Journal of Genetics and Genomics

One striking feature of the large KRAB-containing zinc finger protein (KZFP) family is its rapid expansion and divergence since its formation about 400 million years ago. However, the evolutionary characteristics of KRAB domains, C2H2 zinc fingers and the full protein of KZFPs have not been fully analyzed. As for the drivers of the rapid evolution, its partly due to their coevolution with transposable elements (TEs). But their diverse functions besides inhibiting TEs suggest other reasons exist. Here we address these two issues by the systematic analysis of the divergence time and diversification pattern of KZFPs at three aspects and the functional analysis of the potential target genes besides TEs. We found that old-zinc-finger-containing KZFPs tend to have varied and disordered KRAB domains, indicating there are two ways of the evolution of KZFPs, including the variation of KRAB domains and the diversification of zinc fingers. Among them, the divergence of zinc fingers mainly contributes to the rapid evolution of KZFPs. Thus, we mainly focused on the functional requirements of the evolution of zinc fingers. Different from the classical regulation pattern of this family, we found and experimentally confirmed that KZFPs tend to bind to non-TE regions and can positively regulate target genes. Although most young genes tend to be with a low expression level, young-zinc-finger-containing KZFPs tend to be highly expressed in early embryonic development or early mesoderm differentiation, indicating their particular evolutionarily novel functional roles in these two processes. We further validated a young KZFP, ZNF611, can bind to non-TE region of STK38 gene and positively regulates its expression in ESCs. The emergence of new sequence in STK38 promoter may drive the evolution of zinc fingers in ZNF611. Finally, we proposed a two-way evolution model of KZFP family.

The early stages of embryonic development rely on maternal products for proper regulation. However, systematic screening for functional maternal-specific factors has been challenging due to the time- and labor-intensive nature of traditional approaches. Here, we combined a computational pipeline and F0 homozygous mutation technology to screen for functional maternal-specific chromatin regulators in zebrafish embryogenesis and identified Mcm3l, Mcm6l, and Npm2a as playing essential roles in DNA replication and cell division. Our results contribute to understanding the molecular mechanisms underlying early embryo development and highlight the importance of maternal-specific chromatin regulators in this critical stage.

Craniofacial malformation (CFM) is a congenital defect encompassing a wide range of phenotypic presentations and is largely driven by genetics. Despite the discovery of more than 300 causal genes, there are a myriad of CFM cases with unknown genetic etiology. The complex gene regulations and heterogeneous cellular interactions in the developing head complicate disease-gene identification and prenatal genetic diagnosis. Recent progress in multiomic profiling of human embryogenesis enables the discovery of novel candidates from established GWAS data. Here, we developed an approach to prioritize GWAS variants using the epigenomes and single-cell transcriptomes of embryonic tissues and progenitor cells by implementing machine learning classifiers and combinatorial analysis. Systematic evaluation revealed significant improvement in the machine learning model performance after integrating transcriptome of neural crest cells (NCCs) and cranial placodes, as well as epigenomic profile of early craniofacial tissues. We identified 249 genes from the best-performing classifier, which include documented CFM-associated genes. Gene regulatory network (GRN) inference showed that 24 candidate genes were involved in NCC- and placode-specific regulons, of which 15 (F11R, ISL1, KANK4, L1TD1, LAMB1, MIA, PRDM1, S100A10, S100A11, STOM, STT3B, TESK2, USP43, WDR86, ZNF439) were novel candidates for human CFM. Motif analysis revealed putative functional SNPs contributing to CFM pathogenesis by disrupting transcription factor binding motifs in neural crest and placodes. Our analyses suggested that PRDM1 and ISL1 are strong candidates for human CFM, as supported by other animal functional studies. This study demonstrates a successful method for disease gene identification using epigenomic and single-cell transcriptomic profiles, and sheds light on the linkage between early cell lineages and the pathogenic process of CFM. Author SummaryCraniofacial malformation is one of the most common congenital disorders that affects food ingestion, speech and social interaction of the patients. The identification of craniofacial disease genes is difficult due to the dynamic gene expression and contribution from multiple cell types during embryonic development. In this study, we combine artificial intelligence with patient genetic and embryo multiomic information to identify new candidate genes for human craniofacial malformation. Using machine learning classifiers and combinatorial analyses, we prioritized single-nucleotide variants from patient datasets and identified 249 candidate genes. Annotation of the variants and candidate genes showed that some of them overlapped with known disease genes, demonstrating the efficacy of our approach. Further analyses using lineage reconstruction and motif analyses revealed a number of promising novel candidates, in particular PRDM1 and ISL1, are likely to be causative genes for human CFM. Our study has demonstrated a translatable approach for disease gene identification utilizing machine learning algorithm and multiomic data, and provides a gene list for improving diagnostic panels and understanding the pathogenic processes of craniofacial disorders.

