Biology subjects

Merchant, S. S.

Publications and source records attributed to Merchant, S. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Bioengineered algal lipids enriched in structured medium- and long-chain triacylglycerols, linoleate, and sn-2 palmitate for human milk fat substitutes

Triacylglycerols (TAGs) in human milkfat (HMF) provide over 50% of the calories for infant nutrition. Their unique architecture, featuring palmitic acid predominantly esterified at the sn-2 position, facilitates absorption as 2-palmitoyl monoacylglycerol after hydrolysis of the sn-1 and sn-3 fatty acids by gut lipases. Conventional vegetable oil-based infant formulas mimic the HMF fatty acid profile but lack its distinct regioisomeric structure, abundant medium- and long-chain triglycerides (MLCTs), and oleic-to-linoleic acid ratios that vary with maternal diet and geography. We have engineered the oleaginous green alga Auxenochlorella for biosynthesis of TAGs enriched in MLCTs, sn-2 palmitate, and linoleate via heterologous expression of acyl-acyl-carrier-protein (ACP) thioesterases, palmitate-specific lysophosphatidic acid acyltransferases, and lysophosphatidylcholine acyltransferase. These structured algal lipids replicate both the major fatty acid proportions and regioisomeric composition of authentic HMF, providing single-source bioengineered alternatives to plant-derived HMF substitutes for infant formula.

synthetic biology↗

bioRxiv2025

Light on its feet: Acclimation to high and low diurnal light is flexible in Chlamydomonas reinhardtii

Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods (Dupuis & Ojeda et al. 2025). Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL period, and they failed to complete a cell cycle. However, although LL-acclimated cells showed severe photodamage after 6 hours of HL, they recovered chloroplast form and function later that afternoon and successfully divided at nightfall. These cells rapidly altered their thylakoid membrane ultrastructure, increased their photoprotective quenching capacity, and decreased their inventory of photosystem and antenna proteins by the end of the first HL day. Transcriptomic and proteomic analyses revealed rapid induction of thousands of genes, including those encoding proteases, chaperones, and other proteins involved in the chloroplast unfolded protein response. These results show that the alga is highly flexible and competent to rapidly acclimate to changes in diurnal light intensity.

plant biology↗

bioRxiv2025

Mono-mix strategy enables comparative proteomics of a cross-kingdom microbial symbiosis

Cross-kingdom microbial symbioses, such as those between algae and bacteria, are key players in biogeochemical cycles. The molecular changes during initiation and establishment of symbiosis are of great interest, but quantitatively monitoring such changes can be challenging, particularly when the microorganisms differ greatly in size or are intimately associated. Here, we analyze output from data-dependent acquisition (DDA) LC-MS/MS proteomics experiments investigating the well-studied interaction between the alga Chlamydomonas reinhardtii and the heterotrophic bacterium Mesorhizobium japonicum. We found that detection of bacterial proteins decreased in coculture by 50% proteome-wide due to the abundance of algal proteins. As a result, standard differential expression analysis led to numerous false-positive reports of significantly downregulated proteins, where it was not possible to distinguish meaningful biological responses to symbiosis from artifacts of the reduced protein detection in coculture relative to monoculture. We show that data normalization alone does not eliminate the impact of altered detection on differential expression analysis of the cross-kingdom symbiosis. We assessed two additional strategies to overcome this methodological artifact inherent to DDA proteomics. In the first, we combined algal and bacterial monocultures at a relative abundance that mimicked the coculture, creating a "mono-mix" control to which the coculture could be compared. This approach enabled comparable detection of bacterial proteins in the coculture and the monoculture control. In the second strategy, we enhanced detection of lowly abundant bacterial proteins by using sample fractionation upstream of LC-MS/MS analysis. When these simple approaches were combined, they allowed for meaningful comparisons of nearly 10,000 algal proteins and over 4,000 bacterial proteins in response to symbiosis by DDA. They successfully recovered expected changes in the bacterial proteome in response to algal coculture, including upregulation of sugar-binding proteins and transporters. They also revealed novel proteomic responses to coculture that guide hypotheses about algal-bacterial interactions.

systems biology↗

bioRxiv2025

A defined growth medium for trace metal limitation studies in Auxenochlorella sp. (UTEX 250-A) during mixotrophic growth with glucose.

