Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility.

Chromatin accessibility is a hallmark of active regulatory regions and is functionally linked to transcriptional networks and cell identity. However, the molecular mechanisms and networks that govern chromatin accessibility have not been thoroughly studied. Here we conducted a genome-wide CRISPR screening combined with an optimized ATAC-see protocol to identify genes that modulate global chromatin accessibility. In addition to known chromatin regulators like CREBBP and EP400, we discovered a number of previously unrecognized proteins that modulate chromatin accessibility, including TFDP1, HNRNPU, EIF3D and THAP11 belonging to diverse biological pathways. ATAC-seq analysis upon their knockouts revealed their distinct and specific effects on chromatin accessibility. Remarkably, we found that TFDP1, a transcription factor, modulates global chromatin accessibility through transcriptional regulation of canonical histones. In addition, our findings highlight the manipulation of chromatin accessibility as an approach to enhance various cell engineering applications, including genome editing and induced pluripotent stem cell reprogramming.

Generation of chimpanzee induced pluripotent stem cell lines for cross-species comparisons.

As humans' closest living relatives, chimpanzees offer valuable insights into human evolution. However, technical and ethical limitations hinder investigations into the molecular and cellular foundations that distinguish chimpanzee and human traits. Recently, induced pluripotent stem cells (iPSCs) have emerged as a novel model for functional comparative studies and provided a non-invasive alternative for studying embryonic phenomena. In this study, we generated five new chimpanzee iPSC lines from peripheral blood cells and skin fibroblasts with SeV vectors carrying four reprogramming factors (human OCT3/4, SOX2, KLF4, and L-MYC) and characterized their pluripotency and differentiation potential. We also examined the expression of a human-specific non-coding RNA, HSTR1, which is predicted to be involved in human brain development. Our results show that the chimpanzee iPSCs possess pluripotent characteristics and can differentiate into various cell lineages. Moreover, we found that HSTR1 is expressed in human iPSCs and their neural derivatives but not in chimpanzee counterparts, supporting its possible role in human-specific brain development. As iPSCs are inherently variable due to genetic and epigenetic differences in donor cells or reprogramming procedures, it is essential to expand the number of chimpanzee iPSC lines to comprehensively capture the molecular and cellular properties representative of chimpanzees. Hence, our cells provide a valuable resource for investigating the function and regulation of human-specific transcripts such as HSTR1 and for understanding human evolution more generally.

Kangaroo endogenous retrovirus (KERV) forms megasatellite DNA with a simple repetition pattern in which the provirus structure is retained.

The kangaroo endogenous retrovirus (KERV) was previously reported to have undergone a rapid copy number increase in the red-necked wallaby; however, the mode of amplification was left to be clarified. The present study revealed that the long terminal repeat (LTR) (0.6 kb) and internal region (2.0 kb) of a provirus are repeated alternately, forming megasatellite DNA which we named kervRep. This repetition pattern was the same as that observed for walbRep, megasatellite DNA originating from another endogenous retrovirus. Their formation process can be explained using a simple model: pairing slippage followed by homologous recombination. This model features that the initial step is triggered by the presence of two identical sequences within a short distance; the possession of LTRs by endogenous retroviruses fulfills this condition. The discovery of two cases suggests that formation of this type of satellite DNA is one of non-negligible effects of endogenous retroviruses on their host genomes.

Geography-Dependent Horizontal Gene Transfer from Vertebrate Predators to Their Prey.

Horizontal transfer (HT) of genes between multicellular animals, once thought to be extremely rare, is being more commonly detected, but its global geographic trend and transfer mechanism have not been investigated. We discovered a unique HT pattern of Bovine-B (BovB) LINE retrotransposons in vertebrates, with a bizarre transfer direction from predators (snakes) to their prey (frogs). At least 54 instances of BovB HT were detected, which we estimate to have occurred across time between 85 and 1.3 Ma. Using comprehensive transcontinental sampling, our study demonstrates that BovB HT is highly prevalent in one geographical region, Madagascar, suggesting important regional differences in the occurrence of HTs. We discovered parasite vectors that may plausibly transmit BovB and found that the proportion of BovB-positive parasites is also high in Madagascar where BovB thus might be physically transported by parasites to diverse vertebrates, potentially including humans. Remarkably, in two frog lineages, BovB HT occurred after migration from a non-HT area (Africa) to the HT hotspot (Madagascar). These results provide a novel perspective on how the prevalence of parasites influences the occurrence of HT in a region, similar to pathogens and their vectors in some endemic diseases.

