RESUMEN
The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.
RESUMEN
Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300's enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.
Asunto(s)
Cromatina , Neoplasias de la Próstata , Receptores de Glucocorticoides , Humanos , Masculino , Antagonistas de Andrógenos/farmacología , Línea Celular Tumoral , Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMEN
Most common genetic variants associated with disease are located in non-coding regions of the genome. One mechanism by which they function is through altering transcription factor (TF) binding. In this study, we explore how genetic variation is connected to differences in the regulatory landscape of livers from C57BL/6J and 129S1/SvImJ mice fed either chow or a high-fat diet. To identify sites where regulatory variation affects TF binding and nearby gene expression, we employed an integrative analysis of H3K27ac ChIP-seq (active enhancers), ATAC-seq (chromatin accessibility) and RNA-seq (gene expression). We show that, across all these assays, the genetically driven (i.e. strain-specific) differences in the regulatory landscape are more pronounced than those modified by diet. Most notably, our analysis revealed that differentially accessible regions (DARs, N = 29635, FDR < 0.01 and fold change > 50%) are almost always strain-specific and enriched with genetic variation. Moreover, proximal DARs are highly correlated with differentially expressed genes. We also show that TF binding is affected by genetic variation, which we validate experimentally using ChIP-seq for TCF7L2 and CTCF. This study provides detailed insights into how non-coding genetic variation alters the gene regulatory landscape, and demonstrates how this can be used to study the regulatory variation influencing TF binding.
Asunto(s)
Cromatina , Regulación de la Expresión Génica , Ratones , Animales , Cromatina/genética , Ratones Endogámicos C57BL , Ratones Endogámicos , Variación GenéticaRESUMEN
BACKGROUND: Accumulating evidence indicates importance of RNA regulation in cancer. This includes events such as splicing, translation, and regulation of noncoding RNAs, functions which are governed by RNA binding proteins (RBPs). AIMS: To find which RBPs could be relevant for prostate cancer, we performed systematic screening of RBP expression in clinical prostate cancer. METHODS AND RESULTS: We interrogated four proteome-wide proteomics datasets including tumor samples of primary, castration resistant, and metastatic prostate cancer. We found that, while the majority of RBPs are expressed but not significantly altered during prostate cancer development and progression, expression of several RBPs increases in advanced disease. Interestingly, most of the differentially expressed RBPs are not targets of differential posttranscriptional phosphorylation during disease progression. The RBPs undergoing expression changes have functions in, especially, poly(A)-RNA binding, nucleocytoplasmic transport, and cellular stress responses, suggesting that these may play a role in formation of castration resistance. Pathway analyzes indicate that increased ribosome production and chromatin-related functions of RBPs are also linked to castration resistant and metastatic prostate cancers. We selected a group of differentially expressed RBPs and studied their role in cultured prostate cancer cells. With siRNA screens, several of these were indicated in survival (DDX6, EIF4A3, PABPN1), growth (e.g., EIF5A, HNRNPH2, LRRC47, and NVL), and migration (e.g., NOL3 and SLTM) of prostate cancer cells. Our analyzes further show that RRP9, a U3 small nucleolar protein essential for ribosome formation, undergoes changes at protein level during metastasis in prostate cancer. CONCLUSION: In this work, we recognized significant molecular alterations in RBP profiles during development and evolution of prostate cancer. Our study further indicates several functionally significant RBPs warranting further investigation for their functions and possible targetability in prostate cancer.
Asunto(s)
Neoplasias de la Próstata , Proteoma , Masculino , Humanos , Proteoma/metabolismo , Proteómica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Neoplasias de la Próstata/genética , ARN Interferente Pequeño , Factor 4A Eucariótico de Iniciación/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteína I de Unión a Poli(A)RESUMEN
Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.
Asunto(s)
Infecciones por Parvoviridae , Parvovirus , Línea Celular , Cromatina , Humanos , Parvovirus/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4's functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.
