RESUMEN
Mitosis involves intricate steps, such as DNA condensation, nuclear membrane disassembly, and phosphorylation cascades that temporarily halt gene transcription. Despite this disruption, daughter cells remarkably retain the parent cell's gene expression pattern, allowing for efficient transcriptional memory after division. Early studies in mammalian cells suggested that transcription factors (TFs) mark genes for swift reactivation, a phenomenon termed 'mitotic bookmarking', but conflicting data emerged regarding TF presence on mitotic chromosomes. Recent advancements in live-cell imaging and fixation-free genomics challenge the conventional belief in universal formaldehyde fixation, revealing dynamic TF interactions during mitosis. Here, we review recent studies that provide examples of at least four modes of TF-DNA interaction during mitosis and the molecular mechanisms that govern these interactions. Additionally, we explore the impact of these interactions on transcription initiation post-mitosis. Taken together, these recent studies call for a paradigm shift toward a dynamic model of TF behavior during mitosis, underscoring the need for incorporating dynamics in mechanistic models for re-establishing transcription post-mitosis.
Asunto(s)
Mitosis , Factores de Transcripción , Transcripción Genética , Humanos , Factores de Transcripción/metabolismo , Animales , ADN/metabolismo , Regulación de la Expresión GénicaRESUMEN
A large number of transcription factors have been shown to bind and interact with mitotic chromosomes, which may promote the efficient reactivation of transcriptional programs following cell division. Although the DNA-binding domain (DBD) contributes strongly to TF behavior, the mitotic behaviors of TFs from the same DBD family may vary. To define the mechanisms governing TF behavior during mitosis in mouse embryonic stem cells, we examined two related TFs: Heat Shock Factor 1 and 2 (HSF1 and HSF2). We found that HSF2 maintains site-specific binding genome-wide during mitosis, whereas HSF1 binding is somewhat decreased. Surprisingly, live-cell imaging shows that both factors appear excluded from mitotic chromosomes to the same degree, and are similarly more dynamic in mitosis than in interphase. Exclusion from mitotic DNA is not due to extrinsic factors like nuclear import and export mechanisms. Rather, we found that the HSF DBDs can coat mitotic chromosomes, and that HSF2 DBD is able to establish site-specific binding. These data further confirm that site-specific binding and chromosome coating are independent properties, and that for some TFs, mitotic behavior is largely determined by the non-DBD regions.
Asunto(s)
Cromosomas , Proteínas de Choque Térmico , Mitosis , Factores de Transcripción , Animales , Ratones , Cromosomas/genética , Cromosomas/metabolismo , ADN/metabolismo , Factores de Transcripción del Choque Térmico/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the pre-initiation complex onto promoter DNA. Decades of research have shown that the TATA-box binding protein (TBP) is essential for Pol II loading and initiation. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells has no global effect on ongoing Pol II transcription. In contrast, acute TBP depletion severely impairs RNA Polymerase III initiation. Furthermore, Pol II transcriptional induction occurs normally upon TBP depletion. This TBP-independent transcription mechanism is not due to a functional redundancy with the TBP paralog TRF2, though TRF2 also binds to promoters of transcribed genes. Rather, we show that the TFIID complex can form and, despite having reduced TAF4 and TFIIA binding when TBP is depleted, the Pol II machinery is sufficiently robust in sustaining TBP-independent transcription.
Asunto(s)
ARN Polimerasa II , Factores de Transcripción , Animales , Ratones , Factores de Transcripción/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , TATA Box/genética , Células Madre Embrionarias/metabolismo , Transcripción Genética , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , ARN Polimerasa III/genéticaRESUMEN
Topoisomerase 1 (Top1) relieves torsional stress on DNA, including from RNA Polymerase II (Pol II) transcription. A new study by Wiegard et al. uncovers an unexpected role of Top1 in the appropriate clearance of Pol II from mitotic DNA, allowing for a reset of transcriptional memory in the daughter cells.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , ADN , Replicación del ADN , ARN Polimerasa II/metabolismoRESUMEN
At the heart of the transcription process is the specific interaction between transcription factors (TFs) and their target DNA sequences. Decades of molecular biology research have led to unprecedented insights into how TFs access the genome to regulate transcription. In the last 20 years, advances in microscopy have enabled scientists to add imaging as a powerful tool in probing two specific aspects of TF-DNA interactions: structure and dynamics. In this review, we examine how applications of diverse imaging technologies can provide structural and dynamic information that complements insights gained from molecular biology assays. As a case study, we discuss how applications of advanced imaging techniques have reshaped our understanding of TF behavior across the cell cycle, leading to a rethinking in the field of mitotic bookmarking.
