Selection for improved energy use efficiency and drought tolerance in canola results in distinct transcriptome and epigenome changes

To increase both the yield potential and stability of crops, integrated breeding strategies are used that have mostly a direct genetic basis, but the utility of epigenetics to improve complex traits is unclear. A better understanding of the status of the epigenome and its contribution to agronomic performance would help in developing approaches to incorporate the epigenetic component of complex traits into breeding programs. Starting from isogenic canola (Brassica napus) lines, epilines were generated by selecting, repeatedly for three generations, for increased energy use efficiency and drought tolerance. These epilines had an enhanced energy use efficiency, drought tolerance, and nitrogen use efficiency. Transcriptome analysis of the epilines and a line selected for its energy use efficiency solely revealed common differentially expressed genes related to the onset of stress tolerance-regulating signaling events. Genes related to responses to salt, osmotic, abscisic acid, and drought treatments were specifically differentially expressed in the drought-tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine-4 of histone 3, further supported the phenotype by targeting drought-responsive genes and facilitating the transcription of the differentially expressed genes. From these results, we conclude that the canola epigenome can be shaped by selection to increase energy use efficiency and stress tolerance. Hence, these findings warrant the further development of strategies to incorporate epigenetics into breeding

Inflammation Is A Common Factor Between Central And Peripheral Neurodegeneration

The studies in this dissertation investigate neurodegenerative conditions of the central and peripheral nervous system utilizing bioinformatics and systems biology approaches. Various neurodegenerative conditions are associated with neuroinflammation or the inflammation of nervous tissue. We utilized Parkinson’s disease as our system for neuroinflammation in the central nervous system and diabetic peripheral neuropathy for the peripheral nervous system. Parkinson’s disease is associated with loss of dopaminergic neurons in the substantia nigra and consequent loss of dopamine signaling in the striatum of the central nervous system. Characteristics of Parkinson’s Disease include symptoms such as shaking, rigidity, slowness of movement, difficulty walking, dementia, depression, anxiety, sleeping disorders, and hallmark formation of misfolded α-synuclein aggregates called Lewy bodies. Diabetic peripheral neuropathy is a microvascular complication associated with diabetes mellitus. Degeneration of the peripheral nervous system in diabetes presents as neuropathic pain in the periphery with eventual loss of sensation in a stocking and glove like pattern. The loss of sensation is an underlying cause of diabetic foot syndrome which is the leading cause of lower limb amputations. This dissertation consists of three studies. The first study compared multiple murine models of diabetic peripheral neuropathy at different stages of the disease against human subjects in effort to identify an underlying cause of disease using publicly available microarray transcriptomic data. Pathway and network analysis were performed in conjunction on differentially expressed genes identified by comparing healthy controls to diabetic mice and progressive to non-progressive human subjects with diabetic peripheral neuropathy. Clusters of pathways in this network were related to inflammation, degradation, apoptosis, as well as kinase and immune signaling, as conserved changes across multiple time points, models, and species of DPN. These observed pathways, commonly disrupted across progression, species, and various murine models of the disease, are likely the key responses associated with diabetic peripheral neuropathy. The second study further investigated a single high dose streptozotocin model of type 1 diabetes mellitus by comparing tissues related to diabetic peripheral neuropathy (sciatic nerve and dorsal root ganglia) and diabetic nephropathy (renal glomerulus and cortex). RNA-sequencing identified differentially expressed genes in each complication-prone tissue between healthy controls and streptozotocin-treated mice. Genes with a conserved directional change were analysed using network and pathway analysis. Clusters related to DNA-damage response, oxidative stress, and immune response were represented in shared genes between diabeticnephropathy and diabetic peripheral neuropathy tissue experiencing a common directional change. These cluster themes are likely key conserved disruptions in microvascular complication-prone tissue. The third study explored neuroinflammation of the central nervous system utilizing mice overexpressing α-synuclein under the mouse thymidine1 promoter as an animal model of Parkinson’s disease. This murine model exhibits parkinsonian motor and non-motor symptoms as well as α-synuclein aggregation pathology. Early activation of microglia, the resident innate immune cells of the brain, and an inflammatory response can be measured in the brains of these animals as early as one month of age. RNA and DNA were extracted from microglia isolated from these animals at 3 and 13 months of age for RNA-sequencing and reduced representation bisulfite sequencing, respectively. The time points for tissue collection involve the beginning of motor symptoms at 3 months and 13 months is immediately prior to a loss of 40% of dopamine signaling which occurs at 14 months of age. The overexpression of α-synuclein-induced both genomic methylation and gene expression changes that are indicative of an immunologically activated M1 state of microglia. Correlation between gene expression and a change in methylation status were investigated but only intronic CG rich sites held a significant correlation with observed gene expression (r=-0.15, p=0.008). Profiling the changes induced by α-synuclein provides valuable insight into the systems contributing to disease progression. Overall, these results warrant further investigation into the role inflammation plays on the progression of neurodegenerative diseases. Our wide range of models and techniques lends strength to the notion of common immune activation pathways induced by a variety of disease insults in both the central and peripheral nervous systems

