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Nature Communications May 2023Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here,...
Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.
Topics: RNA; RNA Isoforms; High-Throughput Nucleotide Sequencing; Consensus; Protein Isoforms; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 37149708
DOI: 10.1038/s41467-023-38324-9 -
Journal of Hematology & Oncology Sep 2020Acute myeloid leukemia (AML) is a fatal hematopoietic malignancy and has a prognosis that varies with its genetic complexity. However, there has been no appropriate...
BACKGROUND
Acute myeloid leukemia (AML) is a fatal hematopoietic malignancy and has a prognosis that varies with its genetic complexity. However, there has been no appropriate integrative analysis on the hierarchy of different AML subtypes.
METHODS
Using Microwell-seq, a high-throughput single-cell mRNA sequencing platform, we analyzed the cellular hierarchy of bone marrow samples from 40 patients and 3 healthy donors. We also used single-cell single-molecule real-time (SMRT) sequencing to investigate the clonal heterogeneity of AML cells.
RESULTS
From the integrative analysis of 191727 AML cells, we established a single-cell AML landscape and identified an AML progenitor cell cluster with novel AML markers. Patients with ribosomal protein high progenitor cells had a low remission rate. We deduced two types of AML with diverse clinical outcomes. We traced mitochondrial mutations in the AML landscape by combining Microwell-seq with SMRT sequencing. We propose the existence of a phenotypic "cancer attractor" that might help to define a common phenotype for AML progenitor cells. Finally, we explored the potential drug targets by making comparisons between the AML landscape and the Human Cell Landscape.
CONCLUSIONS
We identified a key AML progenitor cell cluster. A high ribosomal protein gene level indicates the poor prognosis. We deduced two types of AML and explored the potential drug targets. Our results suggest the existence of a cancer attractor.
Topics: Bone Marrow Examination; Cell Lineage; Clone Cells; Computer Systems; DNA, Mitochondrial; DNA, Neoplasm; Gene Expression Regulation, Leukemic; Gene Regulatory Networks; High-Throughput Nucleotide Sequencing; Humans; Leukemia, Monocytic, Acute; Leukemia, Myeloid, Acute; Neoplasm Proteins; Neoplastic Stem Cells; Phenotype; Prognosis; RNA, Messenger; RNA, Neoplasm; Recurrence; Ribosomal Proteins; Single-Cell Analysis; Transcription Factors
PubMed: 32977829
DOI: 10.1186/s13045-020-00941-y -
Cell Jul 2020The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that...
The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
Topics: Animals; Basic-Leucine Zipper Transcription Factors; Chromatin; Germinal Center; Histone Deacetylases; Humans; Immunologic Memory; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutagenesis, Site-Directed; Nuclear Proteins; Nuclear Receptor Co-Repressor 2; Precursor Cells, B-Lymphoid; Protein Binding; Proto-Oncogene Proteins c-bcl-6; RNA Interference; RNA, Small Interfering; Receptors, Cytoplasmic and Nuclear; Repressor Proteins; Transcription, Genetic
PubMed: 32619424
DOI: 10.1016/j.cell.2020.05.049 -
Nature Communications Feb 2022Extensive knowledge has been gained on the transcription network controlled by ERα, however, the mechanism underlying ESR1 (encoding ERα) expression is less...
Extensive knowledge has been gained on the transcription network controlled by ERα, however, the mechanism underlying ESR1 (encoding ERα) expression is less understood. We recently discovered that the Hippo pathway is required for the proper expression of ESR1. YAP/TAZ are transcription coactivators that are phosphorylated and inhibited by the Hippo pathway kinase LATS. Here we delineated the molecular mechanisms underlying ESR1 transcription repression by the Hippo pathway. Mechanistically, YAP binds to TEAD to increase local chromatin accessibility to stimulate transcription of nearby genes. Among the YAP target genes, Vestigial-Like Protein 3 (VGLL3) competes with YAP/TAZ for binding to TEAD transcription factor and recruits the NCOR2/SMRT repressor to the super-enhancer of ESR1 gene, leading to epigenetic alteration and transcriptional silencing. We developed a potent LATS inhibitor VT02956. Targeting the Hippo pathway by VT02956 represses ESR1 expression and inhibits the growth of ER breast cancer cells as well as patient-derived tumour organoids. Moreover, histone deacetylase inhibitors, such as Entinostat, induce VGLL3 expression to inhibit ER breast cancer cells. Our study suggests LATS as unexpected cancer therapeutic targets, especially for endocrine-resistant breast cancers.