The inner ear controls hearing and balance, while the temporal molecular signatures and transcriptional regulatory dynamics underlying its development are still unclear. In this study, we investigated time-series transcriptome in the mouse inner ear from embryonic day 11.5 (E11.5) to postnatal day 7 (P7) using bulk RNA-Seq. A total of 10,822 differentially expressed genes were identified between pairwise stages. We identified nine significant temporal expression profiles using time-series expression analysis. The constantly down-regulated profiles throughout the development are related to DNA activity and neurosensory development, while the constantly up-regulated profiles are related to collagen and extracellular matrix. Further co-expression network analysis revealed that several hub genes, such as pnoc1, cd9, and krt27, are related to the neurosensory development, cell adhesion, and keratinization. We uncovered three important transcription regulatory paths during mice inner ear development. Transcription factors related to Hippo/TGF{beta} signaling induced decreased expressions of genes relate to the neurosensory and inner ear development, while a series of INF genes activated the expressions of genes in immunoregulation. In addition to deepening our understanding of the temporal and regulatory mechanisms of inner ear development, our transcriptomic data could fuel future multi-species comparative studies and elucidate the evolutionary trajectory of auditory development.

The recent advance of single-cell copy number variation analysis plays an essential role in addressing intra-tumor heterogeneity, identifying tumor subgroups, and restoring tumor evolving trajectories at single-cell scale. Pleasant visualization of copy number analysis results boosts productive scientific exploration, validation, and sharing. Several single-cell analysis figures have the effectiveness of visualizations for understanding single-cell genomics in published articles and software packages. However, they almost lack real-time interaction, and it is hard to reproduce them. Moreover, existing tools are time-consuming and memory-intensive when they reach large-scale single-cell throughputs. We present an online visualization platform, scSVAS, for real-time interactive single-cell genomics data visualization. scSVAS is specifically designed for large-scale single-cell analysis. Compared with other tools, scSVAS manifests the most comprehensive functionalities. After uploading the specified input files, scSVAS deploys the online interactive visualization automatically. Users may make scientific discoveries, share interactive visualization, and download high-quality publication-ready figures. scSVAS provides versatile utilities for managing, investigating, sharing, and publishing single-cell copy number variation profiles. We envision this online platform will expedite the biological understanding of cancer clonal evolution in single-cell resolution. All visualizations are publicly hosted at https://sc.deepomics.org.

The bam mutant ovary of Drosophila represents a classic tumor model caused by germline stem cell (GSC) differentiation defects. To date, its molecular and genetic features have rarely been characterized in detail at the single-cell resolution. Here, we performed single-cell RNA sequencing (scRNA-seq) to comprehensively delineate the transcriptomic landscape and identify distinct germline cell types in bam mutant ovaries by using in situ hybridization. Differentially expressed gene analysis and PAGA plots reveal different transcriptional profiles and developmental relationships in ovarian cells. Based on the expression pattern of eggpl, a useful marker for undifferentiated germ cell identity, and morphological differences in bam mutant ovarioles, two potentially distinct germ cell states are distinguished. Comparative single-cell analysis reveals the potential regulatory network and cellular communication in subclusters of undifferentiated germ cells, and contributes to the identification of gcrf1 as a novel marker gene for female GSC, which involves in the regulation of early germ cell proliferation and Drosophila fertility. Collectively, our study not only provides insights into tumorigenesis caused by GSC differentiation defects but also offers a valuable transcriptomic resource that can be mined for the reproductive features of bam mutant tumors by community.

