Alvina G. Lai

CIRCADIAN CLOCK-ASSOCIATED 1 regulates ROS homeostasis and oxidative stress responses

Organisms have evolved endogenous biological clocks as internal timekeepers to coordinate metabolic processes with the external environment. Here, we seek to understand the mechanism of synchrony between the oscillator and products of metabolism known as Reactive Oxygen Species (ROS) in Arabidopsis thaliana. ROS-responsive genes exhibit a time-of-day–specific phase of expression under diurnal and circadian conditions, implying a role of the circadian clock in transcriptional regulation of these genes. Hydrogen peroxide production and scavenging also display time-of-day phases. Mutations in the core-clock regulator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), affect the transcriptional regulation of ROS-responsive genes, ROS homeostasis, and tolerance to oxidative stress. Mis-expression of EARLY FLOWERING 3, LUX ARRHYTHMO, and TIMING OF CAB EXPRESSION 1 affect ROS production and transcription, indicating a global effect of the clock on the ROS network. We propose CCA1 as a master regulator of ROS homeostasis through association with the Evening Element in promoters of ROS genes in vivo to coordinate time-dependent responses to oxidative stress. We also find that ROS functions as an input signal that affects the transcriptional output of the clock, revealing an important link between ROS signaling and circadian output. Temporal coordination of ROS signaling by CCA1 and the reciprocal control of circadian output by ROS reveal a mechanistic link that allows plants to master oxidative stress responses.

The abrogation of condensin function provides independent evidence for defining the self-renewing population of pluripotent stem cells

Heterogeneity of planarian stem cells has been categorised on the basis of single cell expression analyses and subsequent experiments to demonstrate lineage relationships. Some data suggest that despite heterogeneity in gene expression amongst cells in the cell cycle, in fact only one sub-population, known as sigma neoblasts, can self-renew. Without the tools to perform live in vivo lineage analysis, we instead took an alternative approach to provide independent evidence for defining the self-renewing stem cell population. We exploited the role of highly conserved condensin family genes to functionally assay neoblast self-renewal properties. Condensins are involved in forming properly condensed chromosomes to allow cell division to proceed during mitosis, and their abrogation inhibits mitosis and can lead to repeated endoreplication of the genome in cells that make repeated attempts to divide. We find that planarians possess only the condensin I complex, and that this is required for normal stem cell function. Abrogation of condensin function led to rapid stem cell depletion accompanied by the appearance of ‘giant’ cells with increased DNA content. Using previously discovered markers of heterogeneity we show that enlarged cells are always from the sigma-class of the neoblast population and we never observe evidence for endoreplication for the other neoblast subclasses. Overall, our data establish that condensins are essential for stem cell maintenance and provide independent evidence that only sigma-neoblasts are capable of multiple rounds of cell division and hence self-renewal.

Epigenetic analyses of the planarian genome reveals conservation of bivalent promoters in animal stem cells.

Planarian flatworms have an indefinite capacity to regenerate missing or damaged body parts owing to a population of pluripotent adult stems cells called neoblasts (NBs). Currently, little is known about the importance of the epigenetic status of NBs and how histone modifications regulate homeostasis and cellular differentiation. We have developed an improved and optimized ChIP-seq protocol for NBs in Schmidtea mediterranea and have generated genome-wide profiles for the active marks H3K4me3 and H3K36me3, and suppressive marks H3K4me1 and H3K27me3. The genome-wide profiles of these marks were found to correlate well with NB gene expression profiles. We found that genes with little transcriptional activity in the NB compartment but which switch on in post-mitotic progeny during differentiation are bivalent, being marked by both H3K4me3 and H3K27me3 at promoter regions. In further support of this hypothesis bivalent genes also have a high level of paused RNA Polymerase II at the promoter-proximal region. Overall, this study confirms that epigenetic control is important for the maintenance of a NB transcriptional program and makes a case for bivalent promoters as a conserved feature of animal stem cells and not a vertebrate specific innovation. By establishing a robust ChIP-seq protocol and analysis methodology, we further promote planarians as a promising model system to investigate histone modification mediated regulation of stem cell function and differentiation.Background Planarian flatworms have an indefinite capacity to regenerate due to a population of pluripotent adult stem cells (neoblasts). Previous studies have suggested that they have features in common with both pluripotent mammalian embryonic stem cells and germ line stem cells. However, little is known about the importance of epigenetic regulation in these cells, which is likely to be crucial for neoblast biology and regeneration. We set out to develop analytical and experimental tools for planarians to allow the study of epigenetic marks in neoblasts and allow direct comparison of this model system with other animals. Results We developed an optimized ChIP-seq protocol for planarian neoblasts that allowed us to generate genome wide profiles for H3K4me1, H3K4me3 and H3K27me3. These were found to correlate as expected with genome wide expression profiles from analyses of planarian RNA-seq data. We found that many genes that are silent in neoblasts and then switch in post-mitotic progeny during differentiation have both H3K4me3 and H3K27me3 at promoter regions and are therefore bivalent. Further analysis suggested that bivalency is present at hundreds of loci in the pluripotent neoblast population. Conclusions We confirm that epigenetic regulation is key to neoblast biology and that bivalent promoters are not confined to vertebrate lineages, but may be a conserved feature of animal stem cells. Our work further establishes planarian neoblasts as a powerful model system for understanding the epigenetic regulation of pluripotency and regeneration.