Replacement of owl monkey centromere satellite by a newly evolved variant was a recent and rapid process.

Alpha satellite DNA is a major DNA component of primate centromeres. We previously reported that Azara's owl monkey has two types of alpha satellite DNA, OwlAlp1 and OwlAlp2. OwlAlp2 (344 bp) exhibits a sequence similarity throughout its entire length with alpha satellite DNA of closely related species. OwlAlp1 (185 bp) corresponds to the part of OwlAlp2. Based on the observation that the CENP-A protein binds to OwlAlp1, we proposed that OwlAlp1 is a relatively new repetitive DNA that replaced OwlAlp2 as the centromeric satellite DNA. However, a detailed picture of the evolutionary process of this centromere DNA replacement remains largely unknown. Here, we performed a phylogenetic analysis of OwlAlp1 and OwlAlp2 sequences, and also compared our results to alpha satellite DNA sequences of other primate species. We found that: (i) OwlAlp1 exhibits a higher similarity to OwlAlp2 than to alpha satellite DNA of other species, (ii) OwlAlp1 has a single origin, and (iii) sequence variation is lower in OwlAlp1 than in OwlAlp2. We conclude that OwlAlp1 underwent a recent and rapid expansion in the owl monkey lineage. This centromere DNA replacement could have been facilitated by the heterochromatin reorganization that is associated with the adaptation of owl monkeys to a nocturnal lifestyle.

Exploring correlations in genetic and cultural variation across language families in northeast Asia.

Culture evolves in ways that are analogous to, but distinct from, genomes. Previous studies examined similarities between cultural variation and genetic variation (population history) at small scales within language families, but few studies have empirically investigated these parallels across language families using diverse cultural data. We report an analysis comparing culture and genomes from in and around northeast Asia spanning 11 language families. We extract and summarize the variation in language (grammar, phonology, lexicon), music (song structure, performance style), and genomes (genome-wide SNPs) and test for correlations. We find that grammatical structure correlates with population history (genetic history). Recent contact and shared descent fail to explain the signal, suggesting relationships that arose before the formation of current families. Our results suggest that grammar might be a cultural indicator of population history while also demonstrating differences among cultural and genetic relationships that highlight the complex nature of human history.

Identification of Chromosome 17 Trisomy in a Cynomolgus Monkey (Macaca fascicularis) by Multicolor FISH Techniques.

A female cynomolgus monkey (Macaca fascicularis) with facial features characteristic of Down syndrome showed abnormal behavior, unwariness toward humans, and poor concentration. The number of metaphase chromosomes in blood lymphocytes was examined and found to be 43, which indicated one extra chromosome to the normal diploid number (2n = 42). We then used Q-banding and multicolor FISH techniques to identify the extra chromosome. The results revealed an additional chromosome 17, with no other chromosomal rearrangements, such as translocations. Since no mosaicism or heterozygous variant chromosomes were observed, full trisomy 17 was assessed in this female cynomolgus monkey. Chromosome 17 corresponds to human chromosome 13, and human trisomy 13, known as Patau syndrome, results in severe clinical signs and, often, a short life span; however, this individual has reached an age of 10 years with only mild clinical signs. Although genomic differences exist between human and macaques, this individual's case could help to reveal the pathological and genetic mechanisms of Patau syndrome.

Progesterone depletion results in Lamin B1 loss and induction of cell death in mouse trophoblast giant cells.

Trophoblast giant cells (TGCs), a mouse trophoblast subtype, have large amounts of cytoplasm and high ploidy levels via endocycles. The diverse functions and gene expression profiles of TGCs have been studied well, but their nuclear structures remain unknown. In this study, we focus on Lamin B1, a nuclear lamina, and clarify its expression dynamics, regulation and roles in TGC functions. TGCs that differentiated from trophoblast stem cells were used. From days 0 to 9 after differentiation, the number of TGCs gradually increased, but the amount of LMNB1 peaked at day 3 and then slightly decreased. An immunostaining experiment showed that LMNB1-depleted TGCs increased after day 6 of differentiation. These LMNB1-depleted TGCs diffused peripheral localization of the heterochromatin marker H3K9me2 in the nuclei. However, LMINB1-knock down was not affected TGCs specific gene expression. We found that the death of TGCs also increased after day 6 of differentiation. Moreover, Lamin B1 loss and the cell death in TGCs were protected by 10-6 M progesterone. Our results conclude that progesterone protects against Lamin B1 loss and prolongs the life and function of TGCs.

