ABSTRACT
Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Transcription Activator-Like Effector Nucleases/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Engineering , HumansABSTRACT
Epithelial-to-mesenchymal transition (EMT) that endows cancer cells with increased invasive and migratory capacity enables cancer dissemination and metastasis. This process is tightly associated with metabolic reprogramming acquired for rewiring cell status and signaling pathways for survival in dietary insufficiency conditions. However, it remains largely unclear how transcription factor (TF)-mediated transcriptional programs are modulated during the EMT process. Here, we reveal that depletion of a key epithelial TF, ELF3 (E74-like factor-3), triggers a transforming growth factor ß (TGF-ß) signaling activation-like mesenchymal transcriptomic profile and metastatic features linked to the aminoacyl-tRNA biogenesis pathway. Moreover, the transcriptome alterations elicited by ELF3 depletion perfectly resemble an ATF4-dependent weak response to amino acid starvation. Intriguingly, we observe an exclusive enrichment of ELF3 and ATF4 in epithelial and TGF-ß-induced or ELF3-depletion-elicited mesenchymal enhancers, respectively, with rare co-binding on altered enhancers. We also find that the upregulation of aminoacyl-tRNA synthetases and some mesenchymal genes upon amino acid deprivation is diminished in ATF4-depleted cells. In sum, the loss of ELF3 binding on epithelial enhancers and the gain of ATF4 binding on the enhancers of mesenchymal factors and amino acid deprivation responsive genes facilitate the loss of epithelial cell features and the gain of TGF-ß-signaling-associated mesenchymal signatures, which further promote lung cancer cell metastasis.
Subject(s)
Activating Transcription Factor 4 , Amino Acids , DNA-Binding Proteins , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Transcription Factors , Transforming Growth Factor beta , Epithelial-Mesenchymal Transition/genetics , Activating Transcription Factor 4/metabolism , Activating Transcription Factor 4/genetics , Humans , Transcription Factors/metabolism , Transcription Factors/genetics , Amino Acids/metabolism , Transforming Growth Factor beta/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cell Line, Tumor , Signal Transduction , Proto-Oncogene Proteins c-ets/metabolism , Proto-Oncogene Proteins c-ets/genetics , Transcriptome , AnimalsABSTRACT
Dysregulation of histone acetylation is widely implicated in tumorigenesis, yet its specific roles in the progression and metastasis of esophageal squamous cell carcinoma (ESCC) remain unclear. Here, we profiled the genome-wide landscapes of H3K9ac for paired adjacent normal (Nor), primary ESCC (EC) and metastatic lymph node (LNC) esophageal tissues from three ESCC patients. Compared to H3K27ac, we identified a distinct epigenetic reprogramming specific to H3K9ac in EC and LNC samples relative to Nor samples. This H3K9ac-related reprogramming contributed to the transcriptomic aberration of targeting genes, which were functionally associated with tumorigenesis and metastasis. Notably, genes with gained H3K9ac signals in both primary and metastatic lymph node samples (common-gained gene) were significantly enriched in oncogenes. Single-cell RNA-seq analysis further revealed that the corresponding top 15 common-gained genes preferred to be enriched in mesenchymal cells with high metastatic potential. Additionally, in vitro experiment demonstrated that the removal of H3K9ac from the common-gained gene MSI1 significantly downregulated its transcription, resulting in deficiencies in ESCC cell proliferation and migration. Together, our findings revealed the distinct characteristics of H3K9ac in esophageal squamous cell carcinogenesis and metastasis, and highlighted the potential therapeutic avenue for intervening ESCC through epigenetic modulation via H3K9ac.
Subject(s)
Carcinoma, Squamous Cell , Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Humans , Esophageal Squamous Cell Carcinoma/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Histones/genetics , Lysine/therapeutic use , Esophageal Neoplasms/pathology , Acetylation , Cell Proliferation/genetics , Carcinogenesis , Nerve Tissue Proteins , RNA-Binding ProteinsABSTRACT
Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.
