Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Comparative genome-scale modelling of the pathogenic Flavobacteriaceae species Riemerella anatipestifer in China.

Riemerella anatipestifer (RA) is a gram-negative bacterium that has a high potential to infect waterfowl. Although more and more genomes of RA have been generated comparaed to genomic analysis of RA still remains at the level of individual species. In this study, we analysed the pan-genome of 27 RA virulent isolates to reveal the intraspecies genomic diversity from various aspects. The multi-locus sequence typing (MLST) analysis suggests that the geographic origin of R. anatipestifer is Guangdong province, China. Results of pan-genome analysis revealed an open pan-genome for all 27 species with the sizes of 2967 genes. We identified 387 genes among 555 unique genes originated by horizontal gene transfer. Further studies showed 204 strain-specific HGT genes were predicted as virulent proteins. Screening the 1113 core genes in RA through subtractive genomic approach, 70 putative vaccine targets out of 125 non-cytoplasmic proteins have been predicted. Further analysis of these non A. platyrhynchos homologous proteins predicted that 56 essential proteins as drug target with more interaction partners were involved in unique metabolic pathways of RA. In conclusion, the present study indicated the essence and the diversity of RA and also provides useful information for identification of vaccine and drugs candidates in future.

Single-cell DNA methylation and 3D genome architecture in the human brain.

Delineating the gene-regulatory programs underlying complex cell types is fundamental for understanding brain function in health and disease. Here, we comprehensively examined human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in 517 thousand cells (399 thousand neurons and 118 thousand non-neurons) from 46 regions of three adult male brains. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. We further developed single-cell methylation barcodes that reliably predict brain cell types using the methylation status of select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell-type-specific gene regulation in adult human brains.

Single-cell DNA Methylome and 3D Multi-omic Atlas of the Adult Mouse Brain.

Cytosine DNA methylation is essential in brain development and has been implicated in various neurological disorders. A comprehensive understanding of DNA methylation diversity across the entire brain in the context of the brain's 3D spatial organization is essential for building a complete molecular atlas of brain cell types and understanding their gene regulatory landscapes. To this end, we employed optimized single-nucleus methylome (snmC-seq3) and multi-omic (snm3C-seq1) sequencing technologies to generate 301,626 methylomes and 176,003 chromatin conformation/methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell type taxonomy that contains 4,673 cell groups and 261 cross-modality-annotated subclasses. We identified millions of differentially methylated regions (DMRs) across the genome, representing potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide multiplexed error-robust fluorescence in situ hybridization (MERFISH2) data validated the association of this spatial epigenetic diversity with transcription and allowed the mapping of the DNA methylation and topology information into anatomical structures more precisely than our dissections. Furthermore, multi-scale chromatin conformation diversities occur in important neuronal genes, highly associated with DNA methylation and transcription changes. Brain-wide cell type comparison allowed us to build a regulatory model for each gene, linking transcription factors, DMRs, chromatin contacts, and downstream genes to establish regulatory networks. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a companion whole-brain SMART-seq3 dataset. Our study establishes the first brain-wide, single-cell resolution DNA methylome and 3D multi-omic atlas, providing an unparalleled resource for comprehending the mouse brain's cellular-spatial and regulatory genome diversity.

Transcriptomic Characterization of a Chicken Embryo Model Infected With Duck Hepatitis A Virus Type 1.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most common and lethal pathogens in young ducklings. Live-attenuated DHAV vaccine (CH60 strain) developed by passaging in chicken embryos provided effective immune protection for ducklings. However, the accurate mechanism for such adaption in chicken embryos is not fully revealed. Here, we utilize RNA-sequencing to perform global transcriptional analysis of DHAV-1-innoculated embryonated livers along with histopathological and ultrastructural analysis. This study revealed that infection with DHAV-1 strain CH60 is associated with enhanced type I and II interferon responses, activated innate immune responses, elevated levels of suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3) accompanied with abnormalities in multiple metabolic pathways. Excessive inflammatory and innate immune responses induced by the CH60 strain are related to severe liver damage. Our study presents a comprehensive characterization of the transcriptome of chicken embryos infected with DHAV-CH60 and provides insight for in-depth exploration of viral adaption and virus-host interactions.

