DNA methylation and gene expression regulation associated with vascularization in Sorghum bicolor.

Plant secondary cell walls constitute the majority of plant biomass. They are predominantly found in xylem cells, which are derived from vascular initials during vascularization. Little is known about these processes in grass species despite their emerging importance as biomass feedstocks. The targeted biofuel crop Sorghum bicolor has a sequenced and well-annotated genome, making it an ideal monocot model for addressing vascularization and biomass deposition. Here we generated tissue-specific transcriptome and DNA methylome data from sorghum shoots, roots and developing root vascular and nonvascular tissues. Many genes associated with vascular development in other species show enriched expression in developing vasculature. However, several transcription factor families varied in vascular expression in sorghum compared with Arabidopsis and maize. Furthermore, differential expression of genes associated with DNA methylation were identified between vascular and nonvascular tissues, implying that changes in DNA methylation are a feature of sorghum root vascularization, which we confirmed using tissue-specific DNA methylome data. Roots treated with a DNA methylation inhibitor also showed a significant decrease in root length. Tissues and organs can be discriminated based on their genomic methylation patterns and methylation context. Consequently, tissue-specific changes in DNA methylation are part of the normal developmental process.

Clinical Actionability of Comprehensive Genomic Profiling for Management of Rare or Refractory Cancers.

BACKGROUND: The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. METHODS: A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. RESULTS: Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0-10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. CONCLUSION: Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. IMPLICATIONS FOR PRACTICE: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents.

The Genomic Landscape of Renal Oncocytoma Identifies a Metabolic Barrier to Tumorigenesis.

Oncocytomas are predominantly benign neoplasms possessing pathogenic mitochondrial mutations and accumulation of respiration-defective mitochondria, characteristics of unknown significance. Using exome and transcriptome sequencing, we identified two main subtypes of renal oncocytoma. Type 1 is diploid with CCND1 rearrangements, whereas type 2 is aneuploid with recurrent loss of chromosome 1, X or Y, and/or 14 and 21, which may proceed to more aggressive eosinophilic chromophobe renal cell carcinoma (ChRCC). Oncocytomas activate 5' adenosine monophosphate-activated protein kinase (AMPK) and Tp53 (p53) and display disruption of Golgi and autophagy/lysosome trafficking, events attributed to defective mitochondrial function. This suggests that the genetic defects in mitochondria activate a metabolic checkpoint, producing autophagy impairment and mitochondrial accumulation that limit tumor progression, revealing a novel tumor-suppressive mechanism for mitochondrial inhibition with metformin. Alleviation of this metabolic checkpoint in type 2 by p53 mutations may allow progression to eosinophilic ChRCC, indicating that they represent higher risk.

Whole-genome sequencing analysis of phenotypic heterogeneity and anticipation in Li-Fraumeni cancer predisposition syndrome.

The Li-Fraumeni syndrome (LFS) and its variant form (LFL) is a familial predisposition to multiple forms of childhood, adolescent, and adult cancers associated with germ-line mutation in the TP53 tumor suppressor gene. Individual disparities in tumor patterns are compounded by acceleration of cancer onset with successive generations. It has been suggested that this apparent anticipation pattern may result from germ-line genomic instability in TP53 mutation carriers, causing increased DNA copy-number variations (CNVs) with successive generations. To address the genetic basis of phenotypic disparities of LFS/LFL, we performed whole-genome sequencing (WGS) of 13 subjects from two generations of an LFS kindred. Neither de novo CNV nor significant difference in total CNV was detected in relation with successive generations or with age at cancer onset. These observations were consistent with an experimental mouse model system showing that trp53 deficiency in the germ line of father or mother did not increase CNV occurrence in the offspring. On the other hand, individual records on 1,771 TP53 mutation carriers from 294 pedigrees were compiled to assess genetic anticipation patterns (International Agency for Research on Cancer TP53 database). No strictly defined anticipation pattern was observed. Rather, in multigeneration families, cancer onset was delayed in older compared with recent generations. These observations support an alternative model for apparent anticipation in which rare variants from noncarrier parents may attenuate constitutive resistance to tumorigenesis in the offspring of TP53 mutation carriers with late cancer onset.

Single temperature for Monte Carlo optimization on complex landscapes.

We propose a new strategy for Monte Carlo (MC) optimization on rugged multidimensional landscapes. The strategy is based on querying the statistical properties of the landscape in order to find the temperature at which the mean first passage time across the current region of the landscape is minimized. Thus, in contrast to other algorithms such as simulated annealing, we explicitly match the temperature schedule to the statistics of landscape irregularities. In cases where these statistics are approximately the same over the entire landscape or where nonlocal moves couple distant parts of the landscape, a single-temperature MC scheme outperforms any other MC algorithm with the same move set. We also find that in strongly anisotropic Coulomb spin glass and traveling salesman problems, the only relevant statistics (which we use to assign a single MC temperature) are those of irregularities in low-energy funnels. Our results may explain why protein folding is efficient at constant temperature.

Statistical mechanics of nucleosome ordering by chromatin-structure-induced two-body interactions.