Auxenochlorella sp. (strain UTEX 250-A) is a fast-growing, oleaginous alga that is an emerging reference organism for basic research, with broad biotechnological applications. Advancing UTEX 250-A as a biotechnological workhorse requires a deeper understanding of its nutritional demands, particularly under different trophic conditions. However, little is known about the specific acclimations that allow UTEX 250-A to thrive when essential nutrients are scarce. Here, we describe the formulation of a defined growth medium that supports the cultivation of UTEX 250-A with controlled trace levels of the essential micronutrients iron, copper, and zinc. Special attention was given to ensure that the medium was compatible with inductively coupled plasma mass spectrometry (ICP-MS) analysis, enabling the detection and minimization of metal contamination in the nanomolar range. The medium was designed to provide sufficient nutrients to support the mixotrophic growth of UTEX 250-A from an initial density of 105 cells/mL to a stationary density of 2.4x108 cells/mL, with additional nutrients supplied to accommodate metabolic and trophic transitions during stationary phase, when photosynthesis is restored due to carbon limitation. This replete medium provides a foundation for robust nutrient limitation studies in Auxenochlorella sp. (UTEX 250-A).

systems biology↗

bioRxiv2025

Allodiploid hybridization, loss-of-heterozygosity and aneuploidy in the green alga Auxenochlorella, an emerging model for discovery research and bioengineering

Auxenochlorella spp. are diploid oleaginous green algae whose streamlined genomes can be readily manipulated by homologous recombination, making them highly amenable to discovery research and bioengineering. Vegetatively diploid organisms experience specific evolutionary phenomena, including allodiploid hybridization, mitotic recombination, loss-of-heterozygosity and aneuploidy; however, studies of these forces have largely focused on yeasts. Here, we present a telomere-to-telomere phased diploid genome assembly of Auxenochlorella UTEX 250-A (haploid length 22 Mb) and introduce a genetic toolkit for site-specific manipulation of the nuclear genome in multiple strains, featuring several selectable markers, inducible promoters, and fluorescent reporters for protein localization. UTEX 250-A is an allodiploid hybrid of Auxenochlorella protothecoides and Auxenochlorella symbiontica, two species differentiated by extensive chromosomal rearrangements. UTEX 250-A haplotypes are a mosaic of each parental species following mitotic recombination, and two chromosomes are trisomic. Loss-of-heterozygosity events are pervasive across Auxenochlorella and can evolve rapidly in the laboratory. High-quality structural annotation yielded [~]7,500 genes per haplotype. Auxenochlorella have experienced gene family loss and reduction, including core photosynthesis genes, and exhibit periodic adenine and cytosine methylation at promoters and gene bodies, respectively. Approximately 10% of genes, especially those involved in DNA repair and sex, overlap antisense long noncoding RNAs, which may participate in a regulatory mechanism. We demonstrate the utility of Auxenochlorella for fundamental research by knockout of a chlorophyll biosynthesis enzyme, and confirm one trisomy by allele-specific transformation. These results demonstrate the generality of several evolutionary forces associated with vegetative diploidy and provide a foundation for use of Auxenochlorella as a reference organism. One-sentence summaryAuxenochlorella, green algae shaped by evolutionary forces acting on vegetative diploids, are amenable to discovery research and bioengineering via efficient site-specific homologous recombination

plant biology↗

bioRxiv2024

An algal nutrient-replete, optimized medium for fast growth and high triacylglycerol accumulation

Microalgae are promising sources to sustainably meet the global needs for energy and products. Algae grow under different trophic conditions, where nutritional status regulates biosynthetic pathways, energy production, and growth. The green alga Chromochloris zofingiensis has strong economic potential because it co-produces biofuel precursors and the high-value antioxidant astaxanthin while accumulating biomass when grown mixotrophically. As an emerging reference alga for photosynthesis, metabolism, and bioproduction, C. zofingiensis needs a defined, optimized medium to standardize experiments during fast growth. Because the interplay of glucose consumption (+Glc) and mineral deficiency influences photosynthesis, growth, and the production of lipids and astaxanthin, we designed a replete nutrient medium tailored to the C. zofingiensis cellular ionome. We combined inductively coupled plasma mass spectrometry (ICP-MS) and +Glc growth curves to determine a medium that is nutrient replete for at least 5 days of +Glc logarithmic growth. We found that there are high nutritional needs for phosphorus and sulfur during mixotrophy. Iron was the only element measured for which the cellular concentration correlated with exogenous concentration and was iteratively adjusted until the internal ionome was consistent through the logarithmic growth phase. This Chromochloris-Optimized Ratio of Elements (CORE) medium supports fast growth and high biomass without causing excess nutrient toxicity. This defined, nutrient-replete standard is important for future C. zofingiensis investigations and can be adapted for other species to support high biomass. The method used to develop CORE medium shows how ionomics informs replicable media design and may be applied in industrial settings to inform cost-effective biofuel production. Significance StatementStudying how carbon sources and mineral nutrients interplay to regulate algal metabolism can be exploited to discover and control pathways in photosynthesis and biofuel production. Here we design a medium from the cellular ionome of Chromochloris zofingiensis, a powerful algal model for photosynthesis, metabolism, and bioproducts, to provide a defined, replete standard for mixotrophic and heterotrophic growth of green algae. These media design principles show how accounting for increased nutritional demands based on carbon substrate can ensure experimental replicability when probing diverse algal metabolisms.