US10 Protein Is Crucial but not Indispensable for Duck Enteritis Virus Infection in Vitro.

To investigate the function of the duck enteritis virus (DEV) tegument protein US10, we generated US10 deletion and revertant mutants (ΔUS10 and US10FRT) via two-step RED recombination based on an infectious BAC clone of DEV CHv-BAC-G (BAC-G). In multistep growth kinetic analyses, ΔUS10 showed an approximately 100-fold reduction in viral titer, while the genome copies decreased only 4-fold compared to those of BAC-G. In one-step growth kinetic analyses, there were no significant differences in genome copies among BAC-G, ΔUS10 and US10FRT, but ΔUS10 still showed a 5- to 20-fold reduction in viral titer, and the replication defect of ΔUS10 was partially reversed by infection of US10-expressing cells. The transcription levels of Mx, OASL, IL-4, IL-6 and IL-10 in ΔUS10-infected duck embryo fibroblasts (DEFs) were significantly upregulated, while TLR3 was downregulated compared with those in BAC-G-infected DEFs. Taken together, these data indicated that US10 is vital for DEV replication and is associated with transcription of some immunity genes.

DHAV-1 2A1 Peptide - A Newly Discovered Co-expression Tool That Mediates the Ribosomal "Skipping" Function.

Duck hepatitis A virus 1 (DHAV-1) belongs to the genus Avihepatovirus in the family Picornaviridae. Little research has been carried out on the non-structural proteins of this virus. This study reports that 2A1 protein, the first non-structural protein on the DHAV-1 genome, has a ribosomal "skipping" function mediated by a "-GxExNPGP-" motif. In addition, we prove that when the sequence is extended 10aa to VP1 from the N-terminal of 2A1, the ribosome "skips" completely. However, as the N-terminus of 2A is shortened, the efficiency of ribosomal "skipping" reduces. When 2A1 is shortened to 10aa, it does not function. In addition, we demonstrate that N18, P19 G20, and P21 have vital roles in this function. We find that the expression of upstream and downstream proteins linked by 2A1 is different, and the expression of the upstream protein is much greater than that of the downstream protein. In addition, we demonstrate that it is the nature of 2A1 that is responsible for the expression imbalance. We also shows that the protein "cleavage" is not due to RNA "cleavage" or RNA transcription abnormalities, and the expressed protein level is independent of RNA transcriptional level. This study provides a systematic analysis of the activity of the DHAV-1 2A1 sequence and, therefore, adds to the "tool-box" that can be deployed for the co-expression applications. It provides a reference for how to apply 2A1 as a co-expression tool.

Suppression of NF-κB Activity: A Viral Immune Evasion Mechanism.

Nuclear factor-κB (NF-κB) is an important transcription factor that induces the expression of antiviral genes and viral genes. NF-κB activation needs the activation of NF-κB upstream molecules, which include receptors, adaptor proteins, NF-κB (IκB) kinases (IKKs), IκBα, and NF-κB dimer p50/p65. To survive, viruses have evolved the capacity to utilize various strategies that inhibit NF-κB activity, including targeting receptors, adaptor proteins, IKKs, IκBα, and p50/p65. To inhibit NF-κB activation, viruses encode several specific NF-κB inhibitors, including NS3/4, 3C and 3C-like proteases, viral deubiquitinating enzymes (DUBs), phosphodegron-like (PDL) motifs, viral protein phosphatase (PPase)-binding proteins, and small hydrophobic (SH) proteins. Finally, we briefly describe the immune evasion mechanism of human immunodeficiency virus 1 (HIV-1) by inhibiting NF-κB activity in productive and latent infections. This paper reviews a viral mechanism of immune evasion that involves the suppression of NF-κB activation to provide new insights into and references for the control and prevention of viral diseases.