Subject(s)
Gene Editing , Trans-Activators , Mice , Animals , Trans-Activators/metabolism , Cytosine , Mutation , DNA, Mitochondrial/genetics , CRISPR-Cas SystemsABSTRACT
CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Mice , Animals , Humans , CRISPR-Cas Systems/genetics , Adenine/metabolism , Cytosine/metabolism , NucleotidesABSTRACT
Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual base editors that can catalyze concurrent C-to-T and A-to-G editing have been developed, while simultaneous C&G-to-T&A and A&T-to-G&C conversions on the same allele have not been achieved at the desirable site. Here we propose a strategy of combining base editors with dual guide RNAs (gRNAs) that target two overlapped neighboring loci on the opposite strands, which can induce simultaneous C&G-to-T&A and A&T-to-G&C conversions within their overlapping targeting windows. Moreover, one of the paired gRNAs is mutated to perfectly match another gRNA-edited sequence, efficiently facilitating concurrent base conversions on the same allele. To further expand the targeting scopes, PAMless SpRY Cas9-mediated base editors are combined with our optimized dual gRNAs system to induce expected concurrent base editing and to install neighboring pathogenic MNVs in TP53 in cancer cells. In addition, more complex mutation types can be achieved by integrating dual base editors and our dual gRNAs strategy. Thus, we establish a general strategy to efficiently induce MNVs in human genome, helping to dissect the functions of pathogenic MNVs with multifarious types.
ABSTRACT
Selective inhibition of targeted protein kinases is an effective therapeutic approach for treatment of human malignancies, which interferes phosphorylation of cellular substrates. However, a drug-imposed selection creates pressures for tumor cells to acquire chemoresistance-conferring mutations or activating alternative pathways, which can bypass the inhibitory effects of kinase inhibitors. Thus, identifying downstream phospho-substrates conferring drug resistance is of great importance for developing poly-pharmacological and targeted therapies. To identify functional phosphorylation sites involved in 5-fluorouracil (5-FU) resistance during its treatment of colorectal cancer cells, CRISPR-mediated cytosine base editor (CBE) and adenine base editor (ABE) are utilized for functional screens by mutating phosphorylated amino acids with two libraries specifically targeting 7779 and 10 149 phosphorylation sites. Among the top enriched gRNAs-induced gain-of-function mutants, the target genes are involved in cell cycle and post-translational covalent modifications. Moreover, several substrates of RSK2 and PAK4 kinases are discovered as main effectors in responding to 5-FU chemotherapy, and combinational treatment of colorectal cancer cells with 5-FU and RSK2 inhibitor or PAK4 inhibitor can largely inhibit cell growth and enhance cell apoptosis through a RSK2/TP53BP1/γ-H2AX phosphorylation signaling axis. It is proposed that this screen approach can be used for functional phosphoproteomics in chemotherapy of various human diseases.
Subject(s)
Colorectal Neoplasms , Drug Resistance, Neoplasm , Humans , Drug Resistance, Neoplasm/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Fluorouracil/pharmacology , Fluorouracil/therapeutic use , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Kinases/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Adenine/pharmacology , Adenine/therapeutic use , Amino Acids/genetics , Amino Acids/pharmacology , Amino Acids/therapeutic use , Cytosine/pharmacology , Cytosine/therapeutic use , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , p21-Activated Kinases/pharmacologyABSTRACT
CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively.
ABSTRACT
Paternal life experiences impact offspring health via germline, and epigenetic inheritance provides a potential mechanism. However, global reprogramming during offspring embryogenesis and gametogenesis represents the largest hurdle to conceptualize it. Yet, detailed characterization of how sperm epigenetic alterations carrying "environmental memory" can evade offspring embryonic reprogramming remains elusive. Here, mice exposed to long-term restraint stress were employed to study the mechanisms underlying inter- and transgenerational effects of paternal exposure to a long-term psychological stress. We found that stress could induce paternal inheritance of reproductive, behavioral, and metabolic disorders. Bisulfite methylation profiling of 18 sperm and 12 embryo samples of three consecutive generations identified inter- and transgenerational inheritance of paternal Differential DNA Methylation Regions (DMRs) at frequencies ~11.36% and 0.48%, respectively. These DMRs related to genes with functional implications for psychological stress response, and tissue inheritance of these DMRs passed paternal disorders epigenetically to offspring. More importantly, these DMRs evaded offspring embryonic reprogramming through erasure and subsequent reestablishment, but not via un-erasure way. Nonetheless, their reestablishment proportions in the primitive streak (E7.5) stage were altered. Furthermore, sncRNA-seq revealed that stress-induced tsRNA, miRNA and rsRNA dysregulation in paternal sperm might play important roles in DMRs occurrence and paternal inheritance. These finding implied that sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress, and highlighted the possible underlying molecular mechanism.
