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1.
Nat Biotechnol ; 42(1): 52-64, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37037903

ABSTRACT

Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma.


Subject(s)
Chromatin , DNA , Sequence Analysis, DNA
2.
Sci Adv ; 9(13): eabo3789, 2023 03 31.
Article in English | MEDLINE | ID: mdl-37000878

ABSTRACT

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.


Subject(s)
Sarcoma, Ewing , Child , Humans , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Chromatin/genetics , Cell Line, Tumor , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , Binding Sites , Cell Differentiation , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Gene Expression Regulation, Neoplastic
3.
Mol Cell ; 82(15): 2738-2753.e6, 2022 08 04.
Article in English | MEDLINE | ID: mdl-35662392

ABSTRACT

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.


Subject(s)
Chromatin , Transcription, Genetic , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA Breaks, Double-Stranded , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism
4.
Nat Commun ; 13(1): 2267, 2022 04 27.
Article in English | MEDLINE | ID: mdl-35477713

ABSTRACT

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.


Subject(s)
Sarcoma, Clear Cell , Soft Tissue Neoplasms , Carcinogenesis/genetics , Chromatin/genetics , Humans , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Oncogenes , RNA-Binding Protein EWS/genetics , Sarcoma, Clear Cell/genetics , Sarcoma, Clear Cell/pathology , Soft Tissue Neoplasms/genetics
5.
Science ; 373(6554): 547-555, 2021 07 30.
Article in English | MEDLINE | ID: mdl-34326237

ABSTRACT

RNA polymerase I (Pol I) transcription takes place at the border of the fibrillar center (FC) and the dense fibrillar component (DFC) in the nucleolus. Here, we report that individual spherical FC/DFC units are coated by the DEAD-box RNA helicase DDX21 in human cells. The long noncoding RNA (lncRNA) SLERT binds to DDX21 RecA domains to promote DDX21 to adopt a closed conformation at a substoichiometric ratio through a molecular chaperone-like mechanism resulting in the formation of hypomultimerized and loose DDX21 clusters that coat DFCs, which is required for proper FC/DFC liquidity and Pol I processivity. Our results suggest that SLERT is an RNA regulator that controls the biophysical properties of FC/DFCs and thus ribosomal RNA production.


Subject(s)
Cell Nucleolus/metabolism , DEAD-box RNA Helicases/metabolism , RNA Polymerase I/metabolism , RNA, Long Noncoding/metabolism , Cell Line , DEAD-box RNA Helicases/chemistry , DNA, Ribosomal/metabolism , Humans , Molecular Chaperones/metabolism , Protein Conformation , Protein Domains , Protein Multimerization , Transcription, Genetic
6.
Life Sci Alliance ; 4(2)2021 02.
Article in English | MEDLINE | ID: mdl-33361335

ABSTRACT

Synovial sarcoma (SyS) is an aggressive mesenchymal malignancy invariably associated with the chromosomal translocation t(X:18; p11:q11), which results in the in-frame fusion of the BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 to SSX generates an aberrant transcriptional regulator, which, in permissive cells, drives tumor development by initiating major chromatin remodeling events that disrupt the balance between BAF-mediated gene activation and polycomb-dependent repression. Here, we developed SyS organoids and performed genome-wide epigenomic profiling of these models and mesenchymal precursors to define SyS-specific chromatin remodeling mechanisms and dependencies. We show that SS18-SSX induces broad BAF domains at its binding sites, which oppose polycomb repressor complex (PRC) 2 activity, while facilitating recruitment of a non-canonical (nc)PRC1 variant. Along with the uncoupling of polycomb complexes, we observed H3K27me3 eviction, H2AK119ub deposition and the establishment of de novo active regulatory elements that drive SyS identity. These alterations are completely reversible upon SS18-SSX depletion and are associated with vulnerability to USP7 loss, a core member of ncPRC1.1. Using the power of primary tumor organoids, our work helps define the mechanisms of epigenetic dysregulation on which SyS cells are dependent.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin/genetics , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Sarcoma, Synovial/genetics , Binding Sites , Chromatin/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Gene Expression Profiling , Histones/metabolism , Humans , Multiprotein Complexes/metabolism , Organoids , Protein Binding , Protein Transport , Sarcoma, Synovial/metabolism , Transcriptome
7.
Cell ; 181(3): 621-636.e22, 2020 04 30.
Article in English | MEDLINE | ID: mdl-32259487

ABSTRACT

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.