척추동물아문 내 다른 계통 간 대진화를 이해하기 위한 생물정보학적 접근

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 협동과정 생물정보학전공, 2022. 8. 김희발.생물정보학은 디지털화된 유전서열정보를 토대로 다양한 생명현상의 원리를 규명하고 이를 활용해 인류의 삶의 질을 향상하는 것을 목적으로 할 것이다. 생물정보학적 연구는 각 종을 대표하는 표준유전체 구축으로 일반적으로 시작되고 미소 혹은 대진화에 대한 후속 연구를 진행한다. 비록 짧은 단편 해독 기술이 유전체 시대를 열었지만, 짧은 단편의 조립은 낮은 연결성이나 오류가 포함된 유전자 주석 등의 심각한 문제들을 가진다. 긴 단편 해독 기술은 염색체 수준의 주석 (scaffolds)에 필수적인 보다 긴 컨티그 (contig) 조립을 생산할 수 있다. 짧은 단편에서 긴 단편으로 변화하는 페러다임에 발 맞추어, 본 논문은 표준유전체 구축에서 비교유전체 분석까지 이어지는 일련의 생물정보학적 분석에 대한 집약적 연구를 수행했으며, 이는 다양한 척추동물 종들의 대진화를 이해하는 것이 목적이다. 제 1장에서는 연구의 일반적인 배경지식을 정리하였다. 첫째로, 염색체 수준의 주석을 달성한 표준유전체 구축의 페러다임 변화를 설명했다. 다음으로, 특이적 형질에 관련된 분자 진화를 규명하는 비교유전체 분석 방법 및 사례를 정리했다. 제 2장에서는 표준유전체를 구축한 사례로서, 대한민국의 고유종인 큰볏말뚝망둥어의 염색체 수준 표준유전체를 구축했다. 척추동물 유전체 프로젝트와 국제 협력을 통해 4가지 최신 유전체 해독기술들 (Pacbio CLR, 10X Genomics linked reads, Bionano optical mapping, 그리고 Arima Genomics Hi-C)을 활용하여, 기존 표준유전체와 비교해 연결성 (continuity, Scaffold N50 기준)이 약 100배 향상되고 총 25개의 염색체를 가진 고품질 표준유전체를 완성했다. 또한, Pacbio Isoseq전사체 데이터를 유전자 주석에 활용하여 총 24,744개의 유전자를 발굴했다. 제 3장에서는 표준유전체 품질 평가 방법과 비교유전체학적 분석을 접목한 사례로서, 분화 시기가 오래된 종 간에도 BUSCO 유전자를 활용해 염색체 수준의 진화 양상을 탐색하는 방법과 척추동물 내에서 사례를 제시했다. 또한, 포유류, 조류, 어류 등 다양한 척추동물의 표준유전체에서 후속 분석 상의 문제를 야기하는 허위 소실 및 중복 오류를 탐색하는 방법과 사례를 제시하고 발생원인을 밝혔다. 제 4장에서는 기존의 비교유전체학적 분석을 적용한 사례로서, 실러캔스를 포함하는 육기아강 단계통 파생적 진화에 대한 분석을 통해 육상 적응 및 사지 출현의 분자 기작을 규명했다. 제 5장에서는 새로운 비교유전체학적 분석을 적용한 사례로서, 발성학습 조류 및 대조군 각각의 다계통 수렴 진화에 대한 분석을 통해 아미노산 수렴의 진화적 법칙을 제안하고 발성 학습에 연관된 후보 유전자를 발굴했다. 이러한 표준유전체 구축에서부터 비교유전체 분석으로 이어지는 생물정보학적 접근을 통해 규명된 주요 연구결과 중에, 염색체 상 텔로미어 서열 분포 및 아미노산 수렴 진화의 원리는 척추동물 외에 다른 분류 군에서도 비교될 기준이 될 수 있을 것으로 기대된다. 또한, 사지 발달 및 발성 학습에 연관된 후보 유전자를 발굴한 비교유전체학적 접근법은 전 세계 다양한 생물들의 다양한 유용 형질에 연관된 유용 유전자를 발굴하는데 활용될 수 있을 것이다.Bioinformatics aims to improve the quality of life of mankind by decoding molecular mechanisms of biological phenomena based on digitalized sequence information of various species. It generally begins with a construction of reference genomes representing each species and moves on downstream analyses for microevolution within species and macroevolutions between species. Although short-read sequencing technologies initiated genomics era, the short read assemblies had critical problems for lower continuity and erroneous gene annotations causing mis-interpretations. Long read sequencing technologies improved assembly continuities fundamental to chromosome-level scaffolds and corrected false annotations. Following up the paradigm shift from short-reads to long-reads, here, I performed a series of bioinformatic analyses to understand macroevolutions of various vertebrate species from reference genome construction to comparative genome approaches. Chapter 1 summarized the general background of this dissertation. First, it described the paradigm shift of the reference genome constructions achieving chromosome-scale scaffolds. Next, comparative genomic approaches for specific traits were summarized. Chapter 2, as a case of constructing a reference genome, illuminated a chromosome-level reference genome of giant-fin mudskipper, an endemic species in republic of Korea. Based on the four latest genome sequencing technologies (Pacbio