1,721,054 research outputs found
Functional interrogation of the elovl5 gene regulation in Atlantic salmon
Get PDFLong-chain polyunsaturated fatty acids (LC-PUFA) and cholesterol are important for different physiological pathways including immunology. As these cannot be synthesized in the teleosts including Salmon itself, food supplement is considered the prime source in this case. Two duplicated genes in salmon elovl5a and elovl5b, derived from the WGD event at about 80 Mya, are important in the synthesis of LC-PUFAs. Differential gene regulation has been shown for these genes in previous studies, including highly divergent tissue regulation. This tissue-specific expression might be the result of different binding patterns of major lipid-metabolism transcriptional regulators. According to Carmona-Antoñanzas et al., 2016, elovl5a equally responded to the two sterol regulatory element-binding proteins (srebp-1 and Srebp-2) while elovl5b responded stronger to only Srebp-2. In this thesis, we investigated the roles of different transcription factors in the gene expression for the two salmon elovl5 gene copies. We first conducted a reporter-promoter assay to measure the effect of the Lxr-Srebp regulatory pathway on elovl5a and elovl5b regulation. Next, we conducted a CRISPR-Cas9 knockout experiment to assess the importance of predicted binding sites of Lxr, Srebp-1, Srebp-2, and NY-F for the transcriptional regulation of elovl5b.
Though our findings were not completely conclusive, induction of the Lxr-Srebp pathway increased the expression of both elovl5 promoters in the SHK-1 cells. Elovl5a was showing stronger gene expression compared to elovl5b, moreover synthetic (elovl5a ATAC and elovl5b ATAC) were stronger in upregulation than the native promoters. In the CRISPR-cas knockout experiments, we found that mutating Srebp-1, Srebp-2, and NF-Y binding motifs in the elovl5 promoter decreased elovl5b expression. In addition, our experiments indicate that Srebp binding sites were potentially important for regulating elovl5b gene through the Lxr-pathway as knock out of these binding sites led to ablation of the Lxr-agonist effect. In conclusion, our experiments shed light on the gene regulatory mechanisms of elovl5 genes, supporting previous findings regarding the Srebp pathway-mediated regulation in non-salmonid cell lines. Moreover, the novel approach tested for targeted CRISPR-based knock-down experiments will pave the way for more effective interrogation of gene regulatory mechanisms in the years to come.M-G
The effect of chromatin structure on duplicate gene expression in Atlantic salmon
No full textThe Atlantic salmon has gone through several WGD events and is left with almost half of its gene as duplicates. How expression differs in the duplicates is extra intriguing in regards to epigenetics. If the regulatory environment was inherited together with the gene after the WGD and the duplicates are similarly expressed can chromatin structure surrounding the gene give some insight into the gene regulation. ATAC-seq data has been used to determine open chromatin regions that might facilitate transcription. Does the open chromatin structure detected by ATAC-seq data affect the expression of genes in the Atlantic Salmon? There was a relationship found between the open chromatin regions surrounding the genes and their expression. A connection was found between the percentage of the promoter covered in peaks and an increase in expression, the expression diminished with a high coverage level in the region.
An EVE analysis of the salmons duplicates has been done to determine up- and downregulated duplicates in regards to an outgroup that has not gone through the salmonid specific WGD .Is the chromatin structure surrounding the gene duplicates similar for the duplicates with a similar expression? Looking into the difference in ATAC-seq peaks and difference in expression for the duplicates there are duplicates that have a high similarity in both expression and number of peaks, but some duplicates also have a high difference in peaks and expression. For the duplicated genes a higher number of peaks does not seem to give a higher level of expression
Exploring regulatory evolution after whole genome duplication using machine learning
Get PDFThe Atlantic salmon underwent a whole-genome duplication 80 million years ago and has kept around half of the duplicated genes. Over time, some genes have become more active, while others have become less active, due to regulatory changes. This thesis explores if it is possible to separate these genes by the number of nearby transcription factor binding sites.
With previously obtained information about the binding sites for different transcription factors for each gene and the direction of the expression level shift for this gene, a matrix was constructed containing the difference in bound transcription factor binding sites between the gene duplicates. One of the gene duplicates has a significant change in gene expression level, while the other is conserved. The duplicate pairs with increased expression in one copy are called upcons, and the pairs with decreased expression in one copy are called downcons.
Multiple machine learning algorithms were tested to classify upcons vs downcons. Overall, support vector machines performed best, achieving an accuracy of 67%.