plant biology↗

bioRxiv2024

Too dim, too bright, and just right: Systems analysis of the Chlamydomonas diurnal program upon acclimation to light stress and limitation

Photosynthetic organisms coordinate their metabolism and growth with diurnal light, which can range in intensity from limiting to inhibitory. To gain a comprehensive understanding of how diurnal regulatory circuits interface with sensing and response to various light intensities, we performed a systems analysis of synchronized Chlamydomonas populations acclimated to low, moderate, and high diurnal light. Transcriptomic and proteomic data revealed that the Chlamydomonas rhythmic gene expression program is resilient to limiting and excess light. Although gene expression and photodamage are dynamic over the diurnal cycle, Chlamydomonas populations acclimated to low and high diurnal light exhibit constitutive phenotypes with respect to photosystem abundance, thylakoid architecture, and non-photochemical quenching that persist through the night. This suggests that cells "remember" or anticipate the daylight environment. The integrated data constitute an excellent resource for understanding gene regulatory mechanisms and photoprotection in eukaryotes under environmentally relevant conditions.

systems biology↗

bioRxiv2024

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae.

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistronic genes, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for polycistronic expression in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed 1) a preference for weak Kozak-like sequences for ORF 1 and 2) an underrepresentation of potential initiation codons before ORF 2, which are suitable conditions for leaky scanning to allow ORF 2 translation. We used mutational analysis in Auxenochlorella protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky ribosome scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames found across eukaryotic genomes and for transgene expression in synthetic biology applications. Significance StatementTextbook dogma states that nucleus-encoded genes are monocistronic, producing transcripts with a single translated open reading frame. However, highly conserved bicistronic loci are pervasive in green algae that are separated by several hundred million years of evolution, speaking to their ancestral origins and functions within the Chlorophyte lineage. A combination of bioinformatic analysis and in vivo gene manipulation supports leaky ribosomal scanning as the primary mechanism for translation of multiple ORFs from bicistronic transcripts. We have successfully tuned synthesis levels of two proteins encoded on one mRNA by modifying the ORF 1 Kozak-like sequence. These findings may have broad applications in synthetic biology.

plant biology↗

bioRxiv2024

Mutation of negative regulatory gene CEHC1 encoding an FBXO3 protein results in normoxic expression of HYDA genes in Chlamydomonas reinhardtii

Oxygen is known to prevent hydrogen production in Chlamydomonas, both by inhibiting the hydrogenase enzyme and by preventing the accumulation of HYDA-encoding transcripts. We developed a screen for mutants showing constitutive accumulation of HYDA1 transcripts in the presence of oxygen. A reporter gene required for ciliary motility, placed under the control of the HYDA1 promoter, conferred motility only in hypoxic conditions. By selecting for mutants able to swim even in the presence of oxygen we obtained strains that express the reporter gene constitutively. One mutant identified a gene encoding an F-box only protein 3 (FBXO3), known to participate in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1, leads to constitutive expression of HYDA1 and other genes regulated by hypoxia, and of many genes known to be targets of CRR1, a transcription factor in the nutritional copper signaling pathway. CRR1 was required for the constitutive expression of the HYDA1 reporter gene in cehc1-1 mutants. The CRR1 protein, which is normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 acts to facilitate the degradation of CRR1. Our results reveal a novel negative regulator in the CRR1 pathway and possibly other pathways leading to complex metabolic changes associated with response to hypoxia.

plant biology↗

bioRxiv2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short- and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.

plant biology↗

bioRxiv2024

Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtiis capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing -proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the algas fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide - a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.

microbiology↗

bioRxiv2023

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the unicellular green alga Chlamydomonas reinhardtii, Cu import is dependent on Copper Response Regulator 1 (CRR1), the master regulator of Cu homeostasis. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble cysteine-rich protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulate the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high affinity Cu(I) uptake. Notably, the over-accumulation of Cu(I) in Zinc (Zn)-deficiency (20 times the quota) depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and is consistent with the lower substrate affinity of CTR2.

biochemistry↗

bioRxiv2023

Cysteine: an ancestral Cu binding ligand in green algae?

