RESUMEN
The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.
Asunto(s)
Inestabilidad Genómica , Regiones Promotoras Genéticas , Estructuras R-Loop , Terminación de la Transcripción Genética , Humanos , ADN de Cadena Simple/metabolismo , Inestabilidad Genómica/genética , Mutación , Estructuras R-Loop/genética , ARN Polimerasa II/metabolismo , Regiones Promotoras Genéticas/genética , Genoma Humano , Proteínas de Unión al ADN/metabolismoRESUMEN
Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos B , Cromatina/química , Cromatina/metabolismo , Fibroblastos/metabolismo , Genoma , Células HEK293 , Antígenos de Histocompatibilidad Clase II , Humanos , Ratones , Mutación , Fosforilación , Regiones Promotoras Genéticas , RNA-Seq , Secuencias Reguladoras de Ácidos Nucleicos , Activación TranscripcionalRESUMEN
The lung metastasis of breast cancer involves complicated regulatory changes driven by chromatin remodelling. However, the epigenetic reprogramming and regulatory mechanisms in lung metastasis of breast cancer remain unclear. Here, we generated and analysed genome-wide profiles of multiple histone modifications (H3K4me3, H3K27ac, H3K27me3, H3K4me1 and H3K9me3), as well as transcriptome data in lung-metastatic and non-lung-metastatic breast cancer cells. Our results showed that the expression changes were correlated with the enrichment of specific histone modifications in promoters and enhancers. Promoter and enhancer reprogramming regulated gene expression in a synergetic way, and involved in multiple important biological processes and pathways. In addition, lots of gained super-enhancers were identified in lung-metastatic cells. We also identified master regulators driving differential gene expression during lung metastasis of breast cancer. We found that the cooperations between regulators were much closer in lung-metastatic cells. Moreover, regulators such as TFAP2C, GTF2I and LMO4 were found to have potential prognostic value for lung metastasis free (LMF) survival of breast cancer. Functional studies motivated by our data analyses uncovered an important role of LMO4 in regulating metastasis. This study provided comprehensive insights into regulatory mechanisms, as well as potential prognostic markers for lung metastasis of breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Epigénesis Genética/genética , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/genética , Mama/patología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Elementos de Facilitación Genéticos/genética , Femenino , Expresión Génica/genética , Código de Histonas/genética , Histonas/genética , Humanos , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia/patología , Regiones Promotoras Genéticas/genética , Transcriptoma/genéticaRESUMEN
BACKGROUND: A hallmark of acute promyelocytic leukemia (APL) is the expression of PML/RARα fusion protein. Treatment with all-trans retinoic acid (ATRA) results in the terminal differentiation of neutrophil granulocytes. However, the underlying mechanisms remain largely unknown. Here, we identify and elucidate a novel differentiation-suppressive model of APL involving the histone demethylase KDM3B, which has been identified as a suppressor of the tumor genes involved in hematopoietic malignancies. METHODS: First, we established a KDM3B knockdown NB4 cell model to determine the functional characteristics of KDM3B by cell proliferation assay and flow cytometry. Then, we performed ChIP-seq and ATAC-seq to search for potential relationships among KDM3B, histone modification (H3K9me1/me2) and the chromatin state. Finally, molecular biological techniques and a multi-omics analysis were used to explore the role of KDM3B in differentiation of the leukemia cells after ATRA treatment. RESULTS: We found that knocking down KDM3B contributed to the growth of NB4 APL cells via the promotion of cell-cycle progression and blocked granulocytic differentiation. Through global and molecular approaches, we provided futher evidence that knocking down KDM3B altered the global distribution of H3K9me1/me2 and increased the chromatin accessibility. Moreover, knocking down KDM3B inhibited the ATRA-induced degradation of the PML/RARα oncoprotein. CONCLUSION: Our study suggested that KDM3B was able to inhibit APL progression by maintaining chromatin in a compact state and facilitating the ATRA-mediated degradation of PML/RARα. Taken together, the results show that KDM3B may be an alternative target for the treatment regimens and the targeted therapy for APL by sustaining the function of PML/RARα fusion protein.
RESUMEN
The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities-RNA cleavage and RNA polymerase II dephosphorylation-are structurally and functionally integrated into the INTAC complex.
