National Institute of Genetics

The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, how cohesin organizes these domains in living cells, especially in active euchromatin, remains elusive. To address this question, we combined single-nucleosome imaging/tracking and super-resolution 3D-structured illumination microscopy (3D-SIM) with euchromatin-specific labeling of histone H3.3. Using this nanoscopic approach, we revealed that euchromatin forms condensed domains that are constrained by cohesin-mediated loops. This organization refines the classical textbook view of euchromatin as largely open, in line with emerging evidence. Transcription machinery appears to be located near the condensed domain surfaces/borders. Cohesin loss increased nucleosome fluidity within these domains without altering their overall compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings uncover an unexpected physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.

The trigeminal nerve consists of three major branches or rami (r) in jawed vertebrates: the ophthalmic (rV1), maxillary (rV2), and mandibular (rV3), with rV2 and rV3 innervating the upper and lower jaws, respectively. This three-branch configuration is conserved in the lamprey, a representative of the cyclostomes, the sole survivor of jawless vertebrates. While the first branch is widely accepted to be homologous to that of jawed vertebrate rV1, the homology of the other two remains unclear, so they are called rV1, rV2/3A, and rV2/3B. In this study, we investigate evolutionary modifications of the trigeminal nerve by comparing its organization in lamprey and jawed vertebrate embryos. By detailed neuronal tracing analysis of rV2/3B, which innervates the velum, we demonstrate that it consists solely of motor components. Conversely, we identify both sensory and motor fibers in rV2/3A. These results make a striking contrast to the trigeminal nerve of the jawed vertebrates, as their rV2 is purely sensory, but rV3 is a mixed nerve. Furthermore, through comparative analyses of sensory projection inside the brain, we clarify that the principal sensory nucleus of the trigeminal nerve (PrV) of anamniotes (shark, catfish, and Xenopus) lacks a somatotopic organization, suggesting that somatotopy evolved in at least the last common ancestor of amniotes. Last, by gene expression analysis of Drg11, a marker for the PrV and the spinal trigeminal nucleus (SpV), we show that a PrV-equivalent region is not present in the lamprey. These findings indicate significant structural changes in the trigeminal nerve associated with jaw acquisition, including shifts in innervation territories, the disappearance of the branch innervating the velum, and the emergence of novel brain nuclei.

Ornamental medaka strains derived from wild Japanese medaka (Oryzias latipes species complex) are bred worldwide. Over 200 years of selective breeding have produced over 700 strains with a wide variety of phenotypes, including diverse body coloration, scales, eyeball morphology, and fin and body shapes. In this study, we first identified and described 34 phenotypes in ornamental medaka strains. To understand the genomic basis of this phenotypic diversity and the domestication process, we performed whole-genome sequencing on 181 individuals of 86 ornamental Japanese medaka strains. Population genomic analyses revealed that modern ornamental medaka strains are genetically closer to the wild Southern Japan population of the Kansai-Setouchi regions, suggesting the origin of ornamental strains. In addition, the gene loci poc1a, tyr, nme2a, and gabrr2b have undergone selection during domestication. We performed genome-wide association studies analysis for 29 phenotypes observed in ornamental medaka strains and identified strong candidate genes for some phenotypes, including kcnq5a for hirenaga and swallow, bmp5 for deme, adcy5 for orochi, and kitlga for aurora, respectively. We found that loss of exon 8 of adcy5 caused melanism, a dark body color phenotype, in medaka, providing a molecular insight into this phenomenon in vertebrates and human familial dyskinesia. In addition, we uncovered the predominant candidate peaks of genome-wide association studies, including a total of 3,328 genes associated with 26 phenotypes. Our findings highlight the potential of population genomics to explore genotype-phenotype correlations and the genomic basis of body coloration and morphogenesis in medaka.

Introduction:Three-spined stickleback fish are famous for their diversity and charismatic social behavior. However, there are few neuroanatomical resources for studying the neural and brain mechanisms underlying their fascinating behavior.Methods and Results:We identify 11 brain areas important for social behavior by referencing brain atlases for six other teleost fishes. Brain regions were identified via neuroanatomical landmarks and we characterized the presence / absence of tyrosine hydroxylase (TH), a key gene product of the dopaminergic system, in those regions. Comparing the neuroanatomical location of these regions in the stickleback brain and the expression of TH therein to that of other fish species highlights similarities and differences and the need for a brain atlas specific to sticklebacks. This resource serves as a map of the location of regions important for social behavior in the stickleback brain.Conclusion:This resource will help guide future studies connecting gene function to social behavior through the brain and will enable future work to understand the evolution of neural mechanisms that contribute to the diversity of social behavior in this emerging model organism.