The Heterochromatin Block That Functions as a Rod Cell Microlens in Owl Monkeys Formed within a 15-Myr Time Span.

In rod cells of many nocturnal mammals, heterochromatin localizes to the central region of the nucleus and serves as a lens to send light efficiently to the photoreceptor region. The genus Aotus (owl monkeys) is commonly considered to have undergone a shift from diurnal to nocturnal lifestyle. We recently demonstrated that rod cells of the Aotus species Aotus azarae possess a heterochromatin block at the center of its nucleus. The purpose of the present study was to estimate the time span in which the formation of the heterochromatin block took place. We performed three-dimensional hybridization analysis of the rod cell of another species, Aotus lemurinus. This analysis revealed the presence of a heterochromatin block that consisted of the same DNA components as those in A. azarae. These results indicate that the formation was complete at or before the separation of the two species. Based on the commonly accepted evolutionary history of New World monkeys and specifically of owl monkeys, the time span for the entire formation process was estimated to be 15 Myr at most.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

Targeting G-quadruplex DNA as cognitive function therapy for ATR-X syndrome.

Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome is caused by mutations in ATRX, which encodes a chromatin-remodeling protein. Genome-wide analyses in mouse and human cells indicate that ATRX tends to bind to G-rich sequences with a high potential to form G-quadruplexes. Here, we report that Atrx mutation induces aberrant upregulation of Xlr3b expression in the mouse brain, an outcome associated with neuronal pathogenesis displayed by ATR-X model mice. We show that ATRX normally binds to G-quadruplexes in CpG islands of the imprinted Xlr3b gene, regulating its expression by recruiting DNA methyltransferases. Xlr3b binds to dendritic mRNAs, and its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α, promoting synaptic dysfunction. Notably, treatment with 5-ALA, which is converted into G-quadruplex-binding metabolites, reduces RNA polymerase II recruitment and represses Xlr3b transcription in ATR-X model mice. 5-ALA treatment also rescues decreased synaptic plasticity and cognitive deficits seen in ATR-X model mice. Our findings suggest a potential therapeutic strategy to target G-quadruplexes and decrease cognitive impairment associated with ATR-X syndrome.

Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish.

BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Co-Opted Megasatellite DNA Drives Evolution of Secondary Night Vision in Azara's Owl Monkey.

Owl monkeys (genus Aotus) are the only taxon in simian primates that consists of nocturnal or otherwise cathemeral species. Their night vision is superior to that of other monkeys, apes, and humans but not as good as that of typical nocturnal mammals. This incomplete night vision has been used to conclude that these monkeys only secondarily adapted to a nocturnal lifestyle, or to their cathemeral lifestyle that involves high night-time activity. It is known that the rod cells of many nocturnal mammals possess a unique nuclear architecture in which heterochromatin is centrally located. This "inverted nuclear architecture", in contrast with "conventional nuclear architecture", provides elevated night vision by passing light efficiently to the outer segments of photoreceptors. Owl monkey rod cells exhibit an intermediate chromatin distribution, which may provide them with less efficient night vision than other nocturnal mammals. Recently, we identified three megasatellite DNAs in the genome of Azara's owl monkey (Aotus azarae). In the present study, we show that one of the three megasatellite DNAs, OwlRep, serves as the primary component of the heterochromatin block located in the central space of the rod nucleus in A. azarae. This satellite DNA is likely to have emerged in the Aotus lineage after its divergence from those of other platyrrhini taxa and underwent a rapid expansion in the genome. Our results indicate that the heterochromatin core in the A. azarae rod nucleus was newly formed in A. azarae or its recent ancestor, and supports the hypothesis that A. azarae, and with all probability other Aotus species, secondarily acquired night vision.

Physical contact and carbon transfer between a lichen-forming Trebouxia alga and a novel Alphaproteobacterium.

