Biology subjects

Miura, H.

Publications and source records attributed to Miura, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Repeat element-anchored enhancer-promoter interactions (REEPIs) shape the regulatory landscape of hormone-resistant breast cancer

Repeat elements (REs) comprise over half of the human genome and are increasingly recognized as regulatory components, yet their role in therapy resistance remains unclear. Here, we integrate CAGE and RADICL-seq to map regulatory elements and RNA-chromatin interactions in estrogen receptor-positive breast cancer cells (MCF7) and their long-term estrogen-deprived (LTED) derivatives. We identify repeat element-anchored enhancer-promoter interactions (REEPIs), defined by the presence of annotated REs at both enhancer and promoter sites. In LTED cells, REEPIs are enriched for ERV1 and ERVL elements and involve specific repeat pairings associated with transcription factor combinations such as SP1-SMAD3 and SP1-NFYC. At the gene level, these REEPIs are preferentially linked to genes involved in chromatin remodeling, DNA repair, and lineage plasticity. Our findings reveal a repeat-guided regulatory architecture associated with transcriptional adaptation in endocrine resistance and identify REEPIs as a previously underexplored component of regulatory reprogramming in hormone-resistant breast cancer.

cancer biology↗

bioRxiv2026

A pistil peptide toxin-pollen antidote system for reproductive barrier

Summary paragraphPrezygotic reproductive barriers in angiosperms are crucial for female-mediated selection of compatible male gametes. In this study, we identified a biological activity within the stylar tissues of Arabidopsis thaliana pistils that inhibits the elongation of heterospecific pollen tubes. Using genome-wide association studies, we identified a cysteine-rich peptide predominantly expressed in the style as the responsible barrier. This secreted peptide, named Femme fatale (FEM), inhibited pollen tube elongation of heterospecific species and some conspecific strains, both in vivo and in vitro. Importantly, FEMs function was independent of the known reproductive barrier component SPRI1 that functions in the stigmatic papilla cell surface. This means two factors act spatially in sequence to create a multilayered reproductive barrier system against pollen in pistils. We also identified a secreted protein expressed in pollen, Homme capable (HOM), which is encoded by a gene next to FEM. HOM interacts with FEM to counteract its barrier effect and allow pollen tube elongation. The FEM-HOM locus has undergone frequent tandem duplications during Brassicaceae evolution. Dosage differences between the two factors can lead to reproductive incompatibility among lineages. Together, our findings suggest that FEM and HOM form a toxin-antidote system that helps establish reproductive barriers.

plant biology↗

bioRxiv2025

Cell cycle-resolved Hi-C reveals unexpected plasticity of A/B compartments across interphase

The spatial organization of chromatin into active (A) and inactive (B) nuclear compartments is fundamental to genome regulation, yet their cell-cycle dynamics remain largely unexplored. Most research on chromatin dynamics during the cell cycle has primarily focused on events surrounding mitosis, providing only limited insight into chromatin behavior during S-phase. To address this gap, we developed a simple, drug-free approach that combines the Fucci cell-cycle indicator with in situ Hi-C to comprehensively analyze A/B compartment dynamics throughout interphase in mouse embryonic stem cells (mESCs). Unexpectedly, and contrary to prevailing views, we found that A/B compartment strength increased abruptly upon S-phase entry, stabilized during S-phase, and subsequently declined in late S/G2. This abrupt strengthening, which we termed compartment maturation, required passage through the G1/S transition but was independent of active DNA synthesis. This maturation involved substantial architectural remodeling, particularly within the A compartment, which consolidated into a more organized structure as individual A domains rearranged to form long-range interactions. Moreover, compartment maturation was not limited to mESCs but was also evident across different developmental contexts in mice. Based on these observations, we propose a revised, stepwise model of nuclear compartmentalization during cell-cycle progression, consisting of four distinct stages: chromosome unfolding (G1), chromatin maturation (G1/S), stabilization (S phase), and refolding (G2). These findings reveal the unexpected plasticity of A/B compartments and underscore the G1/S transition as a critical period for their reorganization.