Asunto(s)
ADN/química , Genoma , Factores de Transcripción/genética , Secuencia de Bases , Microscopía por Crioelectrón , Cristalografía por Rayos X , Regulación de la Expresión Génica , HumanosRESUMEN
The extracellular molecular chaperone heat shock protein 90 (eHSP90) stabilizes protease client the matrix metalloproteinase 2 (MMP2), leading to tumor cell invasion. Although co-chaperones are critical modulators of intracellular HSP90:client function, how the eHSP90:MMP2 complex is regulated remains speculative. Here, we report that the tissue inhibitor of metalloproteinases-2 (TIMP2) is a stress-inducible extracellular co-chaperone that binds to eHSP90, increases eHSP90 binding to ATP, and inhibits its ATPase activity. In addition to disrupting the eHSP90:MMP2 complex and terminally inactivating MMP2, TIMP2 loads the client to eHSP90, keeping the protease in a transient inhibitory state. Secreted activating co-chaperone AHA1 displaces TIMP2 from the complex, providing a "reactivating" mechanism for MMP2. Gene knockout or blocking antibodies targeting TIMP2 and AHA1 released by HT1080 cancer cells modify their gelatinolytic activity. Our data suggest that TIMP2 and AHA1 co-chaperones function as a molecular switch that determines the inhibition and reactivation of the eHSP90 client protein MMP2.
Asunto(s)
Matriz Extracelular/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiología , Proteolisis , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Proteínas HSP90 de Choque Térmico/genética , Humanos , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Inhibidor Tisular de Metaloproteinasa-2/genéticaRESUMEN
Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.
Asunto(s)
Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Sitios de Unión , ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Células HEK293 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Defects in cellular protein homeostasis are associated with many severe and prevalent pathological conditions such as neurodegenerative diseases, muscle dystrophies, and metabolic disorders. One way to counteract these defects is to improve the protein homeostasis capacity through induction of the heat shock response. Despite numerous attempts to develop strategies for chemical activation of the heat shock response by heat shock transcription factor 1 (HSF1), the underlying mechanisms of drug candidates' mode of action are poorly understood. To lower the threshold for the heat shock response activation, we used the chaperone co-inducer BGP-15 that was previously shown to have beneficial effects on several proteinopathic disease models. We found that BGP-15 treatment combined with heat stress caused a substantial increase in HSF1-dependent heat shock protein 70 (HSPA1A/B) expression already at a febrile range of temperatures. Moreover, BGP-15 alone inhibited the activity of histone deacetylases (HDACs), thereby increasing chromatin accessibility at multiple genomic loci including the stress-inducible HSPA1A. Intriguingly, treatment with well-known potent HDAC inhibitors trichostatin A and valproic acid enhanced the heat shock response and improved cytoprotection. These results present a new pharmacological strategy for restoring protein homeostasis by inhibiting HDACs, increasing chromatin accessibility, and lowering the threshold for heat shock response activation.
Asunto(s)
Cromatina/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Oximas/farmacología , Piperidinas/farmacología , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Proteínas Co-Represoras , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Histona Desacetilasas/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Unión Proteica , Receptor Notch4/metabolismoRESUMEN
In mammals the stress-inducible expression of genes encoding heat shock proteins is under the control of the heat shock transcription factor 1 (HSF1). Activation of HSF1 is a multistep process, involving trimerization, acquisition of DNA-binding and transcriptional activities, which coincide with several posttranslational modifications. Stress-inducible phosphorylation of HSF1, or hyperphosphorylation, which occurs mainly within the regulatory domain (RD), has been proposed as a requirement for HSF-driven transcription and is widely used for assessing HSF1 activation. Nonetheless, the contribution of hyperphosphorylation to the activity of HSF1 remains unknown. In this study, we generated a phosphorylation-deficient HSF1 mutant (HSF1Δâ¼PRD), where the 15 known phosphorylation sites within the RD were disrupted. Our results show that the phosphorylation status of the RD does not affect the subcellular localization and DNA-binding activity of HSF1. Surprisingly, under stress conditions, HSF1Δâ¼PRD is a potent transactivator of both endogenous targets and a reporter gene, and HSF1Δâ¼PRD has a reduced activation threshold. Our results provide the first direct evidence for uncoupling stress-inducible phosphorylation of HSF1 from its activation, and we propose that the phosphorylation signature alone is not an appropriate marker for HSF1 activity.