Recent progress in molecular techniques has begun to alter traditional recognition of lichens as symbiotic organisms comprised of a fungus and photosynthetic partners (green algae and/or cyanobacteria). Diverse organisms, especially various non-photosynthetic bacteria, are now indicated to be integral components of lichen symbiosis. Although lichen-associated bacteria are inferred to have functions that could support the symbiosis, little is known about their physical and nutritional interaction with fungi and algae. In the present study, we identified specific interaction between a lichen-forming alga and a novel bacterium. Trebouxia alga was isolated from a lichen, Usnea hakonensis, and kept as a strain for 8 years. Although no visible bacterial colonies were observed in this culture, high-throughput sequencing of DNA isolated from the culture revealed that the strain is composed of a Trebouxia alga and an Alphaproteobacterium species. In situ hybridization showed that bacterial cells were localized on the surface of the algal cells. Physiological assays revealed that the bacterium was able to use ribitol, glucose and mannitol, all of which are known to exist abundantly in lichens. It was resistant to three antibiotics. Bacteria closely related to this species were also identified in lichen specimens, indicating that U. hakonensis may commonly associate with this group of bacteria. These features of the novel bacterium suggest that it may be involved in carbon cycling of U. hakonensis as a member of lichen symbiosis and less likely to have become associated with the alga after isolation from a lichen.

A Pair of Maternal Chromosomes Derived from Meiotic Nondisjunction in Trisomy 21 Affects Nuclear Architecture and Transcriptional Regulation.

Eukaryotic genomes are organised into complex higher-order structures within the nucleus, and the three-dimensional arrangement of chromosomes is functionally important for global gene regulation. The existence of supernumerary chromosome 21 in Down syndrome may perturb the nuclear architecture at different levels, which is normally optimised to maintain the physiological balance of gene expression. However, it has not been clearly elucidated whether and how aberrant configuration of chromosomes affects gene activities. To investigate the effects of trisomy 21 on nuclear organisation and gene expression, we performed three-dimensional fluorescent imaging analysis of chromosome-edited human induced pluripotent stem cells (iPSCs), which enabled identification of the parental origin of the three copies of chromosome 21. We found that two copies of maternal chromosomes resulting from meiotic nondisjunction had a higher tendency to form an adjacent pair and were located relatively distant from the nuclear membrane, suggesting the conserved interaction between these homologous chromosomes. Transcriptional profiling of parental-origin-specific corrected disomy 21 iPSC lines indicated upregulated expression of the maternal alleles for a group of genes, which was accompanied by a fluctuating expression pattern. These results suggest the unique effects of a pair of maternal chromosomes in trisomy 21, which may contribute to the pathological phenotype.

Visualization of the spatial arrangement of nuclear organization using three-dimensional fluorescence in situ hybridization in early mouse embryos: A new "EASI-FISH chamber glass" for mammalian embryos.

The fertilized oocyte begins cleavage, leading to zygotic gene activation (ZGA), which re-activates the resting genome to acquire totipotency. In this process, genomic function is regulated by the dynamic structural conversion in the nucleus. Indeed, a considerable number of genes that are essential for embryonic development are located near the pericentromeric regions, wherein the heterochromatin is formed. These genes are repressed transcriptionally in somatic cells. Three-dimensional fluorescence in situ hybridization (3D-FISH) enables the visualization of the intranuclear spatial arrangement, such as gene loci, chromosomal domains, and chromosome territories (CTs). However, the 3D-FISH approach in mammalian embryos has been limited to certain repeated sequences because of its unfavorable properties. In this study, we developed an easy-to-use chamber device (EASI-FISH chamber) for 3D-FISH in early embryos, and visualized, for the first time, the spatial arrangements of pericentromeric regions, the ZGA-activated gene (Zscan4) loci, and CTs (chromosome 7), simultaneously during the early cleavage stage of mouse embryos by 3D-FISH. As a result, it was revealed that morphological changes of the pericentromeric regions and CTs, and relocation of the Zscan4 loci in CTs, occurred in the 1- to 4-cell stage embryos, which was different from those in somatic cells. This convenient and reproducible 3D-FISH technique for mammalian embryos represents a valuable tool that will provide insights into the nuclear dynamics of development.

Phylogenomics and Morphology of Extinct Paleognaths Reveal the Origin and Evolution of the Ratites.