DNA methylation plays a critical role in early embryonic skin development by controlling gene expression. Act as an indirect regulator, long non-coding RNA (lncRNA) recruit DNA methyltransferases to specific genomic sites to methylate DNA. However, the molecular regulation mechanisms underlying hair follicle morphogenesis is unclear in cashmere goat. In this study, RNA-seq and Whole-genome bisulfite sequencing (WGBS) in embryonic day 65 (E65) and E120 skin tissues of cashmere goat were used to reveal this complex regulatory process. RNA-seq, qRT-PCR and immunohistochemistry results showed that Wnt signaling played an important role in both hair follicle induction and differentiation stage, transcriptional factors (TFs) including Hoxc13, Sox9, Sox21, Junb, Lhx2, Vdr and Gata3 participated in hair follicle differentiation via specific expression at E120. Subsequently, combination of WGBS and RNA-seq analysis showed that the expression of hair follicle differentiation genes and TFs genes was negatively correlated with DNA methylation level generally. A portion of hair follicle differentiation genes were methylated and repressed in hair follicle induction stage but were subsequently demethylated and expressed during hair follicle differentiation stage, suggesting DNA methylation play an important role in hair morphogenesis through regulating associated gene expression. Furthermore, the potential differentially expressed lncRNAs associated with DNA methylation on target gene were revealed. LncRNA XR_001918556 may affect the DNA methylation of TFs gene Gata3, lnc-003786 may affect the DNA methylation of signaling gene Fgfr2. In conclusion, differentiation genes were governed by DNA methylation, resulting in repressed expression in hair follicle induction stage and high expression in hair follicle differentiation stage. Furtherly, potential lncRNAs associated with DNA methylation on target genes were delineated. This study would enrich the regulatory network and molecular mechanisms on hair morphogenesis.

Papillary thyroid cancer (PTC) is the most common thyroid malignancy. Although PTC usually has a favorable prognosis, some aggressive PTC subtypes and lymph node (LN) metastasis contribute to high rates of recurrence and poor clinical outcomes. We analyzed single-cell RNA sequencing (scRNA-seq) data from 15 samples, including primary tumors of PTC, metastatic LNs, and paracancerous tissues. After quality filtering, 28,205 cells were detected. Of these, 13,390 cells originated from 7 tumor tissues, 2,869 cells from 2 metastatic LNs, and 11,945 cells from 6 paracancerous tissues. The increase in the proportion of CD4+ Tregs may be a key factor responsible for the immunosuppressive property of PTC. A novel cell type was identified, named Protective EGR1+CD4+ T cell, which might be antagonistic to the CD4+ Tregs and inhibit the formation of the immunosuppressive microenvironment and tumor immune evasion. Inhibitory checkpoints TIGIT and CD96 were found to be better targets than PD-1 for immune therapy in PTC patients with LN metastasis. For PTC patients without LN metastasis, however, PD-1, TIGIT, and CD96 could be suitable targets of immunotherapy. These findings would contribute to the further understanding of molecular mechanisms resulting in occurrence and development of PTC, and provide a theoretical rationale for targeted therapy and immunotherapy.

In mammals, the transcription of transposable elements (TEs) is important for maintaining early embryonic development. Here, we systematically analyzed the expression characteristics of TE-derived transcripts in early embryos by constructing a database of TEs and transcriptome data from goats and using it to study the function of endogenous retroviruses (ERVs) in regulating early embryo development. We found that ERV1 made up the highest proportion of TE sequences and exhibited a stage-specific expression pattern during early embryonic development. Among ERV elements, ERV1 had the potential to encode the Gag protein domain to form virus-like particles (VLPs) in early goat embryos. Knockdown of ERV1_1_574 significantly reduced the embryo development rate and the number of trophoblast cells (P< 0.05). Transcriptome sequencing analysis of morula embryos showed that ERV1_1_574 mainly regulated the expression of genes related to embryo compaction and trophoblast cell differentiation, such as CX43 and CDX2. In summary, we found that ERV1 expression was essential for early embryonic development in goats through regulation of trophoblast cell differentiation.

