and tolerance for expression variability of downstream genes may be a result of evolutionary adaptation, which allows reconciling mutagenicity of non-B DNA structures with their location in functionally important regions and their potential regulatory role.

Competitive superhelical transitions involving cruciform extrusion.

A DNA molecule under negative superhelical stress becomes susceptible to transitions to alternate structures. The accessible alternate conformations depend on base sequence and compete for occupancy. We have developed a method to calculate equilibrium distributions among the states available to such systems, as well as their average thermodynamic properties. Here we extend this approach to include superhelical cruciform extrusion at both perfect and imperfect inverted repeat (IR) sequences. We find that short IRs do not extrude cruciforms, even in the absence of competition. But as the length of an IR increases, its extrusion can come to dominate both strand separation and B-Z transitions. Although many IRs are present in human genomic DNA, we find that extrusion-susceptible ones occur infrequently. Moreover, their avoidance of transcription start sites in eukaryotes suggests that cruciform formation is rarely involved in mechanisms of gene regulation. We examine a set of clinically important chromosomal translocation breakpoints that occur at long IRs, whose rearrangement has been proposed to be driven by cruciform extrusion. Our results show that the susceptibilities of these IRs to cruciform formation correspond closely with their observed translocation frequencies.

Theoretical analysis of competing conformational transitions in superhelical DNA.

We develop a statistical mechanical model to analyze the competitive behavior of transitions to multiple alternate conformations in a negatively supercoiled DNA molecule of kilobase length and specified base sequence. Since DNA superhelicity topologically couples together the transition behaviors of all base pairs, a unified model is required to analyze all the transitions to which the DNA sequence is susceptible. Here we present a first model of this type. Our numerical approach generalizes the strategy of previously developed algorithms, which studied superhelical transitions to a single alternate conformation. We apply our multi-state model to study the competition between strand separation and B-Z transitions in superhelical DNA. We show this competition to be highly sensitive to temperature and to the imposed level of supercoiling. Comparison of our results with experimental data shows that, when the energetics appropriate to the experimental conditions are used, the competition between these two transitions is accurately captured by our algorithm. We analyze the superhelical competition between B-Z transitions and denaturation around the c-myc oncogene, where both transitions are known to occur when this gene is transcribing. We apply our model to explore the correlation between stress-induced transitions and transcriptional activity in various organisms. In higher eukaryotes we find a strong enhancement of Z-forming regions immediately 5' to their transcription start sites (TSS), and a depletion of strand separating sites in a broad region around the TSS. The opposite patterns occur around transcript end locations. We also show that susceptibility to each type of transition is different in eukaryotes and prokaryotes. By analyzing a set of untranscribed pseudogenes we show that the Z-susceptibility just downstream of the TSS is not preserved, suggesting it may be under selection pressure.

Theoretical analysis of the stress induced B-Z transition in superhelical DNA.

We present a method to calculate the propensities of regions within a DNA molecule to transition from B-form to Z-form under negative superhelical stresses. We use statistical mechanics to analyze the competition that occurs among all susceptible Z-forming regions at thermodynamic equilibrium in a superhelically stressed DNA of specified sequence. This method, which we call SIBZ, is similar to the SIDD algorithm that was previously developed to analyze superhelical duplex destabilization. A state of the system is determined by assigning to each base pair either the B- or the Z-conformation, accounting for the dinucleotide repeat unit of Z-DNA. The free energy of a state is comprised of the nucleation energy, the sequence-dependent B-Z transition energy, and the energy associated with the residual superhelicity remaining after the change of twist due to transition. Using this information, SIBZ calculates the equilibrium B-Z transition probability of each base pair in the sequence. This can be done at any physiologically reasonable level of negative superhelicity. We use SIBZ to analyze a variety of representative genomic DNA sequences. We show that the dominant Z-DNA forming regions in a sequence can compete in highly complex ways as the superhelicity level changes. Despite having no tunable parameters, the predictions of SIBZ agree precisely with experimental results, both for the onset of transition in plasmids containing introduced Z-forming sequences and for the locations of Z-forming regions in genomic sequences. We calculate the transition profiles of 5 kb regions taken from each of 12,841 mouse genes and centered on the transcription start site (TSS). We find a substantial increase in the frequency of Z-forming regions immediately upstream from the TSS. The approach developed here has the potential to illuminate the occurrence of Z-form regions in vivo, and the possible roles this transition may play in biological processes.