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GECKO Conference 2021

Epigenetic Inheritance S2

technical paper

**Epigenetic Inheritance across species**

We are happy to invite you to register for the GECKO Conference – inspired by the Gametic Epigenetics Consortium Against Obesity (hence, GECKO). Professor Romain Barrès Head of the GECKO Consortium, and his research group will be hosting the conference, which will take place virtually between **May 25-28, 2021**. **Sessions will be live daily from 8.00-10.30 and 17.00-19.30 CET.**

The theme of the conference is intentionally broad. We will have sessions dedicated to the technical aspects of studying epigenetic inheritance, how we can use a comparative approach to understand inheritance mechanisms, the evolutionary explanations for epigenetic inheritance and a session focused on how our research could influence reproductive biology. 

We already have a fantastic line-up of speakers, including Noora Kotaja (University of Turku), Qi Chen (University of California), Michael Wilson (University of Toronto), Isabelle Mansuy (University and ETH Zurich), Oliver Rando (University of Massachusetts), Anita Öst (Linköping University) and Tobias Uller (Lund University).

The conference format will include panel discussions, short talks selected from submitted abstracts, poster sessions and a set of short and sharp talks to accompany these.

The deadline for submitting an abstract to be considered for a talk or poster is **May 4th .** 
Those selected to present will be informed in the following week, with **final posters due May 17th .**
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### ** Program at a Glance **
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# About GECKO

The Gametic Epigenetics Consortium Against Obesity (GECKO) is a research consortium funded by a Novo Nordisk Foundation Challenge grant from 2019 to 2025. The main goal of the consortium is to understand the effect of diet on the epigenetic signatures that are passed through sperm to offspring, and the effect these signatures have on the developing offspring and future generations. 

The GECKO is comprised of three leading research groups headed by Professor Romain Barrès at the University of Copenhagen, Professor Stephen Simpson at the University of Sydney and Professor Marcelo Nobrega at the University of Chicago.



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Find out more information and register to the event **for free** by making an account in Underline.

Evolutionary Biology

Comparative Biology

Paternal pre-conception nutrition has been associated with metabolic dysfunction in the offspring of several animal species. A likely mechanism contributing to this phenomenon is epigenetic inheritance. Several research groups, including ours, have shown that environmental factors, such as diet, physical activity and metabolic condition, can alter the epigenetic information carried in spermatozoa. Since the epigenetic signature of gametes plays an important role in embryonic development, we hypothesize that nutrition-induced changes of the sperm epigenome can modulate health of the next generation offspring. While the underlying mechanisms of epigenetic inheritance show broad evolutionary conservation, the specificity of epigenetic responses to nutritional challenge in different species remains unknown.
Among selected animal species, we are using the mini pig as a representative model of human obesity and whole-body metabolism and aim to determine the impact of a chronic Western-style dietary intake on the epigenetic programming of spermatozoa. Young, sexually mature male mini pigs are exposed to a 3-month dietary intervention of either a control diet or high-caloric Western-style diet, and semen samples are collected at different time points during the intervention. A washout period of the diets is followed to examine if nutrition-induced epigenetic variation in sperm is reversible. Isolated spermatozoa are examined for alterations in their epigenetic signature and downstream gene set enrichment analysis is performed to determine molecular pathways affected by the differential dietary intakes.


Western diet effects on metabolism and epigenetic reprogramming of spermatozoa in mini pigs

Small non-coding RNA sequencing has become increasingly popular in epigenetic studies and several families of sncRNAs has proven to have pivotal roles for intergenerational responses, embryonic development, genome integrity and other epigenetic processes. Despite recent progress, the analysis of sncRNA data is complicated and there are few bioinformatic tools available that are optimized for sncRNA. Instead, analysis is often built on workflows optimized for mRNA-seq. We believe that this undermines the results, as it does not extract the full potential of the data and often introduces transparency issues. Moreover, tools that are available usually requires the user to maneuver numerous software over multiple programming languages.