cell biology↗

bioRxiv2023

KSHV Terminal Repeat Regulates Inducible Lytic Gene Promoters

The Kaposis sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by multiple copies of 0.8 kb terminal repeat (TR) sequence. While TRs function in plasmid maintenance is well-established, TRs transcription regulatory roles have not been fully explored. Here, we show KSHV TR is a large transcription regulatory domain. A series of Cleavage Under Targets & Release Using Nuclease demonstrated that TR fragments are occupied by histone modifying enzymes that are known to interact with LANA in naturally infected cells, and the TR possessed characteristic enhancer histone modifications. The H3K4me3 and H3K27Ac modifications were conserved in unique region of the KSHV genome among naturally infected cells, and the KSHV Origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with TRs. In the Ori-Lyt region, the LANA complex colocalizes with H3K27Ac-modified nucleosome along with paused RNA polymerase II, and two K-Rta recruitment sites frank the nucleosome. The isolated reporter assays demonstrated that neighboring TR fragments enhanced viral lytic gene promoter activity independent of orientation in KSHV-infected and non-infected 293FT cells. K-Rta transactivation function was drastically enhanced with TR, while LANA acquired promoter repression function with TR. KSHV TR is, therefore a regulatory domain for KSHV inducible genes. However, in contrast to cellular enhancers that are bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body with TR. KSHV evolved a clever mechanism to tightly control the latency-lytic switch with the TR/LANA complex. ImportanceEnhancers are a crucial regulator of differential gene expression programs. Enhancer is the cis-regulatory sequences that determine target genes spatiotemporal and quantitative expression. Here, we show that KSHV terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. KSHV enhancer is occupied by LANA and its interacting proteins, such as CHD4, and CHD4 is known to restrict enhancers to access promoters for activation. This study thus proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates lytic gene transcription.

microbiology↗

bioRxiv2023

Prolonged Kaposi's Sarcoma-associated Herpesvirus vIL-6 Exposure Enhances Inflammatory Responses by Epigenetic Reprogramming

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and possesses greater risks of having other complications, which include malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol-Linked Alkylation for the Metabolic Sequencing and Cleavage Under Target & Release Using Nuclease analysis, we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNAPII with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-KB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitor, OTX015, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals. Author summaryCombined and continuous cytokine stimulation triggers transcription reprogramming and is often used for specific tissue development. Continuous vIL-6 exposure occurs in KSHV-infected patients and leads to inflammatory cytokine storm with high mortality. However, possible epigenetic reprogramming by the vIL-6 and its association with pathogenesis remain unclear. Here we demonstrate the establishment of a new chromatin landscape mediated by BRD4 through prolonged vIL-6 exposure which contributes to more robust and rapid transcription and increased cytokines production. Inhibition of BRD4 suppressed this inflammatory response. Our results indicate that targeting the epigenetic effect of viral cytokines may lead to novel therapies for KSHV-induced inflammatory cytokine storms.

microbiology↗

bioRxiv2023

Delivering mRNAs to mouse tissues using the SEND system

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo. Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

genomics↗

bioRxiv2022

Impact of reinfection with SARS-CoV-2 Omicron variants in previously infected hamsters

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

microbiology↗

bioRxiv2022

Morphological evolution and diversity of pectoral fin skeletons in teleosts

The Teleostei class has the most species of the fishes. Members of this group have paired rostral appendages and pectoral fins, enabling refined movements in the water. Although teleosts live in a diverse set of environments, the skeletal pattern of pectoral fins in teleosts is considered to show little morphological variability. Here, in order to elucidate variations in pectoral fin skeletons and to identify their evolutionary processes, we compared the pectoral fin skeletons from 27 species of teleosts. We identified several variations and a diversity of pectoral fin skeletal patterns within some teleost groups. Taken together with previous reports on teleost skeletons, our findings reveal that in the course of teleost evolution, there are a mixture of conserved and non- conserved components in the pectoral fin skeletons of teleosts, and that teleosts may have experienced the variation and conservation of the number and shape of the proximal radials, the loss of the mesocoracoid, and the change in the distal radial-fin ray relationship.

