Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation.

Cerebral cortical development in mammals involves a highly complex and organized set of events including the transition of neural stem and progenitor cells (NSCs) from proliferative to differentiative divisions to generate neurons. Despite progress, the spatiotemporal regulation of this proliferation-differentiation switch during neurogenesis and the upstream epigenetic triggers remain poorly known. Here we report a cortex-specific PHD finger protein, Phf21b, which is highly expressed in the neurogenic phase of cortical development and gets induced as NSCs begin to differentiate. Depletion of Phf21b in vivo inhibited neuronal differentiation as cortical progenitors lacking Phf21b were retained in the proliferative zones and underwent faster cell cycles. Mechanistically, Phf21b targets the regulatory regions of cell cycle promoting genes by virtue of its high affinity for monomethylated H3K4. Subsequently, Phf21b recruits the lysine-specific demethylase Lsd1 and histone deacetylase Hdac2, resulting in the simultaneous removal of monomethylation from H3K4 and acetylation from H3K27, respectively. Intriguingly, mutations in the Phf21b locus associate with depression and mental retardation in humans. Taken together, these findings establish how a precisely timed spatiotemporal expression of Phf21b creates an epigenetic program that triggers neural stem cell differentiation during cortical development.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development.

Corticogenesis consists of a series of synchronised events, including fate transition of cortical progenitors, neuronal migration, specification and connectivity. NeuroD1, a basic helix-loop-helix (bHLH) transcription factor (TF), contributes to all of these events, but how it coordinates these independently is still unknown. Here, we demonstrate that NeuroD1 expression is accompanied by a gain of active chromatin at a large number of genomic loci. Interestingly, transcriptional activation of these loci relied on a high local density of adjacent bHLH TFs motifs, including, predominantly, Tcf12. We found that activity and expression levels of Tcf12 were high in cells with induced levels of NeuroD1 that spanned the transition of cortical progenitors from proliferative to neurogenic divisions. Moreover, Tcf12 forms a complex with NeuroD1 and co-occupies a subset of NeuroD1 target loci. This Tcf12-NeuroD1 cooperativity is essential for gaining active chromatin and targeted expression of genes involved in cell migration. By functional manipulation in vivo, we further show that Tcf12 is essential during cortical development for the correct migration of newborn neurons and, hence, for proper cortical lamination.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Disruption of cortical cell type composition and function underlies diabetes-associated cognitive decline.

AIMS/HYPOTHESIS: Type 2 diabetes is associated with increased risk of cognitive decline although the pathogenic basis for this remains obscure. Deciphering diabetes-linked molecular mechanisms in cells of the cerebral cortex could uncover novel therapeutic targets. METHODS: Single-cell transcriptomic sequencing (scRNA-seq) was conducted on the cerebral cortex in a mouse model of type 2 diabetes (db/db mice) and in non-diabetic control mice in order to identify gene expression changes in distinct cell subpopulations and alterations in cell type composition. Immunohistochemistry and metabolic assessment were used to validate the findings from scRNA-seq and to investigate whether these cell-specific dysfunctions impact the neurovascular unit (NVU). Furthermore, the behavioural and cognitive alterations related to these dysfunctions in db/db mice were assessed via Morris water maze and novel object discrimination tests. Finally, results were validated in post-mortem sections and protein isolates from individuals with type 2 diabetes. RESULTS: Compared with non-diabetic control mice, the db/db mice demonstrated disrupted brain function as revealed by losses in episodic and spatial memory and this occurred concomitantly with dysfunctional NVU, neuronal circuitry and cerebral atrophy. scRNA-seq of db/db mouse cerebral cortex revealed cell population changes in neurons, glia and microglia linked to functional regulatory disruption including neuronal maturation and altered metabolism. These changes were validated through immunohistochemistry and protein expression analysis not just in the db/db mouse cerebral cortex but also in post-mortem sections and protein isolates from individuals with type 2 diabetes (74.3 ± 5.5 years) compared with non-diabetic control individuals (87.0 ± 8.5 years). Furthermore, metabolic and synaptic gene disruptions were evident in cortical NVU cell populations and associated with a decrease in vascular density. CONCLUSIONS/INTERPRETATION: Taken together, our data reveal disruption in the cellular and molecular architecture of the cerebral cortex induced by diabetes, which can explain, at least in part, the basis for progressive cognitive decline in individuals with type 2 diabetes. DATA AVAILABILITY: The single-cell sequencing data that supports this study are available at GEO accession GSE217665 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE217665 ).

Dissection of cellular disruptions in autism spectrum disorder comorbidities.