We present seqpac—a flexible R package integrating most part of sRNA-seq analysis into one pipeline. Seqpac provides a hub for adaptor trimming, read counting, sequence annotation, data analysis and visualization. Most importantly, it builds on sequence-based counts that preserves the sequence integrity, allowing users to re-annotate individual sequences at any point in the analysis, keeping track of mismatches and identifying cleavage sites. Seqpac builds a framework of functions around a PAC-object, containing Phenotypic information about samples, Annotations associated with each sequence and Counts for each sequence across all samples. This PAC-object is controlled by different targeting objects, making filtering, ordering and comparisons of samples and sequences quick and comprehensive. 

To demonstrate the power of sequence-based counting, the first release of seqpac contains a bundle of functions for advanced tRNA derived small RNA analysis, which can be applied on any species. In addition, to provide a foundation for analysis of all types of RNA families, a family of functions for fast alignment against both reference sequences (fasta) and genomic coordinates (gtf/gff) are included. For convenience we also provided wrapper functions for popular packages such as DESeq2 (differential expression) and FactorMiner (PCA). Seqpac comes with a step-by-step guide to make it accessible even for users with limited experience with R. In our recently published preprint we also compare seqpac to traditional sequencing pipelines and reanalyze previously published data, showing how seqpac may aid in addressing unreported bias and discovering novel rRNA derived fragments. Seqpac is now available at github (https://github.com/Danis102/seqpac).

Seqpac – a biologists guide to sncRNA-seq analysis

Regular physical exercise improves health by reducing the risk of a plethora of chronic disorders. We hypothesized that endurance exercise remodels the activity of gene enhancers in skeletal muscle, and that remodeling of enhancers contributes to the beneficial effects of exercise on human health. Here, we mapped the genome-wide positions and activities of enhancers in skeletal muscle biopsies collected from young sedentary men before and after an endurance exercise program. We identified profound remodeling of enhancer activity after training, with a large subset of the remodeled enhancers located in the proximity of genes transcriptionally regulated after exercise. We found an enrichment of disease-associated genetic variants within this enhancer subset, which supports the view that enhancers regulated by exercise training in the skeletal muscle target genes that control whole-body function. Our data provide evidence of a functional link between epigenetic rewiring after exercise training and the modulation of disease risk in humans.

Epigenetic rewiring of skeletal muscle enhancers after exercise training supports a role in whole-body function and human health

I work within research of how fetal and placental development is regulated through epigenetic mechanisms and am investigating how epigenetic changes established in early life are associated to health and disease later in life.
I am PI and project leader of a large cohort study in Tanzania that we are initiating, in January 2021. By taking advantage of two already established preconception and pregnancy cohorts (FOETALforNCD and STOPPAM) in rural, North-Eastern Tanzania, the overall objective is to conduct a follow-up examination of 500 children and their mothers, in order to evaluate the effect of anemia and malaria during pregnancy on the mother-child pair´s metabolic and infectious health, and the children´s growth, development and susceptibility to non-communicable diseases (NCDs). Furthermore, we want to investigate if epigenetic mechanisms, including DNA methylation, may be involved in regulating altered fetal/child growth and the susceptibility to NCDs, and how these epigenetic changes may be used as biomarkers to determine children at high disease risk. The two initial studies have already shown evidence of adverse effects of anemia and malaria in pregnancy on the children; therefore, it is greatly important to further follow up the health of these children as they grow. Hereby, new knowledge is gained on how to promote health during pregnancy and prevention of NCDs later in the children’s lives.


The PONA2 study – A rural Tanzanian follow-up study of children born to women with anemia and/or malaria during pregnancy

Diet impacts all aspects of health, including behaviour and reproduction. Obesogenic diets have been linked to increased anxiety in rodent models comparing effects of control to treatment “high-fat/Western” diets. Although the behavioural differences have been interpreted as a consequence of high fat, multiple components of the diets differ in a two-diet design, and therefore it is not clear whether fat is to blame. Inspired by ecological experimental designs, Nutritional Geometry uses a multi-dimensional framework to unpack individual and interactive effects of dietary components on health outcomes. We fed male C57Bl6 mice one of ten different isocaloric diets differing in macronutrient balance from 5 to 15 weeks of age, and assessed risk-taking behaviour using an Elevated-Plus maze. Treatment males were then mated to chow-fed females, and offspring raised on a chow diet. At 15 weeks of age, offspring behaviour was assessed using the Elevated-Plus maze. No influence of dietary fat was found on male risk-taking behaviour, rather, time spent in the closed arms was determined by an interactive effect between dietary protein and carbohydrates. Males fed low-protein, high-carbohydrate diets spent the most time in closed arms, indicating increased anxiety. Male offspring of these anxious fathers also showed increased anxiety, although the pattern of movement around the maze differed from their fathers. Epigenetic analyses of sperm and offspring tissues is currently underway to explore mechanisms driving these changes in offspring.

