RESUMO
During wound healing, different pools of stem cells (SCs) contribute to skin repair. However, how SCs become activated and drive the tissue remodeling essential for skin repair is still poorly understood. Here, by developing a mouse model allowing lineage tracing and basal cell lineage ablation, we monitor SC fate and tissue dynamics during regeneration using confocal and intravital imaging. Analysis of basal cell rearrangements shows dynamic transitions from a solid-like homeostatic state to a fluid-like state allowing tissue remodeling during repair, as predicted by a minimal mathematical modeling of the spatiotemporal dynamics and fate behavior of basal cells. The basal cell layer progressively returns to a solid-like state with re-epithelialization. Bulk, single-cell RNA, and epigenetic profiling of SCs, together with functional experiments, uncover a common regenerative state regulated by the EGFR/AP1 axis activated during tissue fluidization that is essential for skin SC activation and tissue repair.
RESUMO
Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.
Assuntos
Microscopia Crioeletrônica , Transporte Proteico , Peixe-Zebra , Humanos , Animais , Endossomos/metabolismo , Células HEK293 , Células HeLa , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Ligação ProteicaRESUMO
Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.
Assuntos
Antígeno B7-H1 , DNA Helicases , Imunidade Inata , Melanoma , Evasão Tumoral , Animais , Camundongos , Antígeno B7-H1/metabolismo , Instabilidade Genômica , Melanoma/imunologia , Melanoma/metabolismo , DNA Helicases/metabolismoRESUMO
Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.
Assuntos
Sequência Conservada , Desenvolvimento Embrionário/genética , Proteínas Quinases/metabolismo , Retroelementos/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Sequência de Bases , Blastocisto/metabolismo , Proliferação de Células , Evolução Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mamíferos/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismoRESUMO
Type 2 immune responses are critical in tissue homeostasis, anti-helminth immunity, and allergy. T helper 2 (Th2) cells produce interleukin-4 (IL-4), IL-5, and IL-13 from the type 2 gene cluster under regulation by transcription factors (TFs) including GATA3. To better understand transcriptional regulation of Th2 cell differentiation, we performed CRISPR-Cas9 screens targeting 1,131 TFs. We discovered that activity-dependent neuroprotector homeobox protein (ADNP) was indispensable for immune reactions to allergen. Mechanistically, ADNP performed a previously unappreciated role in gene activation, forming a critical bridge in the transition from pioneer TFs to chromatin remodeling by recruiting the helicase CHD4 and ATPase BRG1. Although GATA3 and AP-1 bound the type 2 cytokine locus in the absence of ADNP, they were unable to initiate histone acetylation or DNA accessibility, resulting in highly impaired type 2 cytokine expression. Our results demonstrate an important role for ADNP in promoting immune cell specialization.
Assuntos
Histonas , Fatores de Transcrição , Histonas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Células Th2 , Citocinas/metabolismo , Diferenciação Celular , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismoRESUMO
The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.
Assuntos
Osteoclastos , RNA , Humanos , Camundongos , Animais , Proteínas Correpressoras/genética , Osteoclastos/metabolismo , Ligante RANK/genética , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Expressão GênicaRESUMO
Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.
Assuntos
RNA Longo não Codificante , Homeostase do Telômero , Cromatina/genética , Proteômica , Telômero/genética , Telômero/metabolismo , RNA Longo não Codificante/genética , HomeostaseRESUMO
Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.
Assuntos
RNA Longo não Codificante , Telomerase , Homeostase do Telômero , Telômero/genética , Telômero/metabolismo , Telomerase/genética , Telomerase/metabolismo , Estruturas R-Loop/genética , Reparo do DNARESUMO
The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.
Assuntos
Cromatina , Proteína do Retinoblastoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição AP-1/genéticaRESUMO
Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.
Assuntos
Células Dendríticas/metabolismo , Elementos Facilitadores Genéticos/genética , Fatores Reguladores de Interferon/genética , Animais , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Quimiocinas/genética , Transcrição Gênica/genéticaRESUMO
Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proliferação de Células , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/genética , Ligases/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade Proteica , Proteínas de Ligação a RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Transdução de Sinais , Sumoilação , Telômero/genética , Telômero/patologiaRESUMO
Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.
