Parent-of-origin-specific DNA replication timing is confined to large imprinted regions.

Genomic imprinting involves differential DNA methylation and gene expression between homologous paternal and maternal loci. It remains unclear, however, whether DNA replication also shows parent-of-origin-specific patterns at imprinted or other genomic regions. Here, we investigate genome-wide asynchronous DNA replication utilizing uniparental human embryonic stem cells containing either maternal-only (parthenogenetic) or paternal-only (androgenetic) DNA. Four clusters of imprinted genes exhibited differential replication timing based on parent of origin, while the remainder of the genome, 99.82%, showed no significant replication asynchrony between parental origins. Active alleles in imprinted gene clusters replicated earlier than their inactive counterparts. At the Prader-Willi syndrome locus, replication asynchrony spanned virtually the entirety of S phase. Replication asynchrony was carried through differentiation to neuronal precursor cells in a manner consistent with gene expression. This study establishes asynchronous DNA replication as a hallmark of large imprinted gene clusters.

Does nascent polyploidy employ common mechanisms to stabilize and establish?

In a recent issue of Cell Reports, Bray et al. found genetic adaptation in kinetochore components and ion transporters underlying polyploid stabilization in Cochlearia. This resurrects the issue of whether nascent polyploidy in diverse organisms establish via common biological mechanisms.

Kinetochore and ionomic adaptation to whole-genome duplication in Cochlearia shows evolutionary convergence in three autopolyploids.

Whole-genome duplication (WGD) occurs in all kingdoms and impacts speciation, domestication, and cancer outcome. However, doubled DNA management can be challenging for nascent polyploids. The study of within-species polyploidy (autopolyploidy) permits focus on this DNA management aspect, decoupling it from the confounding effects of hybridization (in allopolyploid hybrids). How is autopolyploidy tolerated, and how do young polyploids stabilize? Here, we introduce a powerful model to address this: the genus Cochlearia, which has experienced many polyploidization events. We assess meiosis and other polyploid-relevant phenotypes, generate a chromosome-scale genome, and sequence 113 individuals from 33 ploidy-contrasting populations. We detect an obvious autopolyploidy-associated selection signal at kinetochore components and ion transporters. Modeling the selected alleles, we detail evidence of the kinetochore complex mediating adaptation to polyploidy. We compare candidates in independent autopolyploids across three genera separated by 40 million years, highlighting a common function at the process and gene levels, indicating evolutionary flexibility in response to polyploidy.

Functional genomic analysis of genes important for Candida albicans fitness in diverse environmental conditions.

Fungal pathogens such as Candida albicans pose a significant threat to human health with limited treatment options available. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leverage a pooled functional genomic screening strategy to identify genes important for fitness of C. albicans in diverse conditions. We identify an essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrate that it encodes subunit 3 of replication factor A (Rfa3). Furthermore, we apply computational analyses to identify functionally coherent gene clusters and predict gene function. Through this approach, we predict the cell-cycle-associated function of C3_06880W, a previously uncharacterized gene required for fitness specifically at elevated temperatures, and follow-up assays confirm that C3_06880W encodes Iml3, a component of the C. albicans kinetochore with roles in virulence in vivo. Overall, this work reveals insights into the vulnerabilities of C. albicans.

A human fetal cerebellar map of the late second trimester reveals developmental molecular characteristics and abnormality in trisomy 21.

Our understanding of human fetal cerebellum development during the late second trimester, a critical period for the generation of astrocytes, oligodendrocytes, and unipolar brush cells (UBCs), remains limited. Here, we performed single-cell RNA sequencing (scRNA-seq) in human fetal cerebellum samples from gestational weeks (GWs) 18-25. We find that proliferating UBC progenitors distribute in the subventricular zone of the rhombic lip (RLSVZ) near white matter (WM), forming a layer structure. We also delineate two trajectories from astrogenic radial glia (ARGs) to Bergmann glial progenitors (BGPs) and recognize oligodendrogenic radial glia (ORGs) as one source of primitive oligodendrocyte progenitor cells (PriOPCs). Additionally, our scRNA-seq analysis of the trisomy 21 fetal cerebellum at this stage reveals abnormal upregulated genes in pathways such as the cell adhesion pathway and focal adhesion pathway, which potentially promote neuronal differentiation. Overall, our research provides valuable insights into normal and abnormal development of the human fetal cerebellum.