One-dimensional arrays of nucleosomes (DNA-bound histone octamers separated by stretches of linker DNA) fold into higher-order chromatin structures which ultimately make up eukaryotic chromosomes. Chromatin structure formation leads to 10-11 base pair (bp) discretization of linker lengths caused by the smaller free energy cost of packaging nucleosomes into regular chromatin fibers if their rotational setting (defined by the DNA helical twist) is conserved. We describe nucleosome positions along the fiber using a thermodynamic model of finite-size particles with both intrinsic histone-DNA interactions and an effective two-body potential. We infer one- and two-body energies directly from high-throughput maps of nucleosome positions. We show that higher-order chromatin structure helps explains in vitro and in vivo nucleosome ordering in transcribed regions, and plays a leading role in establishing well-known 10-11 bp genome-wide periodicity of nucleosome positions.

Chromatin remodelers clear nucleosomes from intrinsically unfavorable sites to establish nucleosome-depleted regions at promoters.

Most promoters in yeast contain a nucleosome-depleted region (NDR), but the mechanisms by which NDRs are established and maintained in vivo are currently unclear. We have examined how genome-wide nucleosome placement is altered in the absence of two distinct types of nucleosome remodeling activity. In mutants of both SNF2, which encodes the ATPase component of the Swi/Snf remodeling complex, and ASF1, which encodes a histone chaperone, distinct sets of gene promoters carry excess nucleosomes in their NDRs relative to wild-type. In snf2 mutants, excess promoter nucleosomes correlate with reduced gene expression. In both mutants, the excess nucleosomes occupy DNA sequences that are energetically less favorable for nucleosome formation, indicating that intrinsic histone-DNA interactions are not sufficient for nucleosome positioning in vivo, and that Snf2 and Asf1 promote thermodynamic equilibration of nucleosomal arrays. Cells lacking SNF2 or ASF1 still accomplish the changes in promoter nucleosome structure associated with large-scale transcriptional reprogramming. However, chromatin reorganization in the mutants is reduced in extent compared to wild-type cells, even though transcriptional changes proceed normally. In summary, active remodeling is required for distributing nucleosomes to energetically favorable positions in vivo and for reorganizing chromatin in response to changes in transcriptional activity.

High-throughput sequencing reveals a simple model of nucleosome energetics.

We use genome-wide nucleosome maps to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a minor role. Furthermore, similar sequence signatures are exhibited by control experiments in which nucleosome-free genomic DNA is either sonicated or digested with micrococcal nuclease, making it possible that current predictions based on high-throughput nucleosome-positioning maps are biased by experimental artifacts.

Genomic studies and computational predictions of nucleosome positions and formation energies.

Chromatin is a complex of DNA, RNA, and proteins whose primary function is to package genomic DNA into the tight confines of a cell nucleus. A fundamental repeating unit of chromatin is the nucleosome, an octamer of histone proteins around which 147 base pairs of DNA are wound in almost two turns of a left-handed superhelix. Chromatin is a dynamic structure that exerts profound influence on regulation of gene expression and other cellular functions. These chromatin-directed processes are facilitated by optimizing nucleosome positions throughout the genome and by remodeling nucleosomes in response to various external and internal signals such as environmental perturbations. Here we discuss the large-scale maps of nucleosome positions made available through recent advances in parallel high-throughput sequencing and microarray technologies. We show that these maps reveal common features of nucleosome organization in eukaryotic genomes. We also survey the computational models designed to predict nucleosome formation scores or energies and demonstrate how these predictions can be used to position multiple nucleosomes on the genome without steric overlap.

Power spectrum scale invariance quantifies limbic dysregulation in trait anxious adults using fMRI: adapting methods optimized for characterizing autonomic dysregulation to neural dynamic time series.

In a well-regulated control system, excitatory and inhibitory components work closely together with minimum lag; in response to inputs of finite duration, outputs should show rapid rise and, following the input's termination, immediate return to baseline. The efficiency of this response can be quantified using the power spectrum density's scaling parameter beta, a measure of self-similarity, applied to the first derivative of the raw signal. In this study, we adapted power spectrum density methods, previously used to quantify autonomic dysregulation (heart rate variability), to neural time series obtained via functional MRI. The negative feedback loop we investigated was the limbic system, using affect-valent faces as stimuli. We hypothesized that trait anxiety would be related to efficiency of regulation of limbic responses, as quantified by power-law scaling of fMRI time series. Our results supported this hypothesis, showing moderate to strong correlations of trait anxiety and beta (r=0.45-0.54) for the amygdala, orbitofrontal cortex, hippocampus, superior temporal gyrus, posterior insula, and anterior cingulate. Strong anticorrelations were also found between the amygdala's beta and wake heart rate variability (r=-0.61), suggesting a robust relationship between dysregulated limbic outputs and their autonomic consequences.

Chemosensory cues to conspecific emotional stress activate amygdala in humans.

Alarm substances are airborne chemical signals, released by an individual into the environment, which communicate emotional stress between conspecifics. Here we tested whether humans, like other mammals, are able to detect emotional stress in others by chemosensory cues. Sweat samples collected from individuals undergoing an acute emotional stressor, with exercise as a control, were pooled and presented to a separate group of participants (blind to condition) during four experiments. In an fMRI experiment and its replication, we showed that scanned participants showed amygdala activation in response to samples obtained from donors undergoing an emotional, but not physical, stressor. An odor-discrimination experiment suggested the effect was primarily due to emotional, and not odor, differences between the two stimuli. A fourth experiment investigated behavioral effects, demonstrating that stress samples sharpened emotion-perception of ambiguous facial stimuli. Together, our findings suggest human chemosensory signaling of emotional stress, with neurobiological and behavioral effects.