In conclusion, the results are indicative that classification of the evolutionary direction of genes based on nearby transcription factor binding sites can be done
Insights into the regulation of LC-PUFA biosynthesis in wild and aquaculture Atlantic salmon in response to a vegetable oil diet
Get PDFThe aim of this master thesis was to use transcriptomics to evaluate if lipid metabolism of wild and aquaculture salmon is different. In recent years, there has been extensive research on lipid metabolism in salmonids related to the changes in lipid content of their commercial diet over the past decade (Bell et al. 2003, Bell et al. 2004, Naylor et al. 2009, Sprague et al. 2016, Tacon and Metian 2013). Fish and other vertebrates are not able to produce poly-unsaturated fatty acids (PUFA) de novo, so they are required in the diet (Tocher et al. 2003). When Linolenic acid (LA) and alpha-Linolenic acid (ALA) are available, these essential fatty acids (EFAs) can then be converted to long-chain poly-unsaturated fatty acids (LC-PUFA), which are required for fish health, but also important for the health benefits of human salmon consumption. Because salmon is a very important source of LC-PUFA in humans, there is much interest in understanding the workings of the salmonid apparatus for endogenous LC-PUFA synthesis. Globally, half of all fish consumed comes from aquaculture and especially in Europe has the production from fisheries steadily declined while that of aquaculture grows (FAO 2016). As aquaculture is supplying an increasing amount of the total fish for human consumption, the industry is working on maintaining or improving the LC-PUFA content of fish products while also switching to more sustainable feeds (Sprague et al. 2016). The ability of the fish to endogenously synthesise LC-PUFA from EFAs is an important part of this objective. One aspect to understanding and perhaps improving LC-PUFA synthesis in aquaculture salmon, is to understand the difference between the diets of aquaculture and wild salmon (Bell et al. 1994), and how endogenous LC-PUFA synthesis is affected by these. In this project we set out to look for differences between aquaculture and wild salmon in the activity of their endogenous LC-PUFA synthesis in response to two industrial diets with different LC-PUFA content.
We hypothesised that wild salmon are more efficient than aquaculture salmon at converting fatty acid precursors to LC-PUFAs in response to a plant-based diet which is low in LC-PUFA, most notably the particularly healthy omega-3s docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (Gillard et al. 2018). This was based on the fact that wild salmon have a more variable supply of food and eat prey low in DHA and EPA in the river life-stage (Gillard et al. 2018), while aquaculture salmon are kept in environments with enormous surplus of nutrients. Hence, wild populations are experiencing natural selection favouring individuals with the ability to effectively regulate endogenous LC-PUFA synthesis upon insufficient dietary levels. Such selection pressure for maintaining a likely costly metabolic response has not been present for the past 15 or so generations of domestication in aquaculture salmon.
Genes required for the synthesis of LC-PUFA are found to be more highly expressed in salmon fed VO than those fed FO, regardless of fish strain. Further, aquaculture salmon have higher expression of a set of genes important to LC-PUFA synthesis. It appears that the response to a low LC-PUFA diet is more pronounced in aquaculture salmon than wild, contrary to our hypothesis. However, the evidence from transcriptomics is limited and there is some variation in the results over time.submittedVersionM-BIO
Optimization of transfection of primary hepatocytes from Atlantic salmon for functional studies
No full textGene expression in eukaryotes are regulated through complex interactions between regulatory proteins, cis-regulatory sequences as well as chemical modifications and structure of chromatin. The function of cis-regulatory regions can be studied through expression of reporter genes in cell cultures. Ideally such reporter gene expression should be studied in biologically relevant cells, like primary cell cultures. The primary aim of this work was to optimize a protocol for transfection of primary hepatocytes from Atlantic salmon, to be able to perform functional studies on gene expression in liver. The secondary aim was to test this protocol by performing a promoter reporter assay on the cis-regulatory elements driving the expression in two duplicated Atlantic salmon fatty acyl elongase 5 genes involved in the endogenous synthesis of long-chain polyunsaturated fatty acid (LC-PUFA) synthesis in liver.
Transfection optimizations were done using both cationic lipid-mediated transfection and electroporation. Results from these optimization experiments suggests that electroporation is the superior option for transfecting primary Atlantic salmon hepatocytes, with transfection efficiency up to 30%. We also found that perfusion is an important, but technically challenging element for successful transfection in these cells. Finally, our experiment with the elovl5 promoter sequences showed induced reporter expression in hepatocytes.