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding "first responder" proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, {gamma}-glutamylcysteine and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ~80-fold, corresponding to ~ 2.8 x 109 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

biochemistry↗

bioRxiv2023

Pumping Iron: A Multi-omics Analysis of Two Extremophilic Algae Reveals Mechanisms of Iron Economy

Marine algae are responsible for half of the worlds primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hyper-saline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multi-omics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a novel family of siderophore-iron uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, an LHCA-related protein found also in other chlorophytes. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms. Significance StatementDespite their small size, microalgae play a huge role in CO2 uptake via photosynthesis, and represent an important target for climate crisis mitigation efforts. Most photosynthesis proteins require iron as a co-factor so that insufficient iron often limits algal CO2 sequestration. With this in mind, we examined a genus of microalgae called Dunaliella that is exceptionally well-adapted to low iron environments. We produced complete genomes, transcriptomes, and proteomes for two species of Dunaliella that hail from radically different environments: one from coastal ocean waters and the other from salt flats. We identified dozens of genes and multiple, complementary strategies that both species utilize for iron-uptake and management that explain Dunaliellas remarkable ability to thrive on low iron.

systems biology↗

bioRxiv2022

The Chlamydomonas Genome Project, version 6: reference assemblies for mating type plus and minus strains reveal extensive structural mutation in the laboratory

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of RECQ3 helicase, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results across laboratories and over time. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference.

genomics↗

bioRxiv2021

An optimized ChIP-Seq framework for profiling of histone modifications in Chromochloris zofingiensis

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin Immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein crosslinking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

genomics↗

bioRxiv2021

Genome wide profiling of histone H3 lysine 4 methylation during the Chlamydomonas cell cycle reveals stable and dynamic properties of lysine 4 trimethylation at gene promoters and near ubiquitous lysine 4 monomethylation

Chromatin modifications are key epigenetic regulatory features with roles in various cellular events, yet histone mark identification, gene wide distribution and relationship to gene expression remains understudied in green algae. Histone lysine methylation is regarded as an active chromatin mark in many organisms, and is implicated in mediating active euchromatin. We interrogated the genome wide distribution pattern of mono- and trimethylated H3K4 using Chromatin-Immunoprecipitation followed by deep-sequencing (ChIP-Seq) during key phases of the Chlamydomonas cell cycle: early G1 phase (ZT1) when cells initiate biomass accumulation, S/M phase (ZT13) when cells are undergoing DNA replication and mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at TSSs of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at one or more time points during the cell cycle but not necessarily by continuous active transcription. This finding even applied to early zygotic genes whose expression may be dormant for hundreds or thousands of generations between sexual cycles; but core meiotic genes were completely missing H3K4me3 peaks at their TSS. In addition, bi-directional promoters regulating expression of replication dependent histone genes, had transient H3K4me3 peaks that were present only during S/M phase when their expression peaked. In agreement with biochemical studies, mono-methylated H3K4 was the default state for the vast majority of histones that were outside of TSS and terminator regions of genes. A small fraction of the genome which was depleted of any H3 lysine methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Genome wide H3K4me3 ChIP-Seq data will be a valuable resource, facilitating gene structural annotation, as exemplified by our validation of hundreds of long non-coding RNA genes.

genomics↗

bioRxiv2021

Chlamydomonas ATX1 is essential for Cu distribution towards the secretory pathway and maintenance of biomass in conditions demanding cupro-enzyme dependent metabolic pathways

Copper (Cu) chaperones, of which yeast ATX1 is a prototype, are small proteins with a Cu(I) binding Mx-CxxC motif, and are responsible for directing intracellular Cu towards specific client protein targets that use Cu as a cofactor. The Chlamydomonas reinhardtii ATX1 (CrATX1) was identified because of its high sequence similarity with yeast ATX1. Like the yeast homologue, CrATX1 accumulates in iron-deficient cells (but is not impacted by other metal-deficiencies), and YFP-ATX1 is distributed in the cytoplasm. Reverse genetic analysis using artificial microRNA (amiRNA) to generate lines with reduced CrATX1 abundance and CRISPR/CPF1 to generate ATX1 knock out lines validated a function for ATX1 in iron-poor cells, most likely because of an impact on metalation of the multicopper oxidase FOX1, which is an important component in high-affinity iron uptake. A more general impact on the secretory pathway is indicated by reduced growth of ATX1 mutant lines on guanine as a sole nitrogen source, which we attribute to loss of function of UOX1, a urate oxidase involved in guanine assimilation. The block of Cu trafficking towards the secretory pathway in ATX1 mutants is strikingly evident by a reduced amount of intracellular Cu in all conditions probed in this work.

biochemistry↗