Asunto(s)
Receptores Androgénicos , Animales , Embrión de Pollo , Masculino , Próstata , ProteómicaRESUMEN
Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.
Asunto(s)
Proteínas Proto-Oncogénicas/metabolismo , Receptores de Glucocorticoides/genética , Proteínas Represoras/metabolismo , Sitios de Unión , Movimiento Celular/genética , Proliferación Celular/genética , Inmunoprecipitación de Cromatina , Dexametasona/farmacología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Represoras/genéticaRESUMEN
Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
Asunto(s)
Cromatina/metabolismo , Receptores de Glucocorticoides/metabolismo , Sumoilación , Sitios de Unión , Regulación de la Expresión Génica , Células HEK293 , Humanos , Co-Represor 1 de Receptor Nuclear/metabolismo , Coactivador 1 de Receptor Nuclear , Mapeo de Interacción de Proteínas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/efectos de los fármacosRESUMEN
In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
Asunto(s)
Respuesta al Choque Térmico/genética , Proteínas Intrínsecamente Desordenadas/genética , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/genética , Transcripción Genética , Factores de Elongación Transcripcional/genética , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonales , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Genes Reporteros , Células HEK293 , Células HeLa , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fosforilación , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transducción de Señal , Estrés Fisiológico , Sumoilación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Proteína Fluorescente RojaRESUMEN
We have identified BCL6 corepressor (BCOR) as a hormone-dependent interaction partner of androgen receptor (AR), a key transcription factor in the development of normal and cancerous prostate. BCOR is often mutated in cancers and hematological diseases and as a component of a non-canonical polycomb repressive complex 1 (ncPRC1.1) required for arranging many facets of cellular differentiation. However, its role in androgen signaling or prostate cancer cells remains unknown. Here, our genome-wide analyses reveal that BCOR is recruited in an androgen-dependent fashion to majority of AR-binding chromatin sites in castration-resistant prostate cancer (CRPC) cells. Interestingly, depletion of BCOR has a significant effect on the expression of androgen-repressed genes linked to regulation of cell proliferation, differentiation and development. At many of these genes, such as HOX genes, the depletion leads to a decrease in H2A K119 monoubiquitination and an increase in mRNA expression. Consistently, BCOR depletion impairs the proliferation and viability of CRPC cells, inducing their apoptosis. Collectively, our data indicate a key role for the BCOR-ncPRC1.1 complex in the corepression of an important subset of AR target genes and the regulation of prostate cancer cell proliferation.
Asunto(s)
Histonas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Androgénicos/metabolismo , Proteínas Represoras/metabolismo , Andrógenos/farmacología , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/fisiología , Cromatina/genética , Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Humanos , Masculino , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/genética , Receptores Androgénicos/genética , Proteínas Represoras/genética , UbiquitinaciónRESUMEN
IRF2BP2 (interferon regulatory factor-2 binding protein-2) is an uncharacterized interaction partner of glucocorticoid (GC) receptor (GR), an anti-inflammatory and metabolic transcription factor. Here, we show that GC changes the chromatin binding of IRF2BP2 in natural chromatin milieu. The GC-induced IRF2BP2-binding sites co-occur with GR binding sites and are associated with GC-induced genes. Moreover, the depletion of IRF2BP2 modulates transcription of GC-regulated genes, represses cell proliferation and increases cell movement in HEK293 cells. In A549 cells, the depletion extensively alters the responses to GC and tumor necrosis factor α (TNF), including metabolic and inflammatory pathways. Taken together, our data support the role of IRF2BP2 as a coregulator of both GR and NF-κB, potentially modulating the crosstalk between GC and TNF signaling.
