ATR Protects the Genome against R Loops through a MUS81-Triggered Feedback Loop.

R loops arising during transcription induce genomic instability, but how cells respond to the R loop-associated genomic stress is still poorly understood. Here, we show that cells harboring high levels of R loops rely on the ATR kinase for survival. In response to aberrant R loop accumulation, the ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway is activated by R loop-induced reversed replication forks. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation requires the MUS81 endonuclease. ATR protects the genome from R loops by suppressing transcription-replication collisions, promoting replication fork recovery, and enforcing a G2/M cell-cycle arrest. Furthermore, ATR prevents excessive cleavage of reversed forks by MUS81, revealing a MUS81-triggered and ATR-mediated feedback loop that fine-tunes MUS81 activity at replication forks. These results suggest that ATR is a key sensor and suppressor of R loop-induced genomic instability, uncovering a signaling circuitry that safeguards the genome against R loops.

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation.

The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

Daily changes in temperature, not the circadian clock, regulate growth rate in Brachypodium distachyon.

Plant growth is commonly regulated by external cues such as light, temperature, water availability, and internal cues generated by the circadian clock. Changes in the rate of growth within the course of a day have been observed in the leaves, stems, and roots of numerous species. However, the relative impact of the circadian clock on the growth of grasses has not been thoroughly characterized. We examined the influence of diurnal temperature and light changes, and that of the circadian clock on leaf length growth patterns in Brachypodium distachyon using high-resolution time-lapse imaging. Pronounced changes in growth rate were observed under combined photocyles and thermocycles or with thermocycles alone. A considerably more rapid growth rate was observed at 28°C than 12°C, irrespective of the presence or absence of light. In spite of clear circadian clock regulated gene expression, plants exhibited no change in growth rate under conditions of constant light and temperature, and little or no effect under photocycles alone. Therefore, temperature appears to be the primary cue influencing observed oscillations in growth rate and not the circadian clock or photoreceptor activity. Furthermore, the size of the leaf meristem and final cell length did not change in response to changes in temperature. Therefore, the nearly five-fold difference in growth rate observed across thermocycles can be attributed to proportionate changes in the rate of cell division and expansion. A better understanding of the growth cues in B. distachyon will further our ability to model metabolism and biomass accumulation in grasses.

Cell walls and the developmental anatomy of the Brachypodium distachyon stem internode.

While many aspects of plant cell wall polymer structure are known, their spatial and temporal distribution within the stem are not well understood. Here, we studied vascular system and fiber development, which has implication for both biofuel feedstock conversion efficiency and crop yield. The subject of this study, Brachypodium distachyon, has emerged as a grass model for food and energy crop research. Here, we conducted our investigation using B. distachyon by applying various histological approaches and Fourier transform infrared spectroscopy to the stem internode from three key developmental stages. While vascular bundle size and number did not change over time, the size of the interfascicular region increased dramatically, as did cell wall thickness. We also describe internal stem internode anatomy and demonstrate that lignin deposition continues after crystalline cellulose and xylan accumulation ceases. The vascular bundle anatomy of B. distachyon appears to be highly similar to domesticated grasses. While the arrangement of bundles within the stem is highly variable across grasses, B. distachyon appears to be a suitable model for the rind of large C4 grass crops. A better understanding of growth and various anatomical and cell wall features of B. distachyon will further our understanding of plant biomass accumulation processes.

Perturbation of Brachypodium distachyon CELLULOSE SYNTHASE A4 or 7 results in abnormal cell walls.

BACKGROUND: Cellulose is an integral component of the plant cell wall and accounts for approximately forty percent of total plant biomass but understanding its mechanism of synthesis remains elusive. CELLULOSE SYNTHASE A (CESA) proteins function as catalytic subunits of a rosette-shaped complex that synthesizes cellulose at the plasma membrane. Arabidopsis thaliana and rice (Oryza sativa) secondary wall CESA loss-of-function mutants have weak stems and irregular or thin cell walls. RESULTS: Here, we identify candidates for secondary wall CESAs in Brachypodium distachyon as having similar amino acid sequence and expression to those characterized in A. thaliana, namely CESA4/7/8. To functionally characterize BdCESA4 and BdCESA7, we generated loss-of-function mutants using artificial microRNA constructs, specifically targeting each gene driven by a maize (Zea mays) ubiquitin promoter. Presence of the transgenes reduced BdCESA4 and BdCESA7 transcript abundance, as well as stem area, cell wall thickness of xylem and fibers, and the amount of crystalline cellulose in the cell wall. CONCLUSION: These results suggest BdCESA4 and BdCESA7 play a key role in B. distachyon secondary cell wall biosynthesis.

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

BACKGROUND: Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. RESULTS: We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. CONCLUSION: These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.