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1.
Cell ; 177(4): 837-851.e28, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30955886

RESUMO

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.


Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Linhagem Celular , Endonucleases/genética , Endonucleases/metabolismo , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Genômica , Células HeLa , Humanos , Mutagênese Insercional/genética
2.
Cell ; 176(4): 816-830.e18, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30595451

RESUMO

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.


Assuntos
Período de Replicação do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina , DNA/genética , Período de Replicação do DNA/genética , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos/genética , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Proteínas Repressoras/metabolismo , Análise Espaço-Temporal
3.
Mol Cell ; 79(6): 978-990.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32857953

RESUMO

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.


Assuntos
Endorribonucleases/genética , Precursores de RNA/genética , Estresse Fisiológico/genética , Transativadores/genética , Terminação da Transcrição Genética , Animais , Tamanho Celular , Sobrevivência Celular/genética , Humanos , Pressão Osmótica/fisiologia , Proteoma/genética
4.
Genes Dev ; 33(11-12): 641-655, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048544

RESUMO

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of ß-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.


Assuntos
Carcinogênese , Carcinoma Ductal Pancreático/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/fisiologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carcinoma in Situ/patologia , Carcinoma in Situ/fisiopatologia , Carcinoma Ductal Pancreático/patologia , Transdiferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Metaplasia , Camundongos , Camundongos Transgênicos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo
5.
Hum Mol Genet ; 32(6): 934-947, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36219176

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Its complex pathogenesis and phenotypic heterogeneity hinder therapeutic development and early diagnosis. Altered RNA metabolism is a recurrent pathophysiologic theme, including distinct microRNA (miRNA) profiles in ALS tissues. We profiled miRNAs in accessible biosamples, including skin fibroblasts and whole blood and compared them in age- and sex-matched healthy controls versus ALS participants with and without repeat expansions to chromosome 9 open reading frame 72 (C9orf72; C9-ALS and nonC9-ALS), the most frequent ALS mutation. We identified unique and shared profiles of differential miRNA (DmiRNA) levels in each C9-ALS and nonC9-ALS tissues versus controls. Fibroblast DmiRNAs were validated by quantitative real-time PCR and their target mRNAs by 5-bromouridine and 5-bromouridine-chase sequencing. We also performed pathway analysis to infer biological meaning, revealing anticipated, tissue-specific pathways and pathways previously linked to ALS, as well as novel pathways that could inform future research directions. Overall, we report a comprehensive study of a miRNA profile dataset from C9-ALS and nonC9-ALS participants across two accessible biosamples, providing evidence of dysregulated miRNAs in ALS and possible targets of interest. Distinct miRNA patterns in accessible tissues may also be leveraged to distinguish ALS participants from healthy controls for earlier diagnosis. Future directions may look at potential correlations of miRNA profiles with clinical parameters.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , MicroRNAs , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Demência Frontotemporal/genética , Mutação
6.
Blood ; 142(25): 2159-2174, 2023 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-37616559

RESUMO

ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.


Assuntos
5'-Nucleotidase , Leucemia de Células T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Camundongos , Linhagem Celular Tumoral , Cromatina , Dano ao DNA/genética , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fatores de Transcrição/genética , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo
7.
Mol Cell ; 65(2): 272-284, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28107649

RESUMO

The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions, including promoters, enhancers, and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through a short hairpin RNA (shRNA) screening, we identified single-stranded DNA binding protein replication protein A (RPA) as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and it co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Regiões Promotoras Genéticas , Proteína de Replicação A/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Cromatina/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Fase G1 , Células HEK293 , Células HeLa , Chaperonas de Histonas/genética , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteína de Replicação A/genética , Fatores de Transcrição/genética , Transfecção
8.
Genes Dev ; 30(24): 2669-2683, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087712

RESUMO

Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA), making developmental regulators therapeutically attractive. Here we demonstrate diverse functions for pancreatic and duodenal homeobox 1 (PDX1), a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of pancreatic intraepithelial neoplasia (PanIN)-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor-suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing epithelial-to-mesenchymal transition (EMT), and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression.


Assuntos
Carcinoma Ductal Pancreático/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neoplasias Pancreáticas/genética , Transativadores/metabolismo , Células Acinares/patologia , Animais , Carcinoma Ductal Pancreático/fisiopatologia , Deleção de Genes , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Neoplasias Pancreáticas/fisiopatologia , Análise Serial de Tecidos , Transativadores/genética , Células Tumorais Cultivadas
9.
Int J Cancer ; 153(3): 552-570, 2023 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-37140208

RESUMO

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.


Assuntos
MicroRNAs , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Plasticidade Celular , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/patologia , Fator de Crescimento Transformador beta/metabolismo , Ativinas/genética , Neoplasias Pancreáticas
10.
RNA ; 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33975916

RESUMO

Pre-mRNA splicing is carried out by the spliceosome and involves splice site recognition, removal of introns, and ligation of exons. Components of the spliceosome have been shown to interact with the elongating RNA polymerase II (RNAPII) which is thought to allow splicing to occur concurrently with transcription. However, little is known about the regulation and efficiency of co-transcriptional splicing in human cells. In this study, we used Bru-seq and BruChase-seq to determine the co-transcriptional splicing efficiencies of 17,000 introns expressed across 6 human cell lines. We found that less than half of all introns across these 6 cell lines were co-transcriptionally spliced. Splicing efficiencies for individual introns showed variations across cell lines, suggesting that splicing may be regulated in a cell-type specific manner. Moreover, the splicing efficiency of introns varied within genes. The efficiency of co-transcriptional splicing did not correlate with gene length, intron position, splice site strengths, or the intron/neighboring exons GC content. However, we identified binding signals from multiple RNA binding proteins (RBPs) that correlated with splicing efficiency, including core spliceosomal machinery components-such as SF3B4, U2AF1 and U2AF2 showing higher binding signals in poorly spliced introns. In addition, multiple RBPs, such as BUD13, PUM1 and SND1, showed preferential binding in exons that flank introns with high splicing efficiencies. The nascent RNA splicing patterns presented here across multiple cell types add to our understanding of the complexity in RNA splicing, wherein RNA-binding proteins may play important roles in determining splicing outcomes in a cell type- and intron-specific manner.