Intergenerational effects of dietary macronutrient balance on male mouse risk-taking behaviour

Most rodent dietary studies compare effects of a ‘high-fat diet’ versus a control. These two diets often differ in calorie and protein content, making it difficult to determine what aspect of the diet is responsible for changes observed. 

Nutritional Geometry provides an alternative and multi-dimensional framework to examine how macronutrient balance affects health. Here we examine effects of dietary macronutrient balance on body length and fasting blood glucose in young adult male mice and their female offspring.

Using the nutritional geometry framework, we identify that dietary fat is negatively associated with body length and fasting blood glucose levels in adolescent males. Further, females sired from males exposed to the differential diets exhibited contrasting effects to those observed in fathers, with paternal dietary fat intake being positively associated with female offspring body length and fasting blood glucose in some instances. 

Results from this study highlight the indirect inheritence of phenotypes from fathers to daughters on the parameters of body size and blood glucose, along with suggesting the importance of caloric load versus macronutrient makeup on inherited effects.

Macronutrients have differential effects on anthropometry and metabolic health in male mice and their daughters

DNA methylation editing at gametic hotspots of epigenetic variation
Eleonora Manitta, Lars Ingerslev, Mette Hyde Hochreuter, Romain Barrès

Recent evidences indicate that parental pre-conception exposures can influence offspring metabolic and cognitive functions. A mechanism that might contribute to this phenomenon is epigenetic inheritance. In fact, several factors, including diet, physical activity and metabolic condition, have been found to alter DNA methylation and non-coding RNA expression in sperm. Due to the role of gametes in initiating embryogenesis, such epigenetic changes might affect embryo development and the phenotype of the next generation. Interestingly, differentially methylated regions (DMRs) in sperm cells following environmental insults are found to be more frequently associated with genes involved in nervous system development and function. These sites can be therefore considered as gametic hotspots of epigenetic variation(GHEVs). The outcome of such site-specific methylation changes on embryogenesis, and more specifically neurogenesis, is however unclear. To investigate whether aberrant methylation at GHEVs might affect development, we apply a CRISPR-based system to alter methylation at specific loci in mouse embryonic stem cells (mESCs). We identified CpG sites with altered methylation level in human sperm after a 6-week training intervention and which are conserved in the mouse genome. The selected loci are associated with genes involved in neural development. A dCAS9-DNMT3A complex is used to methylate these CpGs in mESCs. The potential consequences of increased methylation are assessed by driving neuronal lineage commitment and evaluating differentiation efficiency. 


DNA methylation editing at gametic hotspots of epigenetic variation

The ‘western’ diet is an example of a diet used widely in diet research and is usually characterised as high in fat and sugar, although compositions vary greatly between studies. Some factors that are changed often, and to different extents, include the source and proportion of fat and sugar, as well as the total energy content of the diet. Therefore, it is no surprise that that much of the literature in this field is inconsistent and contradictory. In this study, we set out to isolate the effects of dietary sugar and fat on male fertility and metabolism. Additionally, we wanted to ascertain whether offspring food intake and macronutrient selection is altered by paternal diet and further investigate if paternal sugar/fat intake influences offspring susceptibility to obesity and other cardiometabolic disorders. We found that high fat diets in male studs led to the most obesogenic effect. Meanwhile in offspring, sex specific interactions were observed in a variety of outcomes. Male offspring showed a significant increase in body weights and fat mass, but changes were driven by different paternal diets. This work highlights the complex interactions between nutrition, reproduction and overall health and could provide a good foundation for future research.

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