Topics: Female; Humans; Adaptor Proteins, Signal Transducing; Breast Neoplasms; Estrogen Receptor alpha; Hippo Signaling Pathway; Intracellular Signaling Peptides and Proteins; Phosphoproteins; Protein Serine-Threonine Kinases; Signal Transduction; Transcription Factors; YAP-Signaling Proteins
PubMed: 35217640
DOI: 10.1038/s41467-022-28691-0 -
Nature Cancer Jun 2022Resistance to antitumor treatment contributes to patient mortality. Functional proteomic screening of organoids derived from chemotherapy-treated patients with breast...
Resistance to antitumor treatment contributes to patient mortality. Functional proteomic screening of organoids derived from chemotherapy-treated patients with breast cancer identified nuclear receptor corepressor 2 (NCOR2) histone deacetylase as an inhibitor of cytotoxic stress response and antitumor immunity. High NCOR2 in the tumors of patients with breast cancer predicted chemotherapy refractoriness, tumor recurrence and poor prognosis. Molecular studies revealed that NCOR2 inhibits antitumor treatment by regulating histone deacetylase 3 (HDAC3) to repress interferon regulatory factor 1 (IRF-1)-dependent gene expression and interferon (IFN) signaling. Reducing NCOR2 or impeding its epigenetic activity by modifying its interaction with HDAC3 enhanced chemotherapy responsiveness and restored antitumor immunity. An adeno-associated viral NCOR2-HDAC3 competitor potentiated chemotherapy and immune checkpoint therapy in culture and in vivo by permitting transcription of IRF-1-regulated proapoptosis and inflammatory genes to increase IFN-γ signaling. The findings illustrate the utility of patient-derived organoids for drug discovery and suggest that targeting stress and inflammatory-repressor complexes such as NCOR2-HDAC3 could overcome treatment resistance and improve the outcome of patients with cancer.
Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Early Detection of Cancer; Female; Humans; Neoplasm Recurrence, Local; Nuclear Receptor Co-Repressor 2; Organoids; Proteomics
PubMed: 35618935
DOI: 10.1038/s43018-022-00375-0 -
Advanced Science (Weinheim,... May 2021Cullin4B (CUL4B) is a scaffold protein of the CUL4B-Ring E3 ligase (CRL4B) complex. However, the role of CUL4B in the development of breast cancer remains poorly...
Cullin4B (CUL4B) is a scaffold protein of the CUL4B-Ring E3 ligase (CRL4B) complex. However, the role of CUL4B in the development of breast cancer remains poorly understood. Here it is shown that CRL4B interacts with multiple histone deacetylase (HDAC)-containing corepressor complexes, including MTA1/NuRD, SIN3A, CoREST, and NcoR/SMRT complexes. It is demonstrated that CRL4B/NuRD(MTA1) complexes cooccupy the E-cadherin and AXIN2 promoters, and could be recruited by transcription factors including Snail and ZEB2 to promote cell invasion and tumorigenesis both in vitro and in vivo. Remarkably, CUL4B responded to transformation and migration/invasion stimuli and is essential for multiple epithelial-mesenchymal transition (EMT) signaling pathways such as hypoxia. Furthermore, the transcription of CUL4B is directedly activated by hypoxia-inducible factor 1 (HIF1) and repressed by the ER-GATA3 axis. Overexpressing of CUL4B successfully induced CSC-like properties. Strikingly, CUL4B expression is markedly upregulated during breast cancer progression and correlated with poor prognosis. The results suggest that CUL4B lies at a critical crossroads between EMT and stem cell properties, supporting CUL4B as a potential novel target for the development of anti-breast cancer therapy.
Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cullin Proteins; Epithelial-Mesenchymal Transition; Female; Humans; Hypoxia; Mice, SCID; Neoplasm Grading; Repressor Proteins; Signal Transduction; Survival Rate; Xenograft Model Antitumor Assays; Mice
PubMed: 34026424
DOI: 10.1002/advs.202001515 -
Experimental Hematology Dec 2022Over the past 2 decades, the adaptor protein transducin β-like 1 (TBL1X) and its homolog TBL1XR1 have been shown to be upregulated in solid tumors and hematologic... (Review)
Review
Over the past 2 decades, the adaptor protein transducin β-like 1 (TBL1X) and its homolog TBL1XR1 have been shown to be upregulated in solid tumors and hematologic malignancies, and their overexpression is associated with poor clinical outcomes. Moreover, dysregulation of the TBL1 family of proteins has been implicated as a key component of oncogenic prosurvival signaling, cancer progression, and metastasis. Herein, we discuss how TBL1X and TBL1XR1 are required for the regulation of major transcriptional programs through the silencing mediator for tetanoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (NCOR)/ B cell lymphoma 6 (BCL6) complex, Wnt/β catenin, and NF-κB signaling. We outline the utilization of tegavivint (Iterion Therapeutics), a first-in-class small molecule targeting the N-terminus domain of TBL1, as a novel therapeutic strategy in preclinical models of cancer and clinically. Although most published work has focused on the transcriptional role of TBL1X, we recently showed that in diffuse large B-cell lymphoma (DLBCL), the most common lymphoma subtype, genetic knockdown of TBL1X and treatment with tegavivint resulted in decreased expression of critical (onco)-proteins in a posttranscriptional/β-catenin-independent manner by promoting their proteasomal degradation through a Skp1/Cul1/F-box (SCF)/TBL1X supercomplex and potentially through the regulation of protein synthesis. However, given that TBL1X controls multiple oncogenic signaling pathways in cancer, treatment with tegavivint may ultimately result in drug resistance, providing the rationale for combination strategies. Although many questions related to TBL1X function remain to be answered in lymphoma and other diseases, these data provide a growing body of evidence that TBL1X is a promising therapeutic target in oncology.
Topics: Humans; Carcinogenesis; NF-kappa B; Signal Transduction; Transducin; Neoplasms; Gene Expression Regulation, Neoplastic; Receptors, Cytoplasmic and Nuclear; Repressor Proteins
PubMed: 36206873
DOI: 10.1016/j.exphem.2022.09.006 -
Cellular and Molecular Life Sciences :... Aug 2020During embryonic development, one of the two X chromosomes of a mammalian female cell is randomly inactivated by the X chromosome inactivation mechanism, which is mainly... (Review)
Review
During embryonic development, one of the two X chromosomes of a mammalian female cell is randomly inactivated by the X chromosome inactivation mechanism, which is mainly dependent on the regulation of the non-coding RNA X-inactive specific transcript at the X chromosome inactivation center. There are three proteins that are essential for X-inactive specific transcript to function properly: scaffold attachment factor-A, lamin B receptor, and SMRT- and HDAC-associated repressor protein. In addition, the absence of X-inactive specific transcript expression promotes tumor development. During the process of chromosome inactivation, some tumor suppressor genes escape inactivation of the X chromosome and thereby continue to play a role in tumor suppression. A well-functioning tumor suppressor gene on the idle X chromosome in women is one of the reasons they have a lower propensity to develop cancer than men, women thereby benefit from this enhanced tumor suppression. This review will explore the mechanism of X chromosome inactivation, discuss the relationship between X chromosome inactivation and tumorigenesis, and consider the consequent sex differences in cancer.
Topics: Chromosomes, Human, X; Humans; Mutation; Neoplasms; RNA, Long Noncoding; Sex Characteristics; Tumor Suppressor Proteins; X Chromosome Inactivation
PubMed: 32040694
DOI: 10.1007/s00018-020-03469-z -
Proceedings of the National Academy of... Aug 2022Brown adipose tissue (BAT) is a key thermogenic organ whose expression of uncoupling protein 1 (UCP1) and ability to maintain body temperature in response to acute cold...