We generated and sequenced 12,069 viable Caenorhabditis elegans strains produced by ethyl methanesulfonate mutagenesis, identifying 20,315,536 variants, including 541,102 unique missense mutations across 20,914 genes. To organize and visualize this resource, we developed TaoChongBao, an open-access database and strain repository that integrates C. elegans mutation data with AlphaMissense-predicted pathogenicity scores and ClinVar clinical annotations. TaoChongBao enables users to explore worm missense variants, identify conserved residues corresponding to human pathogenic sites, and access viable strains for experimental validation. Compared with the previous Million Mutation Project in C. elegans, TaoChongBao expands mutation coverage over twenty-fold and emphasizes amino acid-altering variants. This resource provides a scalable platform for functional residuomics, variant interpretation, and comparative analyses between C. elegans and human genomes.

As a major origin of evolutionary novelties, gene duplication is a widespread phenomenon across species. However, the evolutionary force that determines the fate of duplicate genes is still under debate. Here, we studied the functional evolution of duplicate genes at both macroevolution and microevolution scales using the genomic sequences of eleven Caenorhabditis species and 773 C. elegans wild isolates. We found that compared to older duplicate genes and single-copy genes, recently duplicated gene copies showed rapid turnover, large genetic diversity, and signs of balancing and positive selection within the species. Young duplicate genes have low basal expression restricted to a few tissues but show highly responsive expression towards pathogenic infections. Recently duplicated genes are enriched in chemosensory perception, protein degradation, and innate immunity, implicating their functions in enhancing adaptability to external perturbations. Importantly, we found that young duplicate genes are rarely essential, while old duplicate genes have the same level of essentiality as singletons, suggesting that essentiality develops over a long time. Together, our work in C. elegans demonstrates that natural selection shapes the dynamic evolutionary trajectory of duplicate genes. SignificanceThe "evolution by gene duplication" theory suggests that gene duplications provide the genetic materials for mutation and selection to act upon, expand the repertoire of molecular functions, and enable evolutionary novelty. Although various models were proposed to describe the fate of duplicate genes, empirical evidence for these models is limited. We analyzed gene duplications in eleven nematode Caenorhabditis species and studied the intraspecific variation of these duplicate genes among C. elegans wild strains. We found that compared to older duplicate gens and single-copy genes, recently duplicated genes show rapid turnover, large genetic diversity, and strong signs of balancing and positive selection but rarely develop essential functions. Our results describe the evolutionary trajectory of duplicate genes shaped by natural selection.

The Hippo pathway is a central regulator of organ size and a key tumor suppressor via coordinating cell proliferation and death. Initially discovered in Drosophila, the Hippo pathway has been implicated as an evolutionarily conserved pathway in mammals; however, how this pathway was evolved to be functional from its origin is still largely unknown. In this study, we traced the Hippo pathway in premetazoan species, characterized the intrinsic functions of its ancestor components, and unveiled the evolutionary history of this key signaling pathway from its unicellular origin. In addition, we elucidated the paralogous gene history for the mammalian Hippo pathway components and characterized their cancer-derived somatic mutations from an evolutionary perspective. Taken together, our findings not only traced the conserved function of the Hippo pathway to its unicellular ancestor components, but also provided novel evolutionary insights into the Hippo pathway organization and oncogenic alteration.

Trisomy 18, commonly known as Edwards syndrome, is the second most common autosomal trisomy among live born neonates. Multiple tissues including cardiac, abdominal, and nervous systems are affected by an extra chromosome 18. To delineate the complexity of anomalies of trisomy 18, we analyzed amniotic fluid cells from two normal and three trisomy 18 samples using single-cell transcriptomics. We identified six cell groups, which function in major tissue development such as kidney, vasculature, and smooth muscle, and display significant alterations in gene expression detected by single-cell RNA-sequencing. Moreover, we demonstrated significant gene expression changes in previously proposed trisomy 18 critical regions, and identified three new regions such as 18p11.32, 18q11, 18q21.32, which are likely associated with trisomy 18 phenotypes. Our results indicate complexity of trisomy 18 at the gene expression level and reveal genetic reasoning of diverse phenotypes in trisomy 18 patients.