Up to 80% of children with autism spectrum disorder have at least one other neuropsychiatric comorbidity. The causes of such disorders are highly genetic, yet many studies fail to take analysis further than risk gene discovery to see cellular and mechanistic changes occurring. Therefore, the goal of this study was to unveil novel gene expression signatures involved in important neurodevelopmental processes that, when disrupted, lead to each of the autism comorbidities of interest. We achieved this by analysing a single-nuclei RNA sequencing dataset with prefrontal cortex samples from autism spectrum disorder plus comorbidities for differentially expressed genes. The highest number of alterations was seen in excitatory neurons, which also showed differential population and cell-cell interactions across disorders and an increase in expression of genes involved in neurodevelopmental pathways. Interestingly, the group without comorbidities displayed an increase in neuron-neuron interactions yet a decrease in population number, suggesting a major rewiring of neuronal connections. Further analysis of the topmost significant genes from this cell type in developing prefrontal cortex datasets revealed interesting expression trajectories corresponding to important time points during corticogenesis. This further identified four novel candidate genes that show a potential link to developmental pathways that may contribute to autism and its comorbidities when dysregulated. The study provides a better understanding of co-occurring conditions at a transcriptomic and cell-type level and thereby aid future research in providing earlier diagnosis, care and intervention.

Tumour invasion and dissemination.

Activating invasion and metastasis are one of the primary hallmarks of cancer, the latter representing the leading cause of death in cancer patients. Whilst many advances in this area have been made in recent years, the process of cancer dissemination and the underlying mechanisms governing invasion are still poorly understood. Cancer cells exhibit multiple invasion strategies, including switching between modes of invasion and plasticity in response to therapies, surgical interventions and environmental stimuli. The ability of cancer cells to switch migratory modes and their inherent plasticity highlights the critical challenge preventing the successful design of cancer and anti-metastatic therapies. This mini-review presents current knowledge on the critical models of tumour invasion and dissemination. We also discuss the current issues surrounding current treatments and arising therapeutic opportunities. We propose that the establishment of novel approaches to study the key biological mechanisms underlying the metastatic cascade is critical in finding novel targets that could ultimately lead to complete inhibition of cancer cell invasion and dissemination.

High-resolution mapping of the Mov-1 locus in wheat by combining radiation hybrid (RH) and recombination-based mapping approaches.

KEY MESSAGE: This work reports a quick method that integrates RH mapping and genetic mapping to map the dominant Mov-1 locus to a 1.1-Mb physical interval with a small number of candidate genes. Bread wheat is an important crop for global human population. Identification of genes and alleles controlling agronomic traits is essential toward sustainably increasing crop production. The unique multi-ovary (MOV) trait in wheat holds potential for improving yields and is characterized by the formation of 2-3 grains per spikelet. The genetic basis of the multi-ovary trait is known to be monogenic and dominant in nature. Its precise mapping and functional characterization is critical to utilizing this trait in a feasible manner. Previous mapping efforts of the locus controlling multiple ovary/pistil formation in the hexaploid wheat have failed to produce a consensus for a particular chromosome. We describe a mapping strategy integrating radiation hybrid mapping and high-resolution genetic mapping to locate the chromosomal position of the Mov-1 locus in hexaploid wheat. We used RH mapping approach using a panel of 188 lines to map the Mov-1 locus in the terminal part of long arm of wheat chromosome 2D with a map resolution of 1.67 Mb/cR1500. Then using a genetic population of MOV × Synthetic wheat of F2 lines, we delineated the Mov-1 locus to a 1.1-Mb physical region with a small number of candidate genes. This demonstrates the value of this integrated strategy to mapping dominant genes in wheat.

NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program.

Cell fate specification relies on the action of critical transcription factors that become available at distinct stages of embryonic development. One such factor is NeuroD1, which is essential for eliciting the neuronal development program and possesses the ability to reprogram other cell types into neurons. Given this capacity, it is important to understand its targets and the mechanism underlying neuronal specification. Here, we show that NeuroD1 directly binds regulatory elements of neuronal genes that are developmentally silenced by epigenetic mechanisms. This targeting is sufficient to initiate events that confer transcriptional competence, including reprogramming of transcription factor landscape, conversion of heterochromatin to euchromatin, and increased chromatin accessibility, indicating potential pioneer factor ability of NeuroD1. The transcriptional induction of neuronal fate genes is maintained via epigenetic memory despite a transient NeuroD1 induction during neurogenesis. NeuroD1 also induces genes involved in the epithelial-to-mesenchymal transition, thereby promoting neuronal migration. Our study not only reveals the NeuroD1-dependent gene regulatory program driving neurogenesis but also increases our understanding of how cell fate specification during development involves a concerted action of transcription factors and epigenetic mechanisms.

JNK-dependent gene regulatory circuitry governs mesenchymal fate.