Comparative genomic analysis of innate immunity reveals novel and conserved components in crustacean food crop species

BackgroundGrowing global demands for crustacean food crop species have driven large investments in aquaculture research worldwide. However, large-scale production is susceptible to pathogen-mediated destruction particularly in developing economies. Thus, a thorough understanding of the immune system components of food crop species is imperative for research to combat pathogens.ResultsThrough a comparative genomics approach utilising extant data from 55 species, we describe the innate immune system of the class Malacostraca, which includes all food crop species. We identify 7407 malacostracan genes from 39 gene families implicated in different aspects of host defence and demonstrate dynamic evolution of innate immunity components within this group. Malacostracans have achieved flexibility in recognising infectious agents through divergent evolution and expansion of pathogen recognition receptors genes. Antiviral RNAi, Toll and JAK-STAT signal transduction pathways have remained conserved within Malacostraca, although the Imd pathway appears to lack several key components. Immune effectors such as the antimicrobial peptides (AMPs) have unique evolutionary profiles, with many malacostracan AMPs not found in other arthropods. Lastly, we describe four putative novel immune gene families, potentially representing important evolutionary novelties of the malacostracan immune system.ConclusionOur analyses across the broader Malacostraca have allowed us to not only draw analogies with other arthropods but also to identify evolutionary novelties in immune modulation components and form strong hypotheses as to when key pathways have evolved or diverged. This will serve as a key resource for future immunology research in crustacean food crops.

Positional Information Resolves Structural Variations and Uncovers an Evolutionarily Divergent Genetic Locus in Accessions of Arabidopsis thaliana

Genome sequencing of closely related individuals has yielded valuable insights that link genome evolution to phenotypic variations. However, advancement in sequencing technology has also led to an escalation in the number of poor quality–drafted genomes assembled based on reference genomes that can have highly divergent or haplotypic regions. The self-fertilizing nature of Arabidopsis thaliana poses an advantage to sequencing projects because its genome is mostly homozygous. To determine the accuracy of an Arabidopsis drafted genome in less conserved regions, we performed a resequencing experiment on a ∼371-kb genomic interval in the Landsberg erecta (Ler-0) accession. We identified novel structural variations (SVs) between Ler-0 and the reference accession Col-0 using a long-range polymerase chain reaction approach to generate an Illumina data set that has positional information, that is, a data set with reads that map to a known location. Positional information is important for accurate genome assembly and the resolution of SVs particularly in highly duplicated or repetitive regions. Sixty-one regions with misassembly signatures were identified from the Ler-0 draft, suggesting the presence of novel SVs that are not represented in the draft sequence. Sixty of those were resolved by iterative mapping using our data set. Fifteen large indels (>100 bp) identified from this study were found to be located either within protein-coding regions or upstream regulatory regions, suggesting the formation of novel alleles or altered regulation of existing genes in Ler-0. We propose future genome-sequencing experiments to follow a clone-based approach that incorporates positional information to ultimately reveal haplotype-specific differences between accessions.