Oral squamous cell carcinoma (OSCC) is widely recognized as an optimal model for precise medicine guided molecular biomarkers of cancer, however, few clinical practices were applied till now. Based on the data from our own studies and published papers, it was found that the expression of MAL was significantly decreased in epithelial cancer as compared with normal tissues, and exhibited a opposite association with pathological grade. To study the molecular events related to deficiency of MAL during carcinogenesis, occurrence and development, a Mal knockout mouse model was constructed and consistently reproduced and bred. The Mal knockout mice are highly vulnerable to tumor induction by carcinogen of 4NQO, evidenced by their extremely earlier carcinogenesis, higher incidence, and more aggressive growth. Analysis of scRNA-seq data indicated that Mal knockout mice lost the ability in maintaining epithelial cell differentiation and get more prone to carcinogen with a remarkably higher incidence of epithelial malignancy. Further analyses identified putative co-functional genes of MAL, including DSG1, AQP3 and S100A8, which are key factors in maintaining epithelial cell differentiation. To conclude, the current study exhibits the clinical significance and explains the tumor suppressing function of MAL. The results also suggest the potential of MAL and its co-functional genes being biomarkers for designing the prevention and/or differentiation therapy strategies in OSCC. SignificanceMAL is found to be strongly opposite with tumor pathological grade from clinical and in vivo studies in OSCC. We propose MAL and its co-functional genes, including DSG1, AQP3 and S100A8, as key factors in maintaining epithelial cell differentiation and are valuable targets for designing prevention and differentiation therapy strategies in OSCC.

Here, we initiated the Westlake BioBank for Chinese (WBBC) pilot project with 4,535 whole-genome sequencing individuals and 5,481 high-density genotyping individuals. We identified 80.99 million SNPs and INDELs, of which 38.6% are novel. The genetic evidence of Chinese population structure supported the corresponding geographical boundaries of the Qinling-Huaihe Line and Nanling Mountains. The genetic architecture within North Han was more homogeneous than South Han, and the history of effective population size of Lingnan began to deviate from the other three regions from 6 thousand years ago. In addition, we identified a novel locus (SNX29) under selection pressure and confirmed several loci associated with alcohol metabolism and histocompatibility systems. We observed significant selection of genes on epidermal cell differentiation and skin development only in southern Chinese. Finally, we provided an online imputation server (https://wbbc.westlake.edu.cn/) which could result in higher imputation accuracy compared to the existing panels, especially for lower frequency variants.

While the number of genome sequences continues to increase, the functions of many detected gene variants remain to be identified. These variants of uncertain significance constitute a major barrier to precision medicine 1-3. Although many computational methods have been developed to predict the function of these variants, they all rely on individual gene features and do not consider complex molecular relationships. Here we develop PathoGN, a molecular network-based approach for predicting variant pathogenicity. PathoGN significantly outperforms existing methods using benchmark datasets. Moreover, PathoGN successfully predicts the pathogenicity of 3,994 variants of uncertain significance in the real-world database ClinVar and designates potential pathogenicity. This is the first computational method for the clinical interpretation of variants using biomolecular networks, and we anticipate our method to be broadly useful for the clinical interpretation of variants and for assigning biological function to unknown variants at the genomic scale.

Drosophila has long been a successful model organism in multiple fields such as genetics and developmental biology. Drosophila genome is relatively smaller and less redundant, yet largely conserved with mammals, making it a productive model in studies of embryogenesis, cell signaling, disease mechanisms, etc. Spatial gene expression pattern is critical for understanding of complex signaling pathways and cell-cell interactions, whereas temporal gene expression changes need to be tracked during highly dynamic activities such as tissue development and disease progression. Systematic studies in Drosophila as a whole are still impeded by lack of these spatiotemporal transcriptomic information. Drosophila embryos and tissues are of relatively small size, limiting the application of current technologies to comprehensively resolve their spatiotemporal gene expression patterns. Here, utilizing SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we dissected the spatiotemporal transcriptomic changes of developing Drosophila with high resolution and sensitivity. Our data recapitulated the spatial transcriptomes of embryonic and larval development in Drosophila. With these data, we identified known and previously undetected subregions in several tissues during development, and revealed known and potential gene regulatory networks of transcription factors within their topographic background. We further demonstrated that Stereo-seq data can be used for 3D reconstruction of Drosophila embryo spatial transcriptomes. Our data provides Drosophila research community with useful resources of spatiotemporally resolved transcriptomic information across developmental stages.