In conclusion, we have developed a robust transfection protocol which paving the way for functional studies of gene regulatory logics in liver cells
Effekten av kromatinstruktur på uttrykket til genduplikater i Atlanterhavslaks
No full textThe Atlantic salmon has gone through several WGD events and is left with almost half of its gene as duplicates. How expression differs in the duplicates is extra intriguing in regards to epigenetics. If the regulatory environment was inherited together with the gene after the WGD and the duplicates are similarly expressed can chromatin structure surrounding the gene give some insight into the gene regulation. ATAC-seq data has been used to determine open chromatin regions that might facilitate transcription. Does the open chromatin structure detected by ATAC-seq data affect the expression of genes in the Atlantic Salmon? There was a relationship found between the open chromatin regions surrounding the genes and their expression. A connection was found between the percentage of the promoter covered in peaks and an increase in expression, the expression diminished with a high coverage level in the region.
An EVE analysis of the salmons duplicates has been done to determine up- and downregulated duplicates in regards to an outgroup that has not gone through the salmonid specific WGD .Is the chromatin structure surrounding the gene duplicates similar for the duplicates with a similar expression? Looking into the difference in ATAC-seq peaks and difference in expression for the duplicates there are duplicates that have a high similarity in both expression and number of peaks, but some duplicates also have a high difference in peaks and expression. For the duplicated genes a higher number of peaks does not seem to give a higher level of expression.Atlanterhavslaksen har gjennomgått flere helgenomduplikasjoner og står igjen med nesten halvparten av sine gener som duplikater. Forskjellen i genuttrykk hos duplikatene er ekstra spennende med tanke på epigenetikk. Dersom det regulatoriske miljøet ble arvet sammen med genet etter helgenomdupliseringen og duplikatene har lignende genuttrykk, så kan kromatinstruktur i nærområdet til genet gi innsikt i genreguleringen. ATAC-seq data har blitt brukt til å bestemme hvilke regioner som har åpen kromatinstruktur som kan legge til rette for transkripsjon. Påvirker kromatinstrukturen som ble detektert av ATAC-seq data genuttrykket i Atlanterhavslaksen? Det var et forhold som ble funnet mellom de åpne kromatinområdene i nærområdet til genene og deres genuttrykk. En sammenheng ble funnet mellom prosentandel av promotoren som var dekket i peaks og en økning i genuttrykk, genuttrykket sank samtidig som en høy andel av regionen var dekket av peaks.
En EVE-analyse av lakseduplikatene ble gjort for å finne ut av opp- og nedregulerte duplikater i forhold til en utgruppe som ikke har vært igjennom den laksespesifikke helgenomdupliseringen. Er kromatinstrukturen rundt genduplikatene lignende for duplikatene som har lignende genuttrykk? Når man ser nærmere på forskjellen i ATAC-seq peaks og forskjellen i genuttrykk for duplikatene, så har noen duplikater en høy likhet i både genuttrykk og antall peaks, men noen duplikater har også en stor forskjell i peaks og genuttrykk. For de dupliserte genene virker det ikke som om et større antall peaks fører til økt genuttrykk.M-BIA
Optimalisering av transfeksjon av primære hepatocytter fra atlanterhavslaks for funksjonelle studier
No full textGene expression in eukaryotes are regulated through complex interactions between regulatory proteins, cis-regulatory sequences as well as chemical modifications and structure of chromatin. The function of cis-regulatory regions can be studied through expression of reporter genes in cell cultures. Ideally such reporter gene expression should be studied in biologically relevant cells, like primary cell cultures. The primary aim of this work was to optimize a protocol for transfection of primary hepatocytes from Atlantic salmon, to be able to perform functional studies on gene expression in liver. The secondary aim was to test this protocol by performing a promoter reporter assay on the cis-regulatory elements driving the expression in two duplicated Atlantic salmon fatty acyl elongase 5 genes involved in the endogenous synthesis of long-chain polyunsaturated fatty acid (LC-PUFA) synthesis in liver.
Transfection optimizations were done using both cationic lipid-mediated transfection and electroporation. Results from these optimization experiments suggests that electroporation is the superior option for transfecting primary Atlantic salmon hepatocytes, with transfection efficiency up to 30%. We also found that perfusion is an important, but technically challenging element for successful transfection in these cells. Finally, our experiment with the elovl5 promoter sequences showed induced reporter expression in hepatocytes.
In conclusion, we have developed a robust transfection protocol which paving the way for functional studies of gene regulatory logics in liver cells.Genuttrykk i eukaryoter er regulert gjennom komplekse interaksjoner mellom regulatoriske proteiner, cisregulatoriske sekvenser og kjemiske og strukturelle endringer av kromatin. Funksjonen av cisregulatoriske sekvenser kan studeres gjennom uttrykk av reportergener i cellekulturer. Ideelt sett bør slike reportergenstudier utføres i biologisk relevante celler, som for eksempel primæcellekulturer. Hovedmålet med dette arbeidet var derfor å optimalisere transfeksjon av primære hepatocytter fra atlanterhavslaks, for å kunne utføre funksjonelle studier av genuttrykk i lever. Det sekundære målet var å teste denne protokollen ved å sammenlikne cis-regulatoriske sekvenser fra to elovl5-genduplikater i atlanterhavslaks. Disse to genene koder for elovl5-enzymer som er involvert i den endogene syntesen av langkjedede flerumettede fettsyrer i lever.