Brown adipose tissue (BAT) is a key thermogenic organ whose expression of uncoupling protein 1 (UCP1) and ability to maintain body temperature in response to acute cold exposure require histone deacetylase 3 (HDAC3). HDAC3 exists in tight association with nuclear receptor corepressors (NCoRs) NCoR1 and NCoR2 (also known as silencing mediator of retinoid and thyroid receptors [SMRT]), but the functions of NCoR1/2 in BAT have not been established. Here we report that as expected, genetic loss of NCoR1/2 in BAT (NCoR1/2 BAT-dKO) leads to loss of HDAC3 activity. In addition, HDAC3 is no longer bound at its physiological genomic sites in the absence of NCoR1/2, leading to a shared deregulation of BAT lipid metabolism between NCoR1/2 BAT-dKO and HDAC3 BAT-KO mice. Despite these commonalities, loss of NCoR1/2 in BAT does not phenocopy the cold sensitivity observed in HDAC3 BAT-KO, nor does loss of either corepressor alone. Instead, BAT lacking NCoR1/2 is inflamed, particularly with respect to the interleukin-17 axis that increases thermogenic capacity by enhancing innervation. Integration of BAT RNA sequencing and chromatin immunoprecipitation sequencing data revealed that NCoR1/2 directly regulate , which integrates extracellular matrix remodeling and inflammation. These findings reveal pleiotropic functions of the NCoR/HDAC3 corepressor complex in BAT, such that HDAC3-independent suppression of BAT inflammation counterbalances stimulation of HDAC3 activity in the control of thermogenesis.
Topics: Adipose Tissue, Brown; Animals; Histone Deacetylases; Inflammation; Mice; Mice, Knockout; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2; Receptors, Retinoic Acid; Thermogenesis; Uncoupling Protein 1
PubMed: 35939699
DOI: 10.1073/pnas.2205276119 -
Frontiers in Immunology 2022Generally, invertebrates were thought to solely rely on their non-specific innate immune system to fight against invading microorganisms. However, increasing studies...
Generally, invertebrates were thought to solely rely on their non-specific innate immune system to fight against invading microorganisms. However, increasing studies have implied that the innate immune response of invertebrates displayed diversity and specificity owing to the hyper-variable immune molecules in organisms. In order to get an insight into the diversity of immune-related genes in , a full-length transcriptome analysis of several immune-related tissues (hemocytes, hepatopancreas and gills) in was performed and the diversity of several immune-related genes was analyzed. The full-length transcriptome analysis of was conducted using a combination of SMRT long-read sequencing and Illumina short-read sequencing. A total of 17,433 nonredundant full-length transcripts with average length of 2,271 bp and N50 length of 2,841 bp were obtained, among which 13,978 (80.18%) transcripts were annotated. Moreover, numerous transcript variants of various immune-related genes were identified, including pattern recognition receptors, antimicrobial peptides, heat shock proteins (HSPs), antioxidant enzymes and vital molecules in prophenoloxidase (proPO)-activating system. Based on the full-length transcriptome analysis, open reading frames (ORFs) of four C-type lectins (CTLs) were cloned, and tissue distributions showed that the four CTLs were ubiquitously expressed in all the tested tissues, and mainly expressed in hepatopancreas and gills. The transcription of the four CTLs significantly increased in several immune-related tissues (hemocytes, hepatopancreas and gills) of challenged with and displayed different profiles. Moreover, the four CTLs displayed distinct bacterial binding and antibacterial activities. The recombinant protein PtCTL-1 (rPtCTL-1) and rPtCTL-3 displayed bacterial binding and antibacterial activities against all tested bacteria. rPtCTL-2 only showed bacterial binding and antibacterial activities against . No obvious bacterial binding or antibacterial activities for PtCTL-4 was observed against the tested bacteria. This study enriches the transcriptomic information on . and provides new insights into the innate immune system of crustaceans. Additionally, our study provided candidates of antibiotic agents for the prevention and treatment of bacteriosis.
Topics: Animals; Anti-Bacterial Agents; Arthropod Proteins; Brachyura; Gene Expression Profiling; Hemocytes; Lectins, C-Type
PubMed: 35464434
DOI: 10.3389/fimmu.2022.843347