Asunto(s)
Complejos Multienzimáticos/química , Proteína Fosfatasa 2/química , ARN Polimerasa II/química , Cromatina/química , Microscopía por Crioelectrón , Holoenzimas/química , Humanos , Dominios ProteicosRESUMEN
Estrogen receptor α (ERα) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcriptional events that promote tumor growth. ERα binding activity largely depends on access to binding sites on chromatin, which is facilitated in part by Pioneer Factors (PFs). Transcription factors operate in complexes through thousands of genomic binding sites in a combinatorial fashion to control the expression of genes. However, the extent of crosstalk and cooperation between ERα pioneer factors and more collaborative transcription factors in breast cancer still remains to be elucidated systematically. Methods: Here, we determined the genomic binding information of 40 transcription-related factors and histone modifications with ChIP-seq in ENCODE and integrated it with other genomic information (RNA-seq, ATAC-seq, Gene microarray, 450k methylation chip, GRO-seq), forming a multi-dimension network to illuminate ERα associated transcription. Results: We show that transcription factor, NR2F2 binds to most sites independently of estrogen. Perturbation of NR2F2 expression decreases ERα DNA binding, chromatin openning, and estrogen-dependent cell growth. In the genome-wide analysis, we show that most binding events of NR2F2 and known pioneer factors FOXA1, GATA3 occur together, covering 85% of the ERα binding sites. Regions bound by all the three TFs appeared to be the most active, to have the strongest ERα binding and to be enriched for the super enhancers. Conclusions: The ERα binds to pre-accessible sites containing ERE elements bound by the three transcription factors (NR2F2, FOXA1 and GATA3).The three genes were also identified to correlate with decreased metastatic potential in patient cohorts and co-regulate each other. Together, our results suggest that NR2F2 is a cofactor with FOXA1 and GATA3 in ERα-mediated transcription.
Asunto(s)
Neoplasias de la Mama/metabolismo , Factor de Transcripción COUP II/metabolismo , Receptor alfa de Estrógeno/metabolismo , Factor de Transcripción GATA3/metabolismo , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Factor de Transcripción COUP II/genética , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Regiones Promotoras GenéticasRESUMEN
Pancreatic cancer is a deadliest type of malignancy and lacks effective intervention. We here report a potential strategy for treatment of this malignancy by the combination of arsenic trioxide (ATO) and BET bromodomain inhibitor JQ1. These two agents synergistically modulate multistages of autophagy and thus induce apoptosis effectively in pancreatic cancer cells. Our genomic and biochemical data have demonstrated that crosstalks between ER stress and autophagy play crucial roles during ATO-induced apoptosis, in which NRF2 may stand at the crossroad between cell death and survival. This has been further strengthened by our finding that NRF2 depletion renders insensitive cells into sensitive ones in regard to ATO treatment-caused cell death. The knockdown of NRF2 and the addition of JQ1 result in similar molecular/cellular effects in promoting effective ATO-induced apoptosis in cells that are insensitive to ATO treatment alone. Thus, the combination of ATO and JQ1 may represent a new treatment strategy for pancreatic cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Trióxido de Arsénico/farmacología , Autofagia/efectos de los fármacos , Azepinas/farmacología , Neoplasias Pancreáticas/patología , Triazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: To analyze bioinformatic datasets for detecting genetic and epigenetic mechanisms shared by chronic periodontitis (CP) and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Datasets from GEO and TCGA databases reporting mRNAs, miRNAs or methylation expression in human CP and OSCC tissues were analyzed. Differential expression, functional enrichment and protein-protein interaction (PPI) network analyses were performed. Differentially expressed miRNAs (DEmiRNAs) and genes (DEG) in CP and OSCC were determined. DEmiRNA-target and DEmiRNA-DEG networks were constructed. Directly and indirectly interacting cross-talk genes were screened, and their prediction accuracy and association with OSCC prognosis was determined. RESULTS: 3 DE-miRNAs (miR-375, miR-3609 and miR-3652) expressed in both CP and OSCC critically regulated most DEGs. Among 12 directly interacting cross-talk genes, NCAPH was significantly related with the prognosis of OSCC. NR2F2 had highest differential expression in CP and OSCC. Among 4 cross-talk genes (FN1, MPPED1, NDEL1, and NR2F2) differentially expressed in CP, 3 (FN1, MPPED1, NDEL1) were also expressed in OSCC. Among 12 indirectly interacting cross-talk genes differentially expressed in OSCC, 3 genes (CDCA8, HIST1H3J, and RAD51) were significantly related to its prognosis. Significant pathways involved in CP and OSCC included: chemokine receptors, class I PI3K signaling events, epithelial-to-mesenchymal transition and signaling events by VEGFR1 and VEGFR2, EGF receptor (ErbB1). CONCLUSION: Bioinformatic analysis of available datasets implicated 1 directly interacting cross-talk gene (NCAPH), 4 indirectly interacting cross-talk genes (NCAPH, NR2F2, FN1, and MPPED1) and 3 DE-miRNAs (hsa-miR-375, miR-3609 and miR-3652) as shared genetic and epigenetic expression patterns between CP and OSCC.