Population genomics uncovers global distribution, antimicrobial resistance, and virulence genes of the opportunistic pathogen Klebsiella aerogenes.

Klebsiella aerogenes is an understudied and clinically important pathogen. We therefore investigate its population structure by genome analysis aligned with metadata. We sequence 130 non-duplicated K. aerogenes clinical isolates and identify two inter-patient transmission events. We then retrieve all publicly available K. aerogenes genomes (n = 1,026, accessed by January 1, 2023) and analyze them with our 130 genomes. We develop a core-genome multi-locus sequence-typing scheme. We find that K. aerogenes is a species complex comprising four phylogroups undergoing evolutionary divergence, likely forming three species. We delineate remarkable clonal diversity and identify three worldwide-distributed carbapenemase-encoding clonal clusters, representing high-risk lineages. We uncover that K. aerogenes has an open genome equipped by a large arsenal of antimicrobial resistance genes. We identify two genetic regions specific for K. aerogenes, encoding a type VI secretion system and flagella/chemotaxis for motility, respectively, both contributing to the virulence. These results provide much-needed insights into the population structure and pan-genomes of K. aerogenes.

The architecture of silk-secreting organs during the final larval stage of silkworms revealed by single-nucleus and spatial transcriptomics.

Natural silks are renewable proteins with impressive mechanical properties and biocompatibility that are useful in various fields. However, the cellular and spatial organization of silk-secreting organs remains unclear. Here, we combined single-nucleus and spatially resolved transcriptomics to systematically map the cellular and spatial composition of the silk glands (SGs) of mulberry silkworms late in larval development. This approach allowed us to profile SG cell types and cell state dynamics and identify regulatory networks and cell-cell communication related to efficient silk protein synthesis; key markers were validated via transgenic approaches. Notably, we demonstrated the indispensable role of the ecdysone receptor (ultraspiracle) in regulating endoreplication in SG cells. Our atlas presents the results of spatiotemporal analysis of silk-secreting organ architecture late in larval development; this atlas provides a valuable reference for elucidating the mechanism of efficient silk protein synthesis and developing sustainable products made from natural silk.

Comparative transcriptomic profiling reveals a role for Olig1 in promoting axon regeneration.

The regenerative potential of injured axons displays considerable heterogeneity. However, the molecular mechanisms underlying the heterogeneity have not been fully elucidated. Here, we establish a method that can separate spinal motor neurons (spMNs) with low and high regenerative capacities and identify a set of transcripts revealing differential expression between two groups of neurons. Interestingly, oligodendrocyte transcription factor 1 (Olig1), which regulates the differentiation of various neuronal progenitors, exhibits recurrent expression in spMNs with enhanced regenerative capabilities. Furthermore, overexpression of Olig1 (Olig1 OE) facilitates axonal regeneration in various models, and down-regulation or deletion of Olig1 exhibits an opposite effect. By analyzing the overlapped differentially expressed genes after expressing individual Olig factor and functional validation, we find that the role of Olig1 is at least partially through the neurite extension factor 1 (Nrsn1). We therefore identify Olig1 as an intrinsic factor that promotes regenerative capacity of injured axons.

Interface-guided phenotyping of coding variants in the transcription factor RUNX1.

Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations.

Five autism-associated transcriptional regulators target shared loci proximal to brain-expressed genes.