MLL3/4 Prevents Stem Cell Hyperplasia And Controls Differentiation Programs In A Planarian Cancer Stem Cell Model

Background The family of Mixed Lineage Leukaemia (MLL) histone methyltransferase proteins are often implicated in disease processes, particularly cancer. Here we focus on the MLL3 and MLL4 which are mutated in a high percentage of cancers implicating them as tumour suppressors, but very little is known about the underlying transcriptional and epigenetic changes that contribute to cancer. Results Here we make use of the highly accessible planarians model system to uncover a role for MLL3/4 in controlling stem cell differentiation and proliferation, that suggests conservation tumour suppressor over a large evolutionary timescale function for this epigenetic regulator. Knockdown of the planarian Mll3/4 orthologs compromises stem cell differentiation and leads to hyper-proliferation and tumour-like outgrowth formation. The planarian system allows as to investigate the epigenetic and transcriptional studies changes in cells that will go on to form tumours at an early stage after loss of MLL3/4 function, identifying genome wide changes that occur early in the development of the pathology. This revealed mis-regulation of both conserved oncogenes and tumour suppressors, that together likely explain the cancer-like phenotype observed in planarians. Conclusions We confirm MLL3/4 tumour suppressor function and uncover a deep conservation of this role in stem cells. We find potentially conserved mis-regulated downstream targets driving the effects of MLL3/4 loss of function. Our work demonstrates the suitability of planarians for the study of epigenetic phenotypes related to cancer and stem cell function, and for capturing early causative changes in a definitive population of tumour forming stem cells in vivo.Currently, little is known about the evolution of epigenetic regulation in animal stem cells. Using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases, we demonstrate that their role as tumour suppressors in stem cells is conserved over a large evolutionary distance in animals. This also suggested the potential conservation of a genome wide epigenetic regulation program in animal stem cells, so we assessed the regulatory effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumour suppressors among the affected genes that are therefore likely candidates for mediating MLL3/4 tumour suppression function in mammals, where little is known about in vivo regulatory targets. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation.

The protein subunit of telomerase displays patterns of dynamic evolution and conservation across different metazoan taxa

BackgroundMost animals employ telomerase, which consists of a catalytic subunit known as the telomerase reverse transcriptase (TERT) and an RNA template, to maintain telomere ends. Given the importance of TERT and telomere biology in core metazoan life history traits, like ageing and the control of somatic cell proliferation, we hypothesised that TERT would have patterns of sequence and regulatory evolution reflecting the diverse life histories across the Animal Kingdom.ResultsWe performed a complete investigation of the evolutionary history of TERT across animals. We show that although TERT is almost ubiquitous across Metazoa, it has undergone substantial sequence evolution within canonical motifs. Beyond the known canonical motifs, we also identify and compare regions that are highly variable between lineages, but show conservation within phyla. Recent data have highlighted the importance of alternative splice forms of TERT in non-canonical functions and although animals may share some conserved introns, we find that the selection of exons for alternative splicing appears to be highly variable, and regulation by alternative splicing appears to be a very dynamic feature of TERT evolution. We show that even within a closely related group of triclad flatworms, where alternative splicing of TERT was previously correlated with reproductive strategy, we observe highly diverse splicing patterns.ConclusionsOur work establishes that the evolutionary history and structural evolution of TERT involves previously unappreciated levels of change and the emergence of lineage specific motifs. The sequence conservation we describe within phyla suggests that these new motifs likely serve essential biological functions of TERT, which along with changes in splicing, underpin diverse functions of TERT important for animal life histories.

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.

Could ROS signals drive tissue-specific clocks?

Circadian clocks have emerged to tune the physiology of organisms to periodic changes in the environment in a dynamic fashion. Negative implications of circadian disruptions in humans, animals and plants have encouraged extensive studies of clock-controlled biological processes in various model species. Recently, it has been shown that the transcription-dependent and -independent biological oscillators are largely driven by cellular oxidative cycles that are intrinsically linked with metabolism. Essentially, the clock is viewed as an integrated network that encompasses cytosolic, genetic and metabolic dimensions. Furthermore, in multicellular organisms, the clock network is organized in a tissue-specific manner. Here we discuss questions that remain unanswered: How do these dimensions communicate with each other and how do tissue-specific clocks exchange temporal information within multicellular organisms?

Interplay between circadian clock and viral infection

The circadian clock underpins most physiological conditions and provides a temporal dimension to our understanding of body and tissue homeostasis. Disruptions of circadian rhythms have been associated with many diseases, including metabolic disorders and cancer. Recent literature highlights a role for the circadian clock to regulate innate and adaptive immune functions that may prime the host response to infectious organisms. Viruses are obligate parasites that rely on host cell synthesis machinery for their own replication, survival and dissemination. Here, we review key findings on how circadian rhythms impact viral infection and how viruses modulate molecular clocks to facilitate their own replication. This emerging area of viral-clock biology research provides a fertile ground for discovering novel anti-viral targets and optimizing immune-based therapies.