Long noncoding RNAs (lncRNAs) are abundantly transcribed in eukaryotes, but most of their physiological roles, especially in neural development, remain unclear due to limited invivo studies. Here we show that Lobe-less (LOL) lncRNA of Drosophila is expressed in developing neuronal cells and is required for the development of the mushroom body, a center of memory and learning in the insect brain. lol mutant flies exhibit defective morphology of the mushroom body and axon branch patterns, as well as misregulation of neurogenic genes. LOL RNA forms nuclear puncta and genetically interacts with Polycomb group genes in the regulation of homeotic genes. These findings demonstrate that this long ncRNA plays a critical role in the epigenetic control of neural circuit formation.

Abstract Precise spatiotemporal control of gene expression is essential for dissecting complex biological systems. Here, we introduce a phage-derived Cro/O R 3 binary system for transcriptional activation that functions robustly in zebrafish and human cells. A Cro repressor fused to the VP16 activation domain efficiently drives expression of O R 3 operator-linked genes, and its activity can be targeted to specific cell types using enhancer-trap and promoter-controlled zebrafish lines. As a proof of concept, we manipulate glutamate transporter Glast/Slc1a3b-expressing cells in the central nervous system by Cro/O R 3-mediated expression of a human ataxia-associated, uptake-deficient Glast/Slc1a3b variant, causing spastic, uncoordinated movement and axial deformation. Concurrent Gal4/UAS-mediated visualization reveals substantially disrupted positioning of motor neurons, highlighting their selective sensitivity to extracellular glutamate homeostasis during spinal circuit assembly. Together, these results establish the Cro/O R 3 system as an orthogonal platform for targeted gene expression, enabling precise mechanistic dissection of how specific cell types and their interactions underlie diverse biological processes.

Microbial translation arrest peptides monitor intracellular environments and feedback-regulate downstream gene expression. Previous studies have identified a class of bacterial arrest peptides with C-terminal RAPP-like sequences, encoded upstream of genes involved in protein localization. In this study, we found that among RAPP-like sequences, RAPP (Arg-Ala-Pro-Pro) and RGPP (Arg-Gly-Pro-Pro) could more readily evolve into translation-impeding sequences with a particularly robust arrest that is refractory to EF-P. RAPP-like motifs were found to be strongly excluded from bacterial proteomes, likely reflecting the risk of disrupting the cellular translation system. Meanwhile, these motifs tended to occur near the C-terminus of relatively small secretory and membrane proteins. Notably, they were encoded upstream of genes with diverse functions beyond protein localization. Indeed, we identified seven RAPP/RGPP-containing arrest peptides from Streptomyces lividans encoded upstream of genes with diverse functions. These findings illustrate the bidirectional evolution of RAPP-containing proteins: their elimination from bacterial proteomes and their adaptation into arrest peptides with various regulatory roles.

Cohesin folds genomes into chromatin loops, the roles of which are under debate. We found that double-strand breaks (DSBs) induce de novo formation of chromatin loops in human cells, with the loop base positioned at the DSB site. These loops form in the S and G2 phases of the cell cycle during homologous recombination repair, concomitantly with DNA end resection and radiation-sensitive protein 51 (RAD51) recruitment. RAD51 shows a broad (megabase-sized) chromatin domain reflective of the homology search. This domain is regulated by cohesin unloader and overlaps with chromatin regions reeled through the break-anchored loop, suggesting that loop extrusion regulates the homology search. Indeed, depletion of the loop-extruding cohesin subunit NIPBL lowers homologous recombination in mouse embryonic stem cells, and this effect is more pronounced when the homologous recombination donor is hundreds of kilobases from the DSB. Our data indicate that loop-extruding cohesin promotes the mammalian homology search by facilitating break-chromatin interactions.

Replication origins in human cells form clusters ranging from tens to hundreds of kilobases called initiation zones (IZs), typically located in intergenic regions between active genes. On a larger megabase scale, chromosomes replicate following temporally defined replication timing (RT), where euchromatic regions replicate early, and heterochromatic regions replicate late. Although the stochastic model of IZ firing with a temporally regulated limiting factor can explain RT formation, this limiting factor in human cells remains unclear. To investigate the relationship between IZ and RT, we map the temporal firing pattern of IZs and examine the genome-wide distributions of replication licensing and firing factors in human cells. We identify TRESLIN-MTBP as a key limiting firing factor for replication initiation. Its loading onto phosphorylated MCM2–7 double hexamer (MCM-DH) is controlled by the opposing phosphorylation events on MCM-DH by Dbf4-dependent kinase and RIF1-Protein Phosphatase 1, which ultimately determine IZs and establish RT.