zoology↗

bioRxiv2021

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.

cell biology↗

bioRxiv2021

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks progress slowly in cells depleted for SAF-A, also contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed that the boundaries between early- and late-replicating domains are blurred in cells depleted for SAF-A. Associated with these defects, SAF-A-depleted cells show elevated {gamma}H2A phosphorylation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

cell biology↗

bioRxiv2020

Functional correlation of H3K9me2 and nuclear compartment formation

BackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation. ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

molecular biology↗

bioRxiv2020

Improvement of phycocyanobilin synthesis for genetically encoded phytochrome-based optogenetics.

Optogenetics is a powerful technique using photoresponsive proteins, and light-inducible dimerization (LID) systems, an optogenetic tool, allow to manipulate intracellular signaling pathways. One of the red/far-red responsive LID system, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phytochromobilin or phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (synPCB), and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated PhyB-PIF and synPCB into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

synthetic biology↗

bioRxiv2019

Endogenization and excision of human herpesvirus 6 in human genomes

The genome of human herpesvirus 6 (HHV-6) is integrated within the nuclear genome of about 1% of humans, but how this came about is not clear. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). piRNAs block integration of transposons in the germline, so piRNA-mediated repression of HHV-6 integration has been suspected. Whether integrated HHV-6 can reactive into an infectious virus is also uncertain. In vitro, recombination of the viral genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected single DR "scar" has not been described in vivo. We analyzed whole-genome sequencing (WGS) data from 13,040 subjects, including 7,485 from Japan. We found an association between integrated HHV-6 and polymorphisms on chr22q in Japanese subjects. However, association with the reported MOV10L1 polymorphism was driven by physical linkage to a single ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. We resolved the junction of the human chromosome with this viral genome using long read sequencing. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a single DR, supporting in vivo excision and viral reactivation. Using WGS data from North American families, we show that the incidence of HHV-6 integration into the germline is lower than its prevalence, and that integrated HHV-6 is not associated with the reported variant in MOV10L1. Together these results explain the recently reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact non-retroviral genome known to be present in human germlines. SIGNIFICANCE STATEMENT Human herpesvirus 6 (HHV-6) infects most people during childhood, usually only causing fever and rash. Reactivation of HHV-6 has been linked to a number of neurological diseases including encephalitis, Alzheimers disease and multiple sclerosis. However, about 1% of people are born with the HHV-6 genome present within their genome, included in the end "cap" of one of their 46 chromosomes. Little is known about how and when HHV-6 genomes entered human genomes, whether or not they still do, and whether or not this poses risk for virus reactivation. We looked for HHV-6 in genome sequences from over 13,000 people. Most HHV-6 variants present in human genomes have been co-evolving with human chromosomes for many generations, and new integration events are rare. Surprisingly, in almost three fourths of Japanese people with HHV-6 in their genome, HHV-6 integrated in the same end of the same chromosome - 22q. Persistence of the HHV-6 genome within the short "cap" that preserves the end of chromosome 22q suggests that the integrated viral sequence may have taken on a useful function for this chromosome. We also found that some human genomes harbor only one part of the HHV-6 genome. This part is the same part that remains after experimental viral reactivation, during which most of the virus is cut out of the genome. This warrants assessment of the risk that integration of HHV-6 into inherited human genomes is not irreversible, and possibly leads to production of infectious virus.

genetics↗

bioRxiv2019

Microrheology for Hi-C Data Reveals the Spectrum of the Dynamic 3D Genome Organization