Asunto(s)
Antiinflamatorios/farmacología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Inflamación/prevención & control , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células A549 , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , FN-kappa B/genética , FN-kappa B/metabolismo , Factores de Transcripción/genéticaRESUMEN
Androgen receptor (AR) is regulated by SUMOylation at its transactivation domain. In vitro, the SUMOylation is linked to transcriptional repression and/or target gene-selective regulation. Here, we generated a mouse model (ArKI) in which the conserved SUMO acceptor lysines of AR are permanently abolished (ArK381R, K500R). ArKI males develop normally, without apparent defects in their systemic androgen action in reproductive tissues. However, the ArKI males are infertile. Their spermatogenesis appears unaffected, but their epididymal sperm maturation is defective, shown by severely compromised motility and fertilization capacity of the sperm. Fittingly, their epididymal AR chromatin-binding and gene expression associated with sperm maturation and function are misregulated. AR is SUMOylated in the wild-type epididymis but not in the testis, which could explain the tissue-specific response to the lack of AR SUMOylation. Our studies thus indicate that epididymal AR SUMOylation is essential for the post-testicular sperm maturation and normal reproductive capability of male mice.
Asunto(s)
Epidídimo/metabolismo , Epidídimo/fisiopatología , Infertilidad Masculina/metabolismo , Infertilidad Masculina/fisiopatología , Receptores Androgénicos/metabolismo , Espermatogénesis/fisiología , Animales , Epidídimo/patología , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Receptores Androgénicos/genética , Espermatogénesis/genética , Sumoilación/genética , Sumoilación/fisiologíaRESUMEN
Glucocorticoid receptor (GR) and androgen receptor (AR) are steroid-inducible transcription factors (TFs). The GR and the AR are central regulators of various metabolic, homeostatic and differentiation processes and hence important therapeutic targets, especially in inflammation and prostate cancer, respectively. Hormone binding to these steroid receptors (SRs) leads to DNA binding and activation or repression of their target genes with the aid of interacting proteins, coregulators. However, protein interactomes of these important drug targets have remained poorly defined. We used proximity-dependent biotin identification to map the protein interaction landscapes of GR and AR in the presence and absence of their cognate agonist (dexamethasone, 5α-dihydrotestosterone) and antagonist (RU486, enzalutamide) in intact human cells. We reproducibly identified more than 30 proteins that interacted with the GR in an agonist-specific manner and whose interactions were significantly influenced by the DNA-binding function of the receptor. Interestingly, the agonist-dependent interactome of the GR overlapped considerably with that of the AR. In addition to known coactivators, corepressors and components of BAF (SWI/SNF) chromatin-remodeling complex, we identified a number of proteins, including lysine methyltransferases and demethylases that have not been previously linked to glucocorticoid or androgen signaling. A substantial number of these novel agonist-dependent GR/AR-interacting proteins, e.g. BCOR, IRF2BP2, RCOR1, and TLE3, have previously been implicated in transcription repression. This together with our data on the effect of BCOR, IRF2BP2, and RCOR1 on GR target gene expression suggests multifaceted functions and roles for SR coregulators. These first high confidence SR interactomes will aid in therapeutic targeting of the GR and the AR.
Asunto(s)
Mapeo de Interacción de Proteínas , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Células A549 , Benzamidas , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Dihidrotestosterona/farmacología , Humanos , Masculino , Mifepristona/farmacología , Nitrilos , Proteínas Nucleares/metabolismo , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Transcripción GenéticaRESUMEN
Post-translational modifications, e.g. SUMO modifications (SUMOylation), provide a mechanism for swiftly changing a protein's activity. Various stress conditions trigger a SUMO stress response (SSR) - a stress-induced rapid change in the conjugation of SUMO to multiple proteins, which predominantly targets nuclear proteins. The SSR has been postulated to protect stressed cells by preserving the functionality of crucial proteins. However, it is unclear how it exerts its protective functions. Interestingly, heat stress (HS) increases SUMOylation of proteins at active promoters and enhancers. In promoters, HS-induced SUMOylation correlates with gene transcription and stress-induced RNA polymerase II (Pol2) pausing. Conversely, a disappearance of SUMOylation in HS occurs at chromatin anchor points that maintain chromatin-looping structures and the spatial organisation of chromatin. In reviewing the literature, we hypothesise that the SSR regulates Pol2 pausing by modulating the interactions of pausing-regulating proteins, whereas deSUMOylation alters the function of chromatin anchors.