Transfeksjonsoptimaliseringen ble gjort både for kationisk lipid-mediert tranfeksjon og elektroporering. Resultatene fra optimaliseringsforsøkene viste at elektroporering er det mest effektive alternativet for å transfektere primære hepatocytter fra atlanterhavslaks, med en transfeksjonseffiktivitet på 30%. I tillegg fant vi at perfusjon er et viktig, men teknisk utfordrende element for vellykket transfeksjon. Til sist viste vi at elovl5-promotere kan indusere transkripsjon i primære leverceller.
Vi konkluderer med at vår nye transfeksjonsprotokoll vil kunne være et viktig verktøy for fremtidige studier av genregulering i leverceller.Norges forskningsråd: FRIMEDBIO ; Småforsk Biovit 2019M-BIOTE
Remodelling of H3K4me3 histone tail modifications across life stages in Atlantic salmon
Get PDFAtlantic salmon undergo a series of preparatory adaptation for a life in the sea, referred to as smoltification. Recent studies have focused on the genetic base of this process and revealed genome-wide transcriptional remodeling related to smoltification and seawater-transfer in different tissues, especially in the liver (Harvey, 2019; Gillard et al., 2018). However, we know very little about the mechanisms that drive the dynamics of genome regulation during smoltification.
A factor that controls the transcriptional level gene expression is Epigenetics, Epigenetics is a common term for various chemical alterations of the DNA molecule or chemical modifications to the histone tail, which is called histone tail modification. one of the well-studied histone tail modification H3K4me3 is reported to associate with active promoter. In this project, we aim to interpret this histone tail modification remodeling of gene expression during different life-stages of salmon.
We generate ChIP-seq data for H3K4me3, profiled the life-stage associated landscape of H3K4me3 in Atlantic salmon liver by using H3K4me specific antibody. 14 fish (4 parr, 6 smolt, and 4 seawater) across three life stages (parr, smolt, and seawater) were sampled.
We found H3K4me3 signals in all life stages are enriched in the promoter and 5UTR of gene region. The life stage-specific genes with the H3K4me3 signal reflecting the physiological characteristics of that stage and associated with smolt liver function and development. By linking the H3K4me3-marked genes with life-stage associated dynamic co-expressed gene cluster from previous study (Harvey, 2019), we find the histone code is weakly correlated with differential gene expression.
In conclusion, the analyses of H3K4me3-signals and gene expression fits with a model where some genes are regulated through histone tails remodeling during parr-smolt transformation, but that this level of genome regulation does not play the major role in the developmental transition from a parr to a smolt.
Unfortunately, we failed to produce high-quality ChIP-seq data from biological replicates. Hence, data analysis based on genome-wide patterns of H3K4me3-signals from pooled replicates, which only allowed us to assess the H3K4me3 signals in a qualitative way
Bidraget frå gjentakande element til evolusjon av laksefiskers genom
Get PDFEukaryotic genomes typically consist of a substantial proportion of repetitive DNA in the form of transposable elements (TEs) and satellite DNA. From studies of mammals, model species, and a few other well studied lineages it is clear that repetitive elements play roles in many important cellular processes and shape evolution of genomes and organisms. However, little is still known about the role of repetitive DNA in biology and genome evolution for most eukaryotic species. Here we use a suite of omics data and genomics analyses to ask the question: What is the role of repeat DNA in genome regulation and structural variation in the Atlantic salmon genome?
In papers 1 and 2 we studied the link between evolution of gene regulation and transposable elements in the context of the salmonid whole genome duplication. We found that gene duplicate copies that had evolved lower gene expression across most tissues had increased TE insertion rates in the promoters. In addition, we found that duplicate copies evolving liver specific increase in gene expression, had gained transcription factor binding sites (TFBS) for liver-specific transcription factors in the promoters, and some of these were found inside TEs. In depth analyses of cis-regulatory elements (CREs) in Paper 2 showed that 15-20% of CRE are within TEs (TE-CREs) and that there were fewer TE-CREs active in brain tissue compared to liver. Interestingly, a small heterogeneous group of TE subfamilies (11%) had contributed ~45% of all TE-CREs, but the ‘superspreader’ activity did not seem to peak in the time shortly following the WGD. CREs donated by ‘superspreaders’ were enriched for many different TFBSs, however, highly brain specific TFBSs were extremely rare in TEs, indicating that strong purifying selection shape TE-CRE evolution.