11.
Cell Commun Signal ; 21(1): 15, 2023 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-36691073

RESUMO

Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract.


Assuntos
Neoplasias da Mama , Proteínas de Ligação a DNA , Humanos , Feminino , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral
12.
Nucleic Acids Res ; 49(13): 7507-7524, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181717

RESUMO

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.


Assuntos
Hidrolases Anidrido Ácido/genética , Variações do Número de Cópias de DNA , Replicação do DNA , Proteínas de Neoplasias/genética , Transcrição Gênica , Hidrolases Anidrido Ácido/biossíntese , Animais , Afidicolina/farmacologia , Linhagem Celular , Sítios Frágeis do Cromossomo , Loci Gênicos , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/biossíntese , Regiões Promotoras Genéticas , Estruturas R-Loop , Ribonuclease H/metabolismo , Estresse Fisiológico
13.
Genes Dev ; 29(2): 171-83, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25593307

RESUMO

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of ß-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.


Assuntos
Carcinoma Ductal Pancreático/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica/genética , Neoplasias Pancreáticas/enzimologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco , beta Catenina/metabolismo
14.
RNA ; 26(11): 1680-1703, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32753408

RESUMO

The human PUF-family proteins, PUM1 and PUM2, posttranscriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3'-UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady-state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a "rulebook" of key contextual features that differentiate functional versus nonfunctional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.


Assuntos
RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Regulação da Expressão Gênica , Células HEK293 , Humanos , Aprendizado de Máquina , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Sequenciamento Completo do Genoma
15.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941784

RESUMO

Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle.IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular "prolytic" state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.


Assuntos
Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno/genética , Transativadores/metabolismo , Transcriptoma , Latência Viral , Apoptose , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Inflamação , Fenótipo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Ativação Transcricional , Carga Viral
16.
J Cell Sci ; 130(11): 1952-1964, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28446540

RESUMO

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/genética , Receptor Notch3/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Diferenciação Celular , Linhagem Celular Transformada , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Genes Reporter , Células HEK293 , Meia-Vida , Humanos , Imidazóis/farmacologia , Luciferases/genética , Luciferases/metabolismo , Masculino , Naftalenos/farmacologia , Cultura Primária de Células , Próstata/citologia , Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Notch3/antagonistas & inibidores , Receptor Notch3/metabolismo , Transdução de Sinais , Tiazóis/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
J Pathol ; 245(3): 324-336, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29672864

RESUMO

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neoplasias Ósseas/metabolismo , Metabolismo Energético , Proteínas Proto-Oncogênicas/metabolismo , Sarcoma de Ewing/metabolismo , Serina/biossíntese , Animais , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Camundongos Nus , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Transdução de Sinais , Transaminases/genética , Transaminases/metabolismo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Genome Res ; 25(2): 189-200, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25373142

RESUMO

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics.


Assuntos
Sítios Frágeis do Cromossomo , Variações do Número de Cópias de DNA , Replicação do DNA , Transcrição Gênica , Animais , Linhagem Celular , Quebra Cromossômica , Mapeamento Cromossômico , Loci Gênicos , Instabilidade Genômica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Especificidade de Órgãos , Reprodutibilidade dos Testes
19.
Proc Natl Acad Sci U S A ; 112(26): 8002-7, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26080430

RESUMO

The 4D organization of the interphase nucleus, or the 4D Nucleome (4DN), reflects a dynamical interaction between 3D genome structure and function and its relationship to phenotype. We present initial analyses of the human 4DN, capturing genome-wide structure using chromosome conformation capture and 3D imaging, and function using RNA-sequencing. We introduce a quantitative index that measures underlying topological stability of a genomic region. Our results show that structural features of genomic regions correlate with function with surprising persistence over time. Furthermore, constructing genome-wide gene-level contact maps aided in identifying gene pairs with high potential for coregulation and colocalization in a manner consistent with expression via transcription factories. We additionally use 2D phase planes to visualize patterns in 4DN data. Finally, we evaluated gene pairs within a circadian gene module using 3D imaging, and found periodicity in the movement of clock circadian regulator and period circadian clock 2 relative to each other that followed a circadian rhythm and entrained with their expression.


Assuntos
Núcleo Celular/metabolismo , Genoma Humano , Interfase , Ritmo Circadiano/genética , Redes Reguladoras de Genes , Humanos
20.
Proc Natl Acad Sci U S A ; 112(11): E1307-16, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25737553

RESUMO

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.


Assuntos
Processamento Alternativo/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Processamento Alternativo/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Éxons/genética , Humanos , Indóis , Íntrons/genética , Proteínas de Fusão Oncogênica/genética , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Transdução de Sinais/efeitos dos fármacos , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Telomerase/metabolismo
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