Many autism spectrum disorder (ASD)-associated genes act as transcriptional regulators (TRs). Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify the regulatory targets of ARID1B, BCL11A, FOXP1, TBR1, and TCF7L2, ASD-associated TRs in the developing human and mouse cortex. These TRs shared substantial overlap in the binding sites, especially within open chromatin. The overlap within a promoter region, 1-2,000 bp upstream of the transcription start site, was highly predictive of brain-expressed genes. This signature was observed in 96 out of 102 ASD-associated genes. In vitro CRISPRi against ARID1B and TBR1 delineated downstream convergent biology in mouse cortical cultures. After 8 days, NeuN+ and CALB+ cells were decreased, GFAP+ cells were increased, and transcriptomic signatures correlated with the postmortem brain samples from individuals with ASD. We suggest that functional convergence across five ASD-associated TRs leads to shared neurodevelopmental outcomes of haploinsufficient disruption.

Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma.

Renal cell carcinoma with sarcomatoid differentiation (sRCC) is associated with poor survival and a heightened response to immune checkpoint inhibitors (ICIs). Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsies due to spatial heterogeneity. Herein, we characterized the epigenomic landscape of sRCC by profiling 107 epigenomic libraries from tissue and plasma samples from 50 patients with RCC and healthy volunteers. By profiling histone modifications and DNA methylation, we identified highly recurrent epigenomic reprogramming enriched in sRCC. Furthermore, CRISPRa experiments implicated the transcription factor FOSL1 in activating sRCC-associated gene regulatory programs, and FOSL1 expression was associated with the response to ICIs in RCC in two randomized clinical trials. Finally, we established a blood-based diagnostic approach using detectable sRCC epigenomic signatures in patient plasma, providing a framework for discovering epigenomic correlates of tumor histology via liquid biopsy.

Dynamic RNA polymerase II occupancy drives differentiation of the intestine under the direction of HNF4.

Terminal differentiation requires massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4,000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identify dynamic occupancy of RNA polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II occupancy and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, chromatin immunoprecipitation sequencing (ChIP-seq), and immunoprecipitation (IP) mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II occupancy at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms that drive differentiation gene expression and find that pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in renewing adult tissue.

The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

Genomic deletions explain the generation of alternative BRAF isoforms conferring resistance to MAPK inhibitors in melanoma.

Resistance to MAPK inhibitors (MAPKi), the main cause of relapse in BRAF-mutant melanoma, is associated with the production of alternative BRAF mRNA isoforms (altBRAFs) in up to 30% of patients receiving BRAF inhibitor monotherapy. These altBRAFs have been described as being generated by alternative pre-mRNA splicing, and splicing modulation has been proposed as a therapeutic strategy to overcome resistance. In contrast, we report that altBRAFs are generated through genomic deletions. Using different in vitro models of altBRAF-mediated melanoma resistance, we demonstrate the production of altBRAFs exclusively from the BRAF V600E allele, correlating with corresponding genomic deletions. Genomic deletions are also detected in tumor samples from melanoma and breast cancer patients expressing altBRAFs. Along with the identification of altBRAFs in BRAF wild-type and in MAPKi-naive melanoma samples, our results represent a major shift in our understanding of mechanisms leading to the generation of BRAF transcripts variants associated with resistance in melanoma.

Genomic signatures of exceptional longevity and negligible aging in the long-lived red sea urchin.

The red sea urchin (Mesocentrotus franciscanus) is one of the Earth's longest-living animals, reported to live more than 100 years with indeterminate growth, life-long reproduction, and no increase in mortality rate with age. To understand the genetic underpinnings of longevity and negligible aging, we constructed a chromosome-level assembly of the red sea urchin genome and compared it to that of short-lived sea urchin species. Genome-wide syntenic alignments identified chromosome rearrangements that distinguish short- and long-lived species. Expanded gene families in long-lived species play a role in innate immunity, sensory nervous system, and genome stability. An integrated network of genes under positive selection in the red sea urchin was involved in genomic regulation, mRNA fidelity, protein homeostasis, and mitochondrial function. Our results implicated known longevity genes in sea urchin longevity but also revealed distinct molecular signatures that may promote long-term maintenance of tissue homeostasis, disease resistance, and negligible aging.

