Showing papers by "Sabeeha S. Merchant published in 2020"

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Abstract: Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

The histone H3-H4 tetramer is a copper reductase enzyme

Abstract: Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts.

Abstract: In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

An atypical short-chain dehydrogenase–reductase functions in the relaxation of photoprotective qH in Arabidopsis

Abstract: Photosynthetic organisms experience wide fluctuations in light intensity and regulate light harvesting accordingly to prevent damage from excess energy. The antenna quenching component qH is a sustained form of energy dissipation that protects the photosynthetic apparatus under stress conditions. This photoprotective mechanism requires the plastid lipocalin LCNP and is prevented by SUPPRESSOR OF QUENCHING1 (SOQ1) under non-stress conditions. However, the molecular mechanism of qH relaxation has yet to be resolved. Here, we isolated and characterized RELAXATION OF QH1 (ROQH1), an atypical short-chain dehydrogenase-reductase that functions as a qH-relaxation factor in Arabidopsis. The ROQH1 gene belongs to the GreenCut2 inventory specific to photosynthetic organisms, and the ROQH1 protein localizes to the chloroplast stroma lamellae membrane. After a cold and high-light treatment, qH does not relax in roqh1 mutants and qH does not occur in leaves overexpressing ROQH1. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed and soq1 roqh1 displays constitutive qH and light-limited growth. We propose that LCNP and ROQH1 perform dosage-dependent, antagonistic functions to protect the photosynthetic apparatus and maintain light-harvesting efficiency in plants.

Single-Cell RNA Sequencing of Batch Chlamydomonas Cultures Reveals Heterogeneity in their Diurnal Cycle Phase

Abstract: The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of the facility with which it can be cultured in the laboratory. Genomic and systems biology approaches have previously been used to describe how the transcriptome responds to environmental changes, but this analysis has been limited to bulk data, representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing in the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample algae communities in the wild. Second, scRNA-seq successfully separated single cells into non-overlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest cell division to economize resources. Notably, these groups of cells recapitulated known patterns observed with bulk RNA-seq, but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We propose that bulk RNA-seq data represent an average of varied cell states that hides underappreciated heterogeneity. One-sentence summary We show that single-cell RNA-seq (scRNA-seq) can be applied to Chlamydomonas cultures to reveal the that heterogenity in bulk cultures is largely driven by diurnal cycle phases The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Matteo Pellegrini (matteop@mcdb.ucla.edu)

Systematic characterization of gene function in a photosynthetic organism

Abstract: Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

Co-Expression Networks in the Green Alga Chlamydomonas reinhardtii Empower Gene Discovery and Functional Exploration

Abstract: The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis, cilium assembly and function, lipid and starch metabolism and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes from manually-curated and community-generated gene lists. We extracted 400 high-confidence cilia-related genes at the intersection of multiple co-expressed lists, illustrating the power of our simple method. Surprisingly, Chlamydomonas experiments did not cluster according to an obvious pattern, suggesting an underappreciated variable during sample collection. One possible source of variation may stem from the strong clustering of nuclear genes as a function of their diurnal phase, even in samples collected in constant conditions, indicating substantial residual synchronization in batch cultures. We provide a step-by-step guide into the analysis of co-expression across Chlamydomonas transcriptome datasets to help foster gene function discovery. One-sentence summary we reveal co-expression potential between Chlamydomonas genes and describe strong synchronization of cells grown in batch cultures as a possible source of underappreciated variation.

Co-expressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

Abstract: Objective To examine the cardiovascular risks of diclofenac initiation compared with initiation of other traditional non-steroidal anti-inflammatory drugs, initiation of paracetamol, and no initiation. Design Series of 252 nationwide cohort studies, each mimicking the strict design criteria of a clinical trial (emulated trial design). Setting Danish, nationwide, population based health registries (1996-2016). Participants Individuals eligible for inclusion were all adults without malignancy; schizophrenia; dementia; or cardiovascular, kidney, liver, or ulcer diseases (that is, with low baseline risk). The study included 1 370 832 diclofenac initiators, 3 878 454 ibuprofen initiators, 291 490 naproxen initiators, 764 781 healthcare seeking paracetamol initiators matched by propensity score, and 1 303 209 healthcare seeking non-initiators also matched by propensity score. Main outcome measures Cox proportional hazards regression was used to compute the intention to treat hazard ratio (as a measure of the incidence rate ratio) of major adverse cardiovascular events within 30 days of initiation. Results The adverse event rate among diclofenac initiators increased by 50% compared with non-initiators (incidence rate ratio 1.5, 95% confidence interval 1.4 to 1.7), 20% compared with paracetamol or ibuprofen initiators (both 1.2, 1.1 to 1.3), and 30% compared with naproxen initiators (1.3, 1.1 to 1.5). The event rate for diclofenac initiators increased for each component of the combined endpoint (1.2 (1.1 to 1.4) for atrial fibrillation/flutter, 1.6 (1.3 to 2.0) for ischaemic stroke, 1.7 (1.4 to 2.0) for heart failure, 1.9 (1.6 to 2.2) for myocardial infarction, and 1.7 (1.4 to 2.1) for cardiac death) as well as for low doses of diclofenac, compared with non-initiators. Although the relative risk of major adverse cardiovascular events was highest in individuals with low or moderate baseline risk (that is, diabetes mellitus), the absolute risk was highest in individuals with high baseline risk (that is, previous myocardial infarction or heart failure). Diclofenac initiation also increased the risk of upper gastrointestinal bleeding at 30 days, by approximately 4.5-fold compared with no initiation, 2.5-fold compared with initiation of ibuprofen or paracetamol, and to a similar extent as naproxen initiation. Conclusions Diclofenac poses a cardiovascular health risk compared with non-use, paracetamol use, and use of other traditional non-steroidal anti-inflammatory drugs.