In Paper 3 we studied the role of repeat-DNA in the evolution of structural genomic variation (SVs) (>50bp). Leveraging seven new long read genome assemblies we find a large number of so far unknown structural variants, and conclude that satellite DNA is highly associated with indel variants. TEs, on the other hand, had contributed comparatively much less to the SV landscape. We conclude that the enormous number of novel SV found in our study is mostly due to satellite expansion and -contraction processes.
This thesis provides an advance in our understanding of the role of repetitive DNA in the evolution of salmonid genomes, and paves the way for future studies into the functional importance of this vast sea of repetitiveness.Ekaryote genom består vanlegvis av ein vesentleg andel gjentakande DNA i form av transposable element (TE-ar) og satelitt-DNA. Frå studiar i pattedyr, modelartar og nokre andre velstuderte organismar er det klart at gjentakande element speler rollar i mange viktige cellulære prosessar og formar evolusjon av genom og organismar. Mykje er imidlertid ukjend om rolla til gjentakande DNA i biologi og genomevolusjon i dei fleste eukaryote artar. Her nyttar vi ein rekke omikk-data og genomiske analysar for å stille spørsmålet: Kva er rolla til gjentakande DNA i genomregulering og strukturell variasjon i genomet til atlantisk laks? I artikkel 1 og 2 studerte vi koplinga mellom evolusjon av genregulering og transposable element i lys av den salmonide heilgenomdupliseringa. Vi fann at dupliserte gener som hadde redusert uttrykk over dei fleste vev hadde større andeler TE i promoteren. I tillegg fann vi at dupliserte gener som evolverte leverspesfikk auke i genuttrykk hadde fått transkripsjonsfaktorbindingsseter (TFBS) for lever-spesifikke transkripsjonsfaktorar, og at somme av desse var inne i TE-ar. Djupare analysar av cis-regulatoriske element (CRE-ar) i Artikkel 2 synte at 15-20% av CRE-ar er inne i TE-ar (TE-CRE-ar) og at det var færre TE-CRE-ar aktive i hjernevev enn i lever. Interessant nok bidro ei lita, heterogren gruppe (11%) med TE-ar med ca 45% av alle TE-CRE-ar, men denne “superspreiaraktiviteten” så ikkje ut til å nå høgda umiddelbart etter heilgenomdupliseringa. CRE-ar gitt av “superspreiarar” var anrika for mange forskjellige TFBS-ar, men svært hjernespesifikke TFBS-ar var svært sjeldne i TE-ar, som antyder at sterk seleksjon har forma TE-CRE-evolusjon.acceptedVersio
Assessment of genetic diversity in plants intended for enrichment planting compared to natural regeneration : a case study in Fjugstad Nature Reserve
Get PDFEuropean ash (Fraxinus excelsior) is facing severe population decline due to ash dieback disease, which not only poses a threat to the existence of this keystone species but also to more than 480 other species associated with it directly or indirectly. The disease's spread rate and extent of damage exceed the rate of regeneration, necessitating management actions to conserve the forest. Enrichment planting with relatively resistant plants can boost evolutionary stability and mitigate the dieback crisis. However, it's essential to genetically assess the enrichment planting materials to ensure their representativeness and sufficiency in diversity compared to the degraded forest.
Our study analyzed the genetic diversity of 100 samples from Danish origin grown in nurseries for enrichment plantation using 12 microsatellite loci. We compared results from these samples with 99 samples from Fjugstad Nature Reserve, 50 collected before the heavy dieback infestation in 2012, and 49 from 2022. The Fjugstad population's genetic diversity did not change before and after the heavy infection, but the coefficient of inbreeding was lower after the infestation, possibly due to the clearing of the canopy and middle layer in the forest, facilitating better pollen flow, seed dispersal and hence gene flow. The genetic diversity of the Fjugstad and Danish populations was also similar, except for higher private allelic richness and expected heterozygosity in the Danish population. moreover, both Fjugstad and Danish population had overlapping allele frequency which complements the genetic diversity of the Fjugstad forest. Additionally, the higher private allelic richness in the Danish population may offer tolerance against dieback, contributing to the forest's genetic tolerance and boosting its population. These findings suggest that Danish reproductive materials are representative and complementary to the genetic diversity of the Fjugstad population, aiding in conservation efforts and evolutionary stability of the declining ash forest in Fjugstad.M-ECO
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