ABSTRACT
Prime editing enables efficient introduction of targeted transversions, insertions, and deletions in mammalian cells and several organisms. However, genetic disease models with base deletions by prime editing have not yet been reported in mice. Here, we successfully generate a mouse model with a cataract disorder through microinjection of prime editor 3 (PE3) plasmids to efficiently induce targeted single-base deletion. Notably, a generated mouse with a high G-deletion rate (38.2%) displays a nuclear cataract phenotype; the PE3-induced deletions in mutant mice achieve high rates of germline transmission to their progenies, with phenotypic inheritance of cataract. Our data propose that modeling a genetic disease with a single nucleotide deletion in mice can be achieved with prime genome editing in vivo.
ABSTRACT
Trimethylation of histone H3 lysine 27 trimethylation (H3K27me3) may be recruited by repressive Polycomb complexes to mediate gene silencing, which is critical for maintaining embryonic stem cell pluripotency and differentiation. However, the roles of aberrant H3K27me3 patterns in tumorigenesis are not fully understood. Here, we discovered that grand silencer domains (breadth > 50 kb) for H3K27me3 were significantly associated with epithelial cell differentiation and exhibited high gene essentiality and conservation in human esophageal epithelial cells. These grand H3K27me3 domains exhibited high modification signals involved in gene silencing, and preferentially occupied the entirety of topologically associating domains and interact with each other. We found that widespread loss of the grand H3K27me3 domains in of esophageal squamous cell carcinomas (ESCCs) were enriched in genes involved in epithelium and endothelium differentiation, which were significantly associated with overexpression with increase of active modifications of H3K4me3, H3K4me1, and H3K27ac marks, as well as DNA hypermethylation in the gene bodies. A total of 208 activated genes with loss of grand H3K27me3 domains in ESCC were identified, where the higher expression and mutation of T-box transcription factor 20 (TBX20) were associated with worse patients' outcomes. Our results showed that knockdown of TBX20 may have led to a striking defect in esophageal cancer cell growth and carcinogenesis-related pathway, including cell cycle and homologous recombination. Together, our results reveal that loss of grand H3K27me3 domains represent a catalog of remarkable activating regulators involved in carcinogenesis.
ABSTRACT
The role of cis-elements and their aberrations remains unclear in esophageal squamous cell carcinoma (ESCC, further abbreviated EC). Here we survey 28 H3K27ac-marked active enhancer profiles and 50 transcriptomes in primary EC, metastatic lymph node cancer (LNC), and adjacent normal (Nor) esophageal tissues. Thousands of gained or lost enhancers and hundreds of altered putative super-enhancers are identified in EC and LNC samples respectively relative to Nor, with a large number of common gained or lost enhancers. Moreover, these differential enhancers contribute to the transcriptomic aberrations in ECs and LNCs. We also reveal putative driver onco-transcription factors, depletion of which diminishes cell proliferation and migration. The administration of chemical inhibitors to suppress the predicted targets of gained super-enhances reveals HSP90AA1 and PDE4B as potential therapeutic targets for ESCC. Thus, our epigenomic profiling reveals a compendium of reprogrammed cis-regulatory elements during ESCC carcinogenesis and metastasis for uncovering promising targets for cancer treatment.
Subject(s)
Enhancer Elements, Genetic , Esophageal Neoplasms/genetics , Esophageal Squamous Cell Carcinoma/genetics , Aged , Carcinogenesis/genetics , Cell Line, Tumor , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/secondary , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Ontology , Histone Code/genetics , Humans , Lymphatic Metastasis/genetics , Male , Middle Aged , Oncogenes , Transcription Factors/geneticsABSTRACT
OBJECTIVES: PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate-limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. MATERIALS AND METHODS: In this study, we used CRISPR-based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA-seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. RESULTS: The splicing sites of PKM, a 5' donor site of GT and a 3' acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax-NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5' donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non-canonical PKM isoforms and produced some novel splicing isoforms. CONCLUSIONS: This work proved that CRISPR-based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.
Subject(s)
Gene Editing , Pyruvate Kinase/metabolism , Alternative Splicing , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Proliferation , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Down-Regulation , Exons , Female , HCT116 Cells , Humans , Mutagenesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pyruvate Kinase/genetics , Up-Regulation , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.