Asunto(s)
Cromatina/metabolismo , Respuesta al Choque Térmico , Proteínas Nucleares/metabolismo , Sumoilación , Animales , Cromatina/fisiología , Humanos , ARN Polimerasa II/metabolismo , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Inflammatory processes and androgen signaling are critical for the growth of prostate cancer (PC), the most common cancer among males in Western countries. To understand the importance of potential interplay between pro-inflammatory and androgen signaling for gene regulation, we have interrogated the crosstalk between androgen receptor (AR) and NF-κB, a key transcriptional mediator of inflammatory responses, by utilizing genome-wide chromatin immunoprecipitation sequencing and global run-on sequencing in PC cells. Co-stimulation of LNCaP cells with androgen and pro-inflammatory cytokine TNFα invoked a transcriptome which was very distinct from that induced by either stimulation alone. The altered transcriptome that included gene programs linked to cell migration and invasiveness was orchestrated by significant remodeling of NF-κB and AR cistrome and enhancer landscape. Although androgen multiplied the NF-κB cistrome and TNFα restrained the AR cistrome, there was no general reciprocal tethering of the AR to the NF-κB on chromatin. Instead, redistribution of FOXA1, PIAS1 and PIAS2 contributed to the exposure of latent NF-κB chromatin-binding sites and masking of AR chromatin-binding sites. Taken together, concomitant androgen and pro-inflammatory signaling significantly remodels especially the NF-κB cistrome, reprogramming the PC cell transcriptome in fashion that may contribute to the progression of PC.
Asunto(s)
Andrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Mediadores de Inflamación/metabolismo , FN-kappa B/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Transcriptoma , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Análisis por Conglomerados , Citocinas/metabolismo , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Masculino , Proteínas Inhibidoras de STAT Activados/metabolismoRESUMEN
Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética , Transcripción Genética , Andrógenos/farmacología , Sitios de Unión/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatina/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
UNLABELLED: The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging andin situproximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCE: Viral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy.
Asunto(s)
Cromatina/virología , Genoma Viral , Histonas/metabolismo , Infecciones por Parvoviridae/virología , Parvovirus Canino/genética , Regiones Promotoras Genéticas , Acetilación , Animales , Gatos , Línea Celular , ADN Viral/metabolismo , Epigénesis Genética , Regulación Viral de la Expresión Génica , Lisina/metabolismo , Microscopía Confocal , Parvovirus Canino/metabolismo , Integración ViralRESUMEN
Canine parvovirus (CPV) infection induces reorganization of nuclear structures. Our studies indicated that late-stage infection induces accumulation of nuclear pore complexes (NPCs) and lamin B1 concomitantly with a decrease of lamin A/C levels on the apical side of the nucleus. Newly formed CPV capsids are located in close proximity to NPCs on the apical side. These results suggest that parvoviruses cause apical enrichment of NPCs and reorganization of nuclear lamina, presumably to facilitate the late-stage infection.
Asunto(s)
Enfermedades de los Perros/virología , Lamina Tipo B/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Infecciones por Parvoviridae/virología , Parvovirus Canino/metabolismo , Animales , Cápside/metabolismo , Núcleo Celular/metabolismo , Perros , Lamina Tipo A/metabolismo , Lámina Nuclear/metabolismoRESUMEN
BACKGROUND: Cells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs (small ubiquitin-like modifier proteins; SUMOylation). While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription. RESULTS: We mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes. CONCLUSIONS: HS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.
Asunto(s)
Cromatina/metabolismo , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Sumoilación , Transcripción Genética , Proteínas de Unión al ADN/fisiología , Factores de Transcripción del Choque Térmico , Humanos , Células K562 , Regiones Promotoras Genéticas , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved ß-loop of the helicase domain.