Smart3-ATAC: a highly sensitive method for joint accessibility and full-length transcriptome analysis in single cells

Joint single-cell measurements of gene expression and DNA regulatory element activity holds great promise as a tool to understand transcriptional regulation. Towards this goal we have developed Smart3-ATAC, a highly sensitive method which allows joint mRNA and chromatin accessibility analysis genome wide in single cells. With Smart3-ATAC, we are able to obtain the highest possible quality measurements per cell. The method combines transcriptomic profiling based on the highly sensitive Smart-seq3 protocol on cytosolic mRNA, with a novel low-loss single-cell ATAC (scATAC) protocol to measure chromatin accessibility. Compared to current droplet multiome methods, the yield of both the scATAC protocol and mRNA-seq protocol is markedly higher.

Quantitative spatial and temporal assessment of regulatory element activity in zebrafish

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

Pathways to polar adaptation in fishes revealed by long-read sequencing

Long-read sequencing is driving a new reality for genome science where highly contiguous assemblies can be produced efficiently with modest resources. Genome assemblies from long-read sequencing are particularly exciting for understanding the evolution of complex genomic regions that are often difficult to assemble. In this study, we leveraged long-read sequencing to generate a high-quality genome assembly for an Antarctic eelpout, Opthalmolycus amberensis, the first for the globally distributed family Zoarcidae. We used this assembly to understand how O. amberensis has adapted to the harsh Southern Ocean and compared it to another group of Antarctic fishes: the notothenioids. We showed that from a genome-wide perspective, selection has largely acted on different targets in eelpouts relative to notothenioids. However, we did find some overlap; in both groups, selection has acted on genes involved in membrane structure and DNA repair. We found evidence for historical shifts of transposable element activity in O. amberensis and other polar fishes, perhaps reflecting a response to environmental change. We were specifically interested in the evolution of two complex genomic regions known to underlie key adaptations to polar seas: hemoglobin and antifreeze proteins (AFPs). We observed unique evolution of the hemoglobin MN cluster in eelpouts and related fishes in the suborder Zoarcoidei relative to other teleosts. For AFPs, we identified the first species in the suborder with no evidence of afpIII sequences (Cebidichthys violaceus), potentially reflecting a lineage-specific loss of this gene cluster. Beyond polar fishes, our results highlight the power of long-read sequencing to understand genome evolution.

In vivo evaluation of GG2–GG1/A2 element activity in the insulin promoter region using the CRISPR–Cas9 system

The insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2–GG1/A2–C1 (bases − 149 to − 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2–GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2–GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR–Cas9 technology. The mice with homozygous mutations in the GG2–GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2–GG1/A2 element in mice is important for Ins transcription in vivo.

Transposable element activity in the transcriptomic analysis of mouse pancreatic tumors

Transposable elements (TEs) are middle-repeated DNA sequences that can move along chromosomes using internal coding and regulatory regions. By their ability to move and because they are repeated, TEs can promote mutations. Especially they can alter the expression pattern of neighboring genes and have been shown to be involved in the mammalian regulatory network evolution. Human and mouse share more than 95% of their genomes and are affected by comparable diseases, which makes the mouse a perfect model in cancer research. However not much investigation concerning the mouse TE content has been made on this topics. In human cancer condition, a global activation of TEs can been observed which may ask the question of their impact on neighboring gene functioning. In this work, we used RNA sequences of highly aggressive pancreatic tumors from mouse to analyze the gene and TE deregulation happening in this condition compared to pancreas from healthy animals. Our results show that several TE families are deregulated and that the presence of TEs is associated with the expression divergence of genes in the tumor condition. These results illustrate the potential role of TEs in the global deregulation at work in the cancer cells.

Insights into the genomic evolution of insects from cricket genomes

Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket Gryllus bimaculatus, and the annotation of the 1.60-Gb genome of the Hawaiian cricket Laupala kohalensis. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the pickpocket class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.

Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells

Genome-wide association studies have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variant(s) within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naive CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.

DNA Modification Patterns within the Transposable Elements of the Fig (Ficus carica L.) Genome

Transposable element activity can be harmful to the host’s genome integrity, but it can also provide selective advantages. One strategy to cope with transposons is epigenetic control through DNA base modifications. We report the non-canonic DNA modification dynamics of fig (Ficus carica L.) by exploiting high-quality genome reference and related N4-methylcytosine (4mC) and N6-methyladenine (6mA) data. Overall, 1.49% of transposon nucleotides showed either 4mC or 6mA modifications: the 4mC/6mA ratio was similar in Class I and Class II transposons, with a prevalence of 4mC, which is comparable to coding genes. Different percentages of 4mC or 6mA were observed among LTR-retrotransposon lineages and sub-lineages. Furthermore, both the Copia and Gypsy retroelements showed higher modification rates in the LTR and coding regions compared with their neighbour regions. Finally, the unconventional methylation of retrotransposons is unrelated to the number of close genes, suggesting that the 4mC and 6mA frequency in LTR-retrotransposons should not be related to transcriptional repression in the adjacency of the element. In conclusion, this study highlighted unconventional DNA modification patterns in fig transposable elements. Further investigations will focus on functional implications, in regards to how modified retroelements affect the expression of neighbouring genes, and whether these epigenetic markers can spread from repeats to genes, shaping the plant phenotype.