Subject(s)
Adenosine Deaminase/genetics , CRISPR-Associated Protein 9/genetics , DNA/genetics , Escherichia coli Proteins/genetics , Gene Editing/methods , Adenine/metabolism , Adenosine Deaminase/metabolism , Bacterial Toxins/genetics , CRISPR-Associated Protein 9/metabolism , Cell Line, Tumor , Cytosine/metabolism , DNA/metabolism , Escherichia coli Proteins/metabolism , Genes, Reporter , HEK293 Cells , Humans , Inosine/genetics , Inosine/metabolism , Protein Engineering , RNA Editing/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA-Seq , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Transcription growth factor ß (TGF-ß) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-ß-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-ß-induced EMT process. Enhancers gained upon TGF-ß treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-ß stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-ß signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-ß signaling-associated cancer metastasis.
Subject(s)
Cellular Reprogramming/drug effects , Enhancer Elements, Genetic/drug effects , Epithelial-Mesenchymal Transition/drug effects , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacology , A549 Cells , Animals , Carcinogenesis/drug effects , Carcinogenesis/genetics , DNA-Binding Proteins/metabolism , HEK293 Cells , Hepatocyte Nuclear Factor 3-beta/metabolism , Hepatocytes/metabolism , Histone Deacetylase 1/metabolism , Histones/metabolism , Humans , Mice , Muscle Proteins/metabolism , Neoplasm Metastasis , Smad2 Protein/metabolism , Smad3 Protein/metabolism , TEA Domain Transcription Factors , Transcription Factors/metabolism , Transcriptional Activation/drug effects , Transforming Growth Factor beta/metabolismABSTRACT
The transcriptome of the preimplantation mouse embryo has been previously annotated by short-read sequencing, with limited coverage and accuracy. Here we utilize a low-cell number transcriptome based on the Smart-seq2 method to perform long-read sequencing. Our analysis describes additional novel transcripts and complexity of the preimplantation transcriptome, identifying 2280 potential novel transcripts from previously unannotated loci and 6289 novel splicing isoforms from previously annotated genes. Notably, these novel transcripts and isoforms with transcription start sites are enriched for an active promoter modification, H3K4me3. Moreover, we generate a more complete and precise transcriptome by combining long-read and short-read data during early embryogenesis. Based on this approach, we identify a previously undescribed isoform of Kdm4dl with a modified mRNA reading frame and a novel noncoding gene designated XLOC_004958. Depletion of Kdm4dl or XLOC_004958 led to abnormal blastocyst development. Thus, our data provide a high-resolution and more precise transcriptome during preimplantation mouse embryogenesis.
Subject(s)
Blastocyst/metabolism , Molecular Sequence Annotation/methods , Transcriptome/genetics , Animals , Embryonic Development/genetics , High-Throughput Nucleotide Sequencing/methods , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Mice , Protein Isoforms/genetics , RNA Splicing/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/geneticsABSTRACT
Primary germ layers have the potential to form all tissues in the mature organism, and their formation during gastrulation requires precise epigenetic modulation of both proximal and distal regulatory elements. Previous studies indicated that spatial and temporal patterns of gene expression in the gastrula predispose individual regions to distinct cell fates. However, the underlying epigenetic mechanisms remain largely unexplored. Here, we profile the spatiotemporal landscape of the epigenome and transcriptome of the mouse gastrula. We reveal the asynchronous dynamics of proximal chromatin states during germ layer formation as well as unique gastrula-specific epigenomic features of regulatory elements, which have strong usage turnover dynamics and clear germ layer-specific signatures. Importantly, we also find that enhancers around organogenetic genes, which are weakly expressed at the gastrulation stage, are frequently pre-marked by histone H3 lysine 27 acetylation (H3K27ac) in the gastrula. By using the transgenic mice and genome editing system, we demonstrate that a pre-marked enhancer, which is located in the intron of a brain-specific gene 2510009E07Rik, exhibits specific enhancer activity in the ectoderm and future brain tissue, and also executes important function during mouse neural differentiation. Taken together, our study provides the comprehensive epigenetic information for embryonic patterning during mouse gastrulation, demonstrates the importance of gastrula pre-marked enhancers in regulating the correct development of the mouse embryo, and thus broadens the current understanding of mammalian embryonic development and related diseases.
Subject(s)
Enhancer Elements, Genetic/physiology , Epigenesis, Genetic , Gastrula/embryology , Gastrulation/genetics , Gene Expression Regulation, Developmental , Germ Layers/embryology , Animals , Brain/embryology , Brain/metabolism , Cells, Cultured , Embryo, Mammalian , Embryonic Stem Cells , Female , Gastrula/cytology , Gastrula/metabolism , Germ Layers/cytology , Germ Layers/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurogenesis/genetics , TranscriptomeABSTRACT
Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.