CLR, 10X Genomics linked reads, Bionano optical mapping, and Arima Genomics Hi-C) in the international cooperation with the Vertebrate genomes project, it improved the 100-fold longer continuity (Scaffold N50) with a total of 25 chromosomal-level scaffolds compared to that of the previous genome. In addition, a total of 24,744 genes were annotated with Pacbio Isoseq transcriptome data. In Chapter 3, as a case of combining the reference genome quality evaluation method and comparative genomic analyses, a method was developed to explore the chromosomal evolution between vertebrate species in distant lineages focusing on the BUSCO genes. In addition, it suggested methods for detecting false loss and duplication errors that cause problems in downstream analyses in reference genomes of various vertebrate lineages, such as, mammals, birds, and fishes, and revealed how those kinds of errors occurred. In Chapter 4, as a case using the existing comparative genomic approaches, the molecular mechanisms of terrestrial adaptation and limb emergence were identified by applying the series of analyses for apormorphic evolution of the monophyletic lineage of lobed-fin fishes including coelacanths and human. In Chapter 5, as a case developing a new comparative genomic approach, the rule of amino acid convergence was proposed and candidate genes related to vocal learning were discovered through the multi-omic analyses for convergent evolution between polyphyletic lineages of vocal learning bird and control groups. Among the major findings of this study based on the bioinformatics approaches from the reference genome construction to comparative genomic researches, telomere sequence distributions on chromosomes and the principles of amino acid convergence would be a standard for comparisons in various lineages. In addition, the systemized comparative genomic approaches that identified candidate genes involved in limb development and vocal learning may be utilized to discover new candidate genes associated with various useful traits of living things in the world.Chapter 1. LITERATURE REVIEW 1 1.1 Paradigm shift in reference genome constructions 2 1.2 Comparative genomics for specific traits 3 Chapter 2. CHROMOSOME-LEVEL GENOME ASSEMBLY OF PERIOPHTHALMUS MAGNUSPINNATUS: AN INDIGENOUS MUDSKIPPER IN THE YELLOW SEA 5 2.1 Abstract 6 2.2 Introduction 7 2.3 Materials and Methods 9 2.4 Results and Discussion 13 Chapter 3. COMPARATIVE GENOMIC APPROACHES TO DETECT ERRONEOUS GENES IN REFERENCE GENOMES AND TO VISUALIZE CHROMOSOME EVOLUTION ACROSS VERTEBRATES 24 3.1 Abstract 25 3.2 Introduction 26 3.3 Materials and Methods 28 3.4 Results and Discussion 32 Chapter 4. COELACANTH-SPECIFIC ADAPTIVE GENES GIVE INSIGHTS INTO PRIMITIVE EVOLUTION FOR WATER-TO-LAND TRANSITION OF TETRAPODS 59 4.1 Abstract 60 4.2 Introduction 61 4.3 Materials and Methods 63 4.4 Results 69 4.5 Discussion 79 Chapter 5. AMINO ACID CONVERGENCES BETWEEN INDEPENDENT LINEAGES IN BIRDS GIVE EVOLUTIONARY INSIGHTS INTO AVIAN VOCAL LEARNING 85 5.1 Abstract 86 5.2 Introduction 87 5.3 Materials and Methods 89 5.4 Results 98 5.5 Discussion 159 GENERAL DISCUSSUSION 167 REFERENCES 168 요약(국문초록) 176박