The epithelial to mesenchymal transition (EMT) is a biological process in which cells lose cell-cell contacts and become motile. EMT is used during development, for example, in triggering neural crest migration, and in cancer metastasis. Despite progress, the dynamics of JNK signaling, its role in genomewide transcriptional reprogramming, and involved downstream effectors during EMT remain largely unknown. Here, we show that JNK is not required for initiation, but progression of phenotypic changes associated with EMT. Such dependency resulted from JNK-driven transcriptional reprogramming of critical EMT genes and involved changes in their chromatin state. Furthermore, we identified eight novel JNK-induced transcription factors that were required for proper EMT. Three of these factors were also highly expressed in invasive cancer cells where they function in gene regulation to maintain mesenchymal identity. These factors were also induced during neuronal development and function in neuronal migration in vivo. These comprehensive findings uncovered a kinetically distinct role for the JNK pathway in defining the transcriptome that underlies mesenchymal identity and revealed novel transcription factors that mediate these responses during development and disease.

Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity.

Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity.

Genetic Labeling of Nuclei-Specific Thalamocortical Neurons Reveals Putative Sensory-Modality Specific Genes.

The thalamus is a central brain structure with topographically ordered long-range axonal projections that convey sensory information to the cortex via distinct nuclei. Although there is an increasing knowledge about genes important for thalamocortical (TC) development, the identification of genetic landmarks of the distinct thalamic nuclei during the embryonic development has not been addressed systematically. Indeed, a more comprehensive understanding of how the axons from the individual nuclei find their way and connect to their corresponding cortical area is called for. Here, we used a genetic dual labeling strategy in mice to purify distinct principal sensory thalamic neurons. Subsequent genome-wide transcriptome profiling revealed genes specifically expressed in each nucleus during embryonic development. Analysis of regulatory regions of the identified genes revealed key transcription factors and networks that likely underlie the specification of individual sensory-modality TC connections. Finally, the importance of correct axon targeting for the specific sensory-modality population transcriptome was evidenced in a Sema6A mutant, in which visual TC axons are derailed at embryonic life. In sum, our data determined the developmental transcriptional profile of the TC neurons that will eventually support sensory processing.

Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes.

During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.

TOX3 regulates neural progenitor identity.

The human genomic locus for the transcription factor TOX3 has been implicated in susceptibility to restless legs syndrome and breast cancer in genome-wide association studies, but the physiological role of TOX3 remains largely unknown. We found Tox3 to be predominantly expressed in the developing mouse brain with a peak at embryonic day E14 where it co-localizes with the neural stem and progenitor markers Nestin and Sox2 in radial glia of the ventricular zone and intermediate progenitors of the subventricular zone. Tox3 is also expressed in neural progenitor cells obtained from the ganglionic eminence of E15 mice that express Nestin, and it specifically binds the Nestin promoter in chromatin immunoprecipitation assays. In line with this, over-expression of Tox3 increased Nestin promoter activity, which was cooperatively enhanced by treatment with the stem cell self-renewal promoting Notch ligand Jagged and repressed by pharmacological inhibition of Notch signaling. Knockdown of Tox3 in the subventricular zone of E12.5 mouse embryos by in utero electroporation of Tox3 shRNA revealed a reduced Nestin expression and decreased proliferation at E14 and a reduced migration to the cortical plate in E16 embryos in electroporated cells. Together, these results argue for a role of Tox3 in the development of the nervous system.

A whole-genome, radiation hybrid mapping resource of hexaploid wheat.

Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR1500 with a total map length of 6866 cR1500 . The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.

Dynamics of chromatin accessibility and epigenetic state in response to UV damage.

Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship.

GSP: a web-based platform for designing genome-specific primers in polyploids.

MOTIVATION: The sequences among subgenomes in a polyploid species have high similarity, making it difficult to design genome-specific primers for sequence analysis. RESULTS: We present GSP, a web-based platform to design genome-specific primers that distinguish subgenome sequences in a polyploid genome. GSP uses BLAST to extract homeologous sequences of the subgenomes in existing databases, performs a multiple sequence alignment, and design primers based on sequence variants in the alignment. An interactive primers diagram, a sequence alignment viewer and a virtual electrophoresis are displayed as parts of the primer design result. GSP also designs specific primers from multiple sequences uploaded by users. AVAILABILITY AND IMPLEMENTATION: GSP is a user-friendly and efficient web platform freely accessible at http://probes.pw.usda.gov/GSP Source code and command-line application are available at https://github.com/bioinfogenome/GSP CONTACTS: yong.gu@ars.usda.gov or devin.coleman-derr@ars.usda.gov SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Determinants and dynamics of genome accessibility.

In eukaryotes, all DNA-templated reactions occur in the context of chromatin. Nucleosome packaging inherently restricts DNA accessibility for regulatory proteins but also provides an opportunity to regulate DNA-based processes through modulating nucleosome positions and local chromatin structure. Recent advances in genome-scale methods are yielding increasingly detailed profiles of the genomic distribution of nucleosomes, their modifications and their modifiers. The picture now emerging is one in which the dynamic control of genome accessibility is governed by contributions from DNA sequence, ATP-dependent chromatin remodelling and nucleosome modifications. Here we discuss the interplay of these processes by reviewing our current understanding of how chromatin access contributes to the regulation of transcription, replication and repair.

DNA-binding factors shape the mouse methylome at distal regulatory regions.

Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.