Assuntos
DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Heterozigoto , Células-Tronco Embrionárias Humanas/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Fácies , Deformidades Congênitas do Pé/genética , Deformidades Congênitas do Pé/metabolismo , Deformidades Congênitas do Pé/patologia , Antígeno 2 Relacionado a Fos/biossíntese , Antígeno 2 Relacionado a Fos/genética , Células HEK293 , Células-Tronco Embrionárias Humanas/patologia , Humanos , Hipotricose/genética , Hipotricose/metabolismo , Hipotricose/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Proteínas Nucleares/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.
Assuntos
Carcinoma Hepatocelular , Antígeno 2 Relacionado a Fos , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , Proteínas Proto-Oncogênicas c-myc , Fator de Transcrição AP-1 , Animais , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Camundongos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Antígeno 2 Relacionado a Fos/metabolismo , Antígeno 2 Relacionado a Fos/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Humanos , Hepatócitos/metabolismo , Multimerização Proteica , Regulação Neoplásica da Expressão Gênica , Camundongos TransgênicosRESUMO
ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transporte Proteico , Endossomos/metabolismo , Corpos Multivesiculares/metabolismo , Transporte BiológicoRESUMO
During chronic stimulation, CD8+ T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Fatores Reguladores de Interferon/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular , Regulação da Expressão Gênica , Células HEK293 , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/imunologia , Humanos , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Ativação Linfocitária , Depleção Linfocítica , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T/genética , Transdução de SinaisRESUMO
Cells respond to temperature stress via up- and downregulation of hundreds of genes. This process is thought to be regulated by the heat shock factor HSF1, which controls the release of RNAPII from promoter-proximal pausing. Here, we analyze the events taking place in hESCs upstream of RNAPII release. We find that temperature stress results in the activation or decommissioning of thousands of enhancers. This process involves alterations in the occupancy of transcription factors HSF1, AP-1, NANOG, KLF4, and OCT4 accompanied by nucleosome remodeling by BRG1 and changes in H3K27ac. Furthermore, redistribution of RAD21 and CTCF results in the formation and disassembly of interactions mediated by these two proteins. These alterations tether and untether enhancers to their cognate promoters or refashion insulated neighborhoods, thus transforming the landscape of enhancer-promoter interactions. Details of the 3D interactome remodeling process support loop extrusion initiating at random sites as a mechanism for the establishment of CTCF/cohesin loops.
Assuntos
Regulação da Expressão Gênica/fisiologia , Resposta ao Choque Térmico/genética , Células-Tronco Embrionárias Humanas/fisiologia , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona , DNA Helicases/genética , Proteínas de Ligação a DNA , Genes Homeobox , Temperatura Alta , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/fisiologia , Regiões Promotoras Genéticas , Proteínas/genética , RNA Polimerase II , Proteínas Repressoras , Estresse Fisiológico/fisiologia , Temperatura , Fator de Transcrição AP-1 , Fatores de Transcrição/genética , CoesinasRESUMO
The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.
Assuntos
Adenovírus Humanos , Proteínas Oncogênicas Virais , Animais , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Transformação Celular Neoplásica/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas Oncogênicas Virais/metabolismo , DNA , Mamíferos/genéticaRESUMO
Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.
Assuntos
Complexo 1 de Proteínas Adaptadoras , ATPases Transportadoras de Cobre , Endossomos , Transporte Proteico , Receptor IGF Tipo 2 , Rede trans-Golgi , Humanos , Endossomos/metabolismo , Células HeLa , Transporte Proteico/genética , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismoRESUMO
RAD51-associated protein 1 (RAD51AP1) is known to promote homologous recombination (HR) repair. However, the precise mechanism of RAD51AP1 in HR repair is unclear. Here, we identify that RAD51AP1 associates with pre-rRNA. Both the N terminus and C terminus of RAD51AP1 recognize pre-rRNA. Pre-rRNA not only colocalizes with RAD51AP1 at double-strand breaks (DSBs) but also facilitates the recruitment of RAD51AP1 to DSBs. Consistently, transient inhibition of pre-rRNA synthesis by RNA polymerase I inhibitor suppresses the recruitment of RAD51AP1 as well as HR repair. Moreover, RAD51AP1 forms liquid-liquid phase separation in the presence of pre-rRNA in vitro, which may be the molecular mechanism of RAD51AP1 foci formation. Taken together, our results demonstrate that pre-rRNA mediates the relocation of RAD51AP1 to DSBs for HR repair.
Assuntos
Proteínas de Ligação a DNA , Recombinação Homóloga , Proteínas de Ligação a RNA , DNA , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Precursores de RNA , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. METHODS AND RESULTS: By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1ß (interleukin-1ß) and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. CONCLUSIONS: Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.