The transcriptome of the marine calanoid copepod Temora longicornis under heat stress and recovery

Understanding the impacts of global change in zooplankton communities is crucial, as alterations in the zooplankton communities can affect entire marine ecosystems. Despite the economic and ecological importance of the calanoid copepod Temora longicornis in the Belgian part of the North Sea, molecular data is still very limited for this species. Using HiSeq Illumina sequencing, we sequenced the whole transcriptome of T. longicornis, after being exposed to realistic temperatures of 14 and 17 °C. After both an acute (1 day) and a more sustained (5 days) thermal exposure to 17 °C, we investigated gene expression differences with animals exposed to 14 °C, which may be critical for the thermal acclimation and resilience of this copepod species. We also studied the possibility of a short term stress recovery of a heat shock. A total of 179,569 transcripts were yielded, of which 44,985 putative ORF transcripts were identified. These transcripts were subsequently annotated into roughly 22,000 genes based on known sequences using Gene Ontology (GO) and KEGG databases. Temora only showed a mild response to both the temperature and the duration of the exposure. We found that the expression of 27 transcripts varied significantly with an increase in temperature of 3 °C, of which eight transcripts were differentially expressed after acute exposure only. Gene set enrichment analysis revealed that, overall, T. longicornis was more impacted by a sustained thermal exposure, rather than an immediate (acute) exposure, with two times as many enriched GO terms in the sustained treatment. We also identified several general stress responses independent of exposure time, such as modified protein synthesis, energy mobilisation, cuticle and chaperone proteins. Finally, we highlighted candidate genes of a possible recovery from heat exposure, identifying similar terms as those enriched in the heat treatments, i.e. related to for example energy metabolism, cuticle genes and extracellular matrix. The data presented in this study provides the first transcriptome available for T. longicornis which can be used for future genomic studies

Altered pathways in methylome and transcriptome longitudinal analysis of normal weight and bariatric surgery women

DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 +/- 1.6 kg/m(2)) and 24 obese women (BMI: 43.3 +/- 5.7 kg/m(2)) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q /=2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated

Altered pathways in methylome and transcriptome longitudinal analysis of normal weight and bariatric surgery women

[Abstract] DNA methylation could provide a link between environmental, genetic factors and weight control and can modify gene expression pattern. This study aimed to identify genes, which are differentially expressed and methylated depending on adiposity state by evaluating normal weight women and obese women before and after bariatric surgery (BS). We enrolled 24 normal weight (BMI: 22.5 ± 1.6 kg/m2) and 24 obese women (BMI: 43.3 ± 5.7 kg/m2) submitted to BS. Genome-wide methylation analysis was conducted using Infinium Human Methylation 450 BeadChip (threshold for significant CpG sites based on delta methylation level with a minimum value of 5%, a false discovery rate correction (FDR) of q < 0.05 was applied). Expression levels were measured using HumanHT-12v4 Expression BeadChip (cutoff of p ≤ 0.05 and fold change ≥2.0 was used to detect differentially expressed probes). The integrative analysis of both array data identified four genes (i.e. TPP2, PSMG6, ARL6IP1 and FAM49B) with higher methylation and lower expression level in pre-surgery women compared to normal weight women: and two genes (i.e. ZFP36L1 and USP32) that were differentially methylated after BS. These methylation changes were in promoter region and gene body. All genes are related to MAPK cascade, NIK/NF-kappaB signaling, cellular response to insulin stimulus, proteolysis and others. Integrating analysis of DNA methylation and gene expression evidenced that there is a set of genes relevant to obesity that changed after BS. A gene ontology analysis showed that these genes were enriched in biological functions related to adipogenesis, orexigenic, oxidative stress and insulin metabolism pathways. Also, our results suggest that although methylation plays a role in gene silencing, the majority of effects were not correlated.São Paulo Research Foundation; 2017/07220-7São Paulo Research Foundation; #2016/05638-1São Paulo Research Foundation; #2013/12819-4São Paulo Research Foundation; #2015/18669-0Instituto de Salud Carlos III; PI17/01287Ministerio de Economía y Empresa; MTM2014-52876-RMinisterio de Economía y Empresa; MTM2017-82724-RXunta de Galicia; ED431C-2016-015Xunta de Galicia; ED431G/0

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions

© 2014 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd. Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host-pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow-derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up-regulated at 24h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment

Seasonal and Form-Specific Gene Expression Signatures Uncover Different Generational Strategies of the Pelagic Tunicate Salpa thompsoni During the Southern Ocean Winter

The pelagic tunicate Salpa thompsoni is recognized as a major metazoan grazer in the Southern Ocean. Long term observations show an increase in this species’ biomass and a southward shift in its distribution both of which are positively correlated with ocean warming and winter sea ice decline around the Antarctic Peninsula. However, our understanding on how salps adapt their life cycle to the extreme seasonality of the Southern Ocean and the putative differences between its two reproductive forms (aggregates, solitaries) is rudimentary. In particular, our current knowledge of whether and how S. thompsoni overwinter is limited, largely due to winter sampling constraints. In this study, we investigated the form-specific gene expression profiles of Salpa thompsoni during the austral autumn and winter. Between the seasons, genes related to translation showed the biggest difference in gene expression. We found more genes were upregulated in solitaries compared to aggregates, indicating a potentially form-specific overwintering strategy. Our data provide first insights into the seasonal and form-specific physiology of salps by considering their complex life cycle, thereby contributing to a more comprehensive understanding of the response of salps to seasonal changes in their environment and to anthropogenic induced global climate change