The Palaeognathae comprise the flightless ratites and the volant tinamous, and together with the Neognathae constitute the extant members of class Aves. It is commonly believed that Palaeognathae originated in Gondwana since most of the living species are found in the Southern Hemisphere [1-3]. However, this hypothesis has been questioned because the fossil paleognaths are mostly from the Northern Hemisphere in their earliest time (Paleocene) and possessed many putative ancestral characters [4]. Uncertainties regarding the origin and evolution of Palaeognathae stem from the difficulty in estimating their divergence times [1, 2] and their remarkable morphological convergence. Here, we recovered nuclear genome fragments from extinct elephant birds, which enabled us to reconstruct a reliable phylogenomic time tree for the Palaeognathae. Based on the tree, we identified homoplasies in morphological traits of paleognaths and reconstructed their morphology-based phylogeny including fossil species without molecular data. In contrast to the prevailing theories, the fossil paleognaths from the Northern Hemisphere were placed as the basal lineages. Combined with our stable divergence time estimates that enabled a valid argument regarding the correlation with geological events, we propose a new evolutionary scenario that contradicts the traditional view. The ancestral Palaeognathae were volant, as estimated from their molecular evolutionary rates, and originated during the Late Cretaceous in the Northern Hemisphere. They migrated to the Southern Hemisphere and speciated explosively around the Cretaceous-Paleogene boundary. They then extended their distribution to the Gondwana-derived landmasses, such as New Zealand and Madagascar, by overseas dispersal. Gigantism subsequently occurred independently on each landmass.

Periostin is required for matricellular localization of CCN3 in periodontal ligament of mice.

CCN3 is a matricellular protein that belongs to the CCN family. CCN3 consists of 4 domains: insulin-like growth factor-binding protein-like domain (IGFBP), von Willebrand type C-like domain (VWC), thrombospondin type 1-like domain (TSP1), and the C-terminal domain (CT) having a cysteine knot motif. Periostin is a secretory protein that binds to extracellular matrix proteins such as fibronectin and collagen. In this study, we found that CCN3 interacted with periostin. Immunoprecipitation analysis revealed that the TSP1-CT interacted with the 4 repeats of the Fas 1 domain of periostin. Immunofluorescence analysis showed co-localization of CCN3 and periostin in the periodontal ligament of mice. In addition, targeted disruption of the periostin gene in mice decreased the matricellular localization of CCN3 in the periodontal ligament. Thus, these results indicate that periostin was required for the matricellular localization of CCN3 in the periodontal ligament, suggesting that periostin mediated an interaction between CCN3 and the extracellular matrix.

Male-specific mortality biases secondary sex ratio in Eurasian tree sparrows Passer montanus.

Sex allocation theory predicts that parents bias the offspring sex ratio strategically. In avian species, the offspring sex ratio can be biased at multiple growth stages, although the mechanisms are not well known. It is crucial to reveal a cause and timing of biased offspring sex ratio. We investigated (i) offspring sex ratio at multiple growth stages, from laying to fledging; and (ii) the stage at which offspring sex ratio became biased; and (iii) the cause of biased offspring sex ratio in Eurasian tree sparrows Passer montanus. Sex determination of 218 offspring, including hatchlings and unhatched eggs from 41 clutches, suggested that the offspring sex ratio was not biased at the egg-laying stage but was significantly female-biased after the laying stage due to higher mortality of male embryos. Half of the unhatched eggs showed no sign of embryo development (37/74, 50.00%), and most undeveloped eggs were male (36/37, 97.30%). Additional experiments using an incubator suggested that the cause of embryo developmental failure was a lack of developmental ability within the egg, rather than a failure of incubation. This study highlights the importance of clarifying offspring sex ratio at multiple stages and suggests that offspring sex ratio is adjusted after fertilization.

Fluorescence-Activated Cell Sorting and Gene Expression Profiling of GFP-Positive Cells from Transgenic Zebrafish Lines.

Gene expression profiling is a useful approach for deeper understanding of the specificity of cells, tissues, and organs in the transcriptional level. Recent development of high-throughput next-generation sequence (NGS) allows the RNA-seq method for this profiling. This method provides precise information of transcripts about the quantitation and the structure such as the splicing variants. In this chapter, we describe a method for gene expression profiling of GFP-positive cells from transgenic zebrafish by RNA-seq. We labeled specific cells in the brain with GFP by crossing a Gal4 driver line with the UAS:GFP line, isolated those cells by fluorescence-activated cell sorting (FACS), and analyzed by RNA-seq.