The 1-dimensional information of genomic DNA is hierarchically packed inside the eukaryotic cell nucleus and organized in 3-dimensional (3D) space. Genome-wide chromosome conformation capture (Hi-C) methods have uncovered the 3D genome organization and revealed multiscale chromatin domains of compartments and topologically associating domains (TADs). Moreover, single-nucleosome live-cell imaging experiments have revealed the dynamic organization of chromatin domains caused by stochastic thermal fluctuations. However, the mechanism underlying the dynamic regulation of such hierarchical and structural chromatin units within the micro-scale thermal medium remains unclear. Microrheology is a way to measure dynamic viscoelastic properties coupling between thermal microenvironment and mechanical response. Here, we propose, to our knowledge, a new microrheology for Hi-C data to analyze the compliance property as a barometer of rigidness and flexibility of genomic regions along with the time evolution. Our method allows conversion of a Hi-C matrix into the spectrum of the rheological property along the genomic coordinate of a single chromosome. To demonstrate the technique, we analyzed Hi-C data during the neural differentiation of mouse embryonic stem cells. We found that TAD boundaries behave as more rigid nodes than the intra-TAD region. The spectrum clearly shows the rheological property of the dynamic chromatin domain formations at an individual time scale. Furthermore, we characterized the appearance of synchronous and liquid-like inter-compartment interactions in differentiated cells. Together, our microrheology provides physical insights revealing the dynamic 3D genome organization from Hi-C data.\n\nSIGNIFICANCEGenomic DNA is hierarchically packed inside the eukaryotic cell nucleus, and the genome organization in 3D contributes to proper genome functions at the multiscale chromatin domains. Although thermal fluctuations inevitably drive movements of the genome molecules in the micro-scale cell environment, there is no method, as yet, to quantify such dynamic 3D genome organization of hierarchical and structural chromatin units. Here, we describe a method to calculate rheological properties as barometers of flexibility and liquid-like behavior of genomic regions. We show that biologically relevant boundaries between chromatin domains are more rigid than the inside at a particular time scale. Our method allows interpretation of static and population-averaged genome conformation data as dynamic and hierarchical 3D genome picture.

biophysics↗

bioRxiv2019

KCa3.1-transgene induction in murine skin produces pruritic eczematous dermatitis with severe epidermal hyperplasia and hyperkeratosis

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-{beta}1 (60-fold), IL-23 (34-fold), IL-6 (33-fold), and TNF (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-{beta}1 (-88%), IL-23 (-77%), and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

physiology↗

bioRxiv2019

Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?

BackgroundHi-C, a derivative of chromosome conformation capture (3C) targeting the whole genome, was originally developed as a means for characterizing chromatin conformation. More recently, this method has also been frequently employed in elongating nucleotide sequences obtained by de novo genome sequencing and assembly, in which the number of resultant sequences rarely converge into the chromosome number. Despite the prevailing and irreplaceable use, sample preparation methods for Hi-C have not been intensively discussed, especially from the standpoint of genome scaffolding.\n\nResultsTo gain insights into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison using vertebrate samples and optimized various factors during sample preparation, sequencing, and computation. As a result, we have identified some key factors that help improve Hi-C scaffolding including the choice and preparation of tissues, library preparation conditions, and restriction enzyme(s), as well as the choice of scaffolding program and its usage.\n\nConclusionsThis study provides the first comparison of multiple sample preparation kits/protocols and computational programs for Hi-C scaffolding, by an academic third party. We introduce a customized protocol designated the inexpensive and controllable Hi-C (iconHi-C) protocol, in which the optimal conditions revealed by this study have been incorporated, and release the resultant chromosome-scale genome assembly of the Chinese softshell turtle Pelodiscus sinensis.

genomics↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2018

Alopecia areata susceptibility variant identified by MHC risk haplotype sequencing reproduces symptomatic patched hair loss in mice

BackgroundAlopecia areata (AA) is a highly heritable multifactorial and complex disease. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a region in the human genome known to be associated with AA as compared to other regions.\n\nResultsBy sequencing MHC risk haplotypes, we identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Using CRISPR/Cas9 for allele-specific genome editing, we then phenocopied AA symptomatic patched hair loss in mice engineered to carry the Cchcr1 risk allele. Skin biopsies of these alopecic mice showed strong up-regulation of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). Using transcriptomics findings, we further identified CCHCR1 as a novel component of hair shafts and cuticles in areas where the engineered alopecic mice displayed fragile and impaired hair.\n\nConclusionsThese results suggest an alternative mechanism for the aetiology of AA based on aberrant keratinization, in addition to generally well-known autoimmune events.

genomics↗