Subject(s)
Intracellular Space/genetics , RNA, Long Noncoding/metabolism , Stem Cells/metabolism , Animals , Cell Differentiation/genetics , Cell Line , Cells, Cultured , Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/metabolism , Humans , Mice , Mouse Embryonic Stem Cells/metabolism , RNA Splicing/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/metabolism , Signal Transduction/genetics , Stem Cells/pathology
8.
Mol Cell ; 76(5): 767-783.e11, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31540874

ABSTRACT

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.


Subject(s)
Cell Nucleolus/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Ribosomal/metabolism , Active Transport, Cell Nucleus , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/ultrastructure , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Female , HEK293 Cells , HeLa Cells , Humans , Nucleic Acid Conformation , Protein Binding , Protein Interaction Domains and Motifs , RNA Precursors/genetics , RNA Precursors/ultrastructure , RNA, Ribosomal/genetics , RNA, Ribosomal/ultrastructure
9.
Crit Rev Biochem Mol Biol ; 53(6): 596-606, 2018 12.
Article in English | MEDLINE | ID: mdl-30252509

ABSTRACT

Small nucleolar RNAs (snoRNAs) are a family of conserved nuclear RNAs that function in the modification of small nuclear RNAs (snRNAs) or ribosomal RNAs (rRNAs), or participate in the processing of rRNAs during ribosome subunit maturation. Eukaryotic DNA transcription and RNA processing produce many long noncoding RNA (lncRNA) species. Although most lncRNAs are processed like typical mRNAs to be 5' capped and 3' polyadenylated, other types of lncRNAs are stabilized from primary Pol II transcripts by alternative mechanisms. One way to generate stable lncRNAs is to co-operate with snoRNA processing to produce snoRNA-ended lncRNAs (sno-lncRNAs) and 5' snoRNA-ended and 3'-polyadenylated lncRNAs (SPAs). Rather than silently accumulating in the nucleus, some sno-lncRNAs and SPAs are involved in the regulation of pre-rRNA transcription and alternative splicing of pre-mRNAs. Here we provide a mini-review to discuss the biogenesis and functions of these unusually processed lncRNAs.


Subject(s)
RNA Processing, Post-Transcriptional/physiology , RNA Stability/physiology , RNA, Long Noncoding/biosynthesis , RNA, Small Nucleolar/biosynthesis , Transcription, Genetic/physiology , Animals , Humans , RNA, Long Noncoding/genetics , RNA, Small Nucleolar/genetics
10.
Gut Liver ; 12(2): 173-182, 2018 Mar 15.
Article in English | MEDLINE | ID: mdl-29291617

ABSTRACT

BACKGROUND/AIMS: Methylation status plays a causal role in carcinogenesis in targeted tissues. However, the relationship between the DNA methylation status of multiple genes in blood leukocytes and colorectal cancer (CRC) susceptibility as well as interactions between dietary factors and CRC risks are unclear. METHODS: We performed a case-control study with 466 CRC patients and 507 cancer-free controls to investigate the association among the methylation status of individual genes, multiple CpG site methylation (MCSM), multiple CpG site heterogeneous methylation and CRC susceptibility. Peripheral blood DNA methylation levels were detected by performing methylation-sensitive high-resolution melting. RESULTS: Total heterogeneous methylation of CA10 and WT1 conferred a significantly higher risk of CRC (adjusted odds ratio [ORadjusted], 5.445; 95% confidence interval [CI], 3.075 to 9.643; ORadjusted, 1.831; 95% CI, 1.100 to 3.047; respectively). Subjects with high-level MCSM (MCSM-H) status demonstrated a higher risk of CRC (ORadjusted, 4.318; 95% CI, 1.529 to 12.197). Additionally, interactions between the high-level intake of fruit and CRH, WT1, and MCSM on CRC were statistically significant. CONCLUSIONS: The gene methylation status of blood leukocytes may be associated with CRC risk. MCSM-H of blood leukocytes was associated with CRC, especially in younger people. Some dietary factors may affect hypermethylation status and influence susceptibility to CRC.