The shoot apical meristem (SAM) is a special category of tissue with pluripotency that forms new organs and individuals, especially floral individuals. However, little is known about the fate of cotton SAMs as a tunica corpus structure. Here, we demonstrate that cotton SAM fate decisions depend on light signals and circadian rhythms, and the genes GhFKF1, GhGI, GhCRY1 and GhCO were responsible for SAM fate decisions and highlighted via RNA sequencing (RNA-seq) analysis of different cotton cultivars, as confirmed by genetic analysis via the CRISPR-Cas9 system. In situ hybridization (ISH) analysis showed that the GhCO gene, induced by a relatively high blue light proportion, was highly upregulated during the initiation of floral meristems (FMs). Further blue light treatment analysis showed that the transition from vegetative to reproductive growth of SAM was promoted by a high proportion of blue light, coupled with high expression of the blue light-responsive genes GhCO and GhCRY1. Taken together, our study suggests that blue light signalling plays a key role in the fate decision of cotton SAM. These results provide a strategy to regulate the SAM differentiation of cotton by using the CRISPR-Cas9 system to change the ratio of red and blue light absorption to breed early-maturity cotton. One-sentence summaryThe SAM differentiation especially the initiation of floral meristem of upland cotton were mediated by genes GhCO and GhCRY1 which in response to blue light.

Ancient DNA advances have reported the complex genetic history of Eurasians, but how the knowledge of ancient subsistence strategy shifts and population movements influenced the fine-scale paternal genetic structure in East Asia has not been assessed. Here, we reported one integrated Y-chromosome genomic database of 15,530 people, including 1753 ancient people and newly-reported 919 individuals genotyped using our recently-developed targeted sequencing YHSeqY3000 panel, to explore Chinese genomic diversity, population evolutionary tracts and their genetic formation mechanism. We identified four major ancient technological innovations and population movements that shaped the landscape of Chinese paternal lineages. First, the expansion of millet farmers and early East Asians from the Yellow River Basin carrying the major O2/D subclades promoted the formation of the Sino-Tibetan peoples major composition and accelerated the Tibetan Plateaus permanent occupation. Second, rice farmers dispersal from the Yangtze River Valley carrying O1 and some sublineages of O2 contributed significantly to Tai-Kadai, Austronesian, Hmong-Mien, Austroasiatic people and southern Han Chinese. Third, Siberian-related paternal lineages of Q and C originated and boomed from Neolithic hunter-gatherers from the Mongolian Plateau and the Amur River Basin and significantly influenced the gene pools of northern Chinese. Fourth, western Eurasian-derived J, G and R lineages initially spread with Yamnaya steppe pastoralists and other proto-Indo-European people and further widely dispersed via the trans-Eurasian cultural communication along the Eurasian Steppe and the ancient Silk Road, remaining genetic trajectories in northwestern Chinese. Our work provided comprehensive modern and ancient genetic evidence to illuminate the impact of population interaction from the ancient farmer or herder-based societies on the genetic diversity patterns of modern people, revised our understandings of ancestral sources of Chinese paternal lineages, underscored the scientific imperative of the large-scale genomic resources of dense spatiotemporal underrepresented sampling populations to understand human evolutionary history.

The 3D architecture of the genome is crucial for controlling gene expression and organ development. Here, we introduce a spatial 3D genomics approach for assessing chromatin conformation in-situ in tissue sections, by integrating microfluidic deterministic barcoding and SPRITE procedures. This method was applied to mouse embryo sections, revealing a hierarchical model of chromatin interactions within and between compartments in various organs. The intra-compartment interactions vary among organs to orchestrate gene expressions, while the inter-compartment interactions remain identical in the most organs. Beyond this, the liver exhibits overwhelmingly packed chromatin with enhanced adjacent-compartment interactions, possibly related to its physiology. These findings highlight the importance of tissue-spatial information in understanding embryonic chromatin organization. The approach presents a powerful tool for investigating these processes in tissues with high heterogeneity. One Sentence SummaryA spatial 3D genomics approach was developed that accesses hierarchical chromatin conformation in-situ in tissue sections.