The Idesia polycarpa genome provides insights into its evolution and oil biosynthesis.

The deciduous tree Idesia polycarpa can provide premium edible oil with high polyunsaturated fatty acid contents. Here, we generate its high-quality reference genome, which is ∼1.21 Gb, comprising 21 pseudochromosomes and 42,086 protein-coding genes. Phylogenetic and genomic synteny analyses show that it diverged with Populus trichocarpa about 16.28 million years ago. Notably, most fatty acid biosynthesis genes are not only increased in number in its genome but are also highly expressed in the fruits. Moreover, we identify, through genome-wide association analysis and RNA sequencing, the I. polycarpa SUGAR TRANSPORTER 5 (IpSTP5) gene as a positive regulator of high oil accumulation in the fruits. Silencing of IpSTP5 by virus-induced gene silencing causes a significant reduction of oil content in the fruits, suggesting it has the potential to be used as a molecular marker to breed the high-oil-content cultivars. Our results collectively lay the foundation for breeding the elite cultivars of I. polycarpa.

Evolution of chromosome-arm aberrations in breast cancer through genetic network rewiring.

The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. Analysis of The Cancer Genome Atlas data shows recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a panel of 23 primary tumor/patient-derived xenograft basal breast cancers reveals early evolution of chr4p deletion. Mechanistically we show that chr4p loss is associated with enhanced proliferation. Gene function studies identify an unknown gene, C4orf19, within chr4p, which suppresses proliferation when overexpressed-a member of the PDCD10-GCKIII kinase module we name PGCKA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames identify chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner, implicating network interactions. Together, these results shed light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes.

Evolutionary analysis of gene ages across TADs associates chromatin topology with whole-genome duplications.

Topologically associated domains (TADs) are interaction subnetworks of chromosomal regions in 3D genomes. TAD boundaries frequently coincide with genome breaks while boundary deletion is under negative selection, suggesting that TADs may facilitate genome rearrangements and evolution. We show that genes co-localize by evolutionary age in humans and mice, resulting in TADs having different proportions of younger and older genes. We observe a major transition in the age co-localization patterns between the genes born during vertebrate whole-genome duplications (WGDs) or before and those born afterward. We also find that genes recently duplicated in primates and rodents are more frequently essential when they are located in old-enriched TADs and interact with genes that last duplicated during the WGD. Therefore, the evolutionary relevance of recent genes may increase when located in TADs with established regulatory networks. Our data suggest that TADs could play a role in organizing ancestral functions and evolutionary novelty.

Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

The genome of Stephania japonica provides insights into the biosynthesis of cepharanthine.

Stephania japonica is an early-diverging eudicotyledon plant with high levels of cepharanthine, proven to be effective in curing coronavirus infections. Here, we report a high-quality S. japonica genome. The genome size is 688.52 Mb, and 97.37% sequences anchor to 11 chromosomes. The genome comprises 67.46% repetitive sequences and 21,036 genes. It is closely related to two Ranunculaceae species, which diverged from their common ancestor 55.90-71.02 million years ago (Mya) with a whole-genome duplication 85.59-96.75 Mya. We further reconstruct ancestral karyotype of Ranunculales. Several cepharanthine biosynthesis genes are identified and verified by western blot. Two genes (Sja03G0243 and Sja03G0241) exhibit catalytic activity as shown by liquid chromatography-mass spectrometry. Then, cepharanthine biosynthesis genes, transcription factors, and CYP450 family genes are used to construct a comprehensive network. Finally, we construct an early-diverging eudicotyledonous genome resources (EEGR) database. As the first genome of the Menispermaceae family to be released, this study provides rich resources for genomic studies.