Subject(s)
Colorectal Neoplasms , DNA Methylation/genetics , Leukocytes/metabolism , Mitochondrial Proteins/genetics , Nerve Tissue Proteins/genetics , WT1 Proteins/genetics , Aged , Biomarkers, Tumor/genetics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Case-Control Studies , China , Colorectal Neoplasms/blood , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , CpG Islands/physiology , Female , Gene Expression Regulation, Neoplastic , Gene-Environment Interaction , Humans , Leukocytes/pathology , Male , Middle Aged , Promoter Regions, Genetic
11.
Cell ; 169(4): 664-678.e16, 2017 05 04.
Article in English | MEDLINE | ID: mdl-28475895

ABSTRACT

Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.


Subject(s)
DEAD-box RNA Helicases/metabolism , RNA Polymerase I/metabolism , RNA Precursors/genetics , RNA, Long Noncoding/metabolism , Allosteric Site , Animals , Carcinogenesis , Cell Line , Cell Line, Tumor , DEAD-box RNA Helicases/chemistry , Female , Gene Knockout Techniques , Humans , Mice , Mice, Nude , RNA Precursors/metabolism , Transcription, Genetic
12.
IUBMB Life ; 68(11): 887-893, 2016 11.
Article in English | MEDLINE | ID: mdl-27699981

ABSTRACT

RNA is essential for all kingdoms of life and exerts important functions beyond transferring genetic information from DNA to protein. With the advent of the state-of-the-art deep sequencing technology, a large portion of noncoding transcripts in eukaryotic genomes has been broadly identified. Among them, long noncoding RNAs (lncRNAs) have been emerged as a new class of RNA molecules that have regulatory potential in a variety of physiological and pathological processes. Here we summarize recent research progresses that have been made by scientists in China on lncRNAs, including their biogenesis, functional implication and the underlying mechanism of action at the current stage. © 2016 IUBMB Life, 68(11):887-893, 2016.


Subject(s)
Biomedical Research/standards , RNA, Long Noncoding/physiology , Animals , Bibliometrics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinogenesis/pathology , China , Gene Expression Regulation, Neoplastic , Humans , Quality Improvement , RNA Interference
13.
Cell Stem Cell ; 17(2): 221-32, 2015 Aug 06.
Article in English | MEDLINE | ID: mdl-26165924

ABSTRACT

Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Library , Genetic Testing , Haploidy , Mouse Embryonic Stem Cells/metabolism , RNA, Guide, Kinetoplastida/metabolism , Alleles , Animals , Base Sequence , Cloning, Organism , DNA Methylation/genetics , Heterozygote , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Molecular Sequence Data
14.
Mol Cell ; 51(6): 792-806, 2013 Sep 26.
Article in English | MEDLINE | ID: mdl-24035497

ABSTRACT

We describe the identification and characterization of circular intronic long noncoding RNAs in human cells, which accumulate owing to a failure in debranching. The formation of such circular intronic RNAs (ciRNAs) can be recapitulated using expression vectors, and their processing depends on a consensus motif containing a 7 nt GU-rich element near the 5' splice site and an 11 nt C-rich element close to the branchpoint site. In addition, we show that ciRNAs are abundant in the nucleus and have little enrichment for microRNA target sites. Importantly, knockdown of ciRNAs led to the reduced expression of their parent genes. One abundant such RNA, ci-ankrd52, largely accumulates to its sites of transcription, associates with elongation Pol II machinery, and acts as a positive regulator of Pol II transcription. This study thus suggests a cis-regulatory role of noncoding intronic transcripts on their parent coding genes.


Subject(s)
DNA Polymerase II/genetics , RNA Polymerase II/genetics , RNA, Long Noncoding/genetics , Transcription, Genetic , Gene Knockdown Techniques , Humans , Introns/genetics , MicroRNAs/genetics , RNA Splice Sites
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