RESUMEN
Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.
Asunto(s)
Cromatina , Proteínas del Grupo de Alta Movilidad , Nucleosomas , Regiones Promotoras Genéticas , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Cromatina/metabolismo , Cromatina/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células HeLa , Ensamble y Desensamble de Cromatina , Células HEK293 , Elongación de la Transcripción Genética , Terminación de la Transcripción GenéticaRESUMEN
The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Proteína Fosfatasa 2 , ARN Polimerasa II , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/ultraestructura , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/ultraestructura , Terminación de la Transcripción Genética , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Unión Proteica , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestructura , Subunidades de Proteína/metabolismo , Subunidades de Proteína/químicaRESUMEN
During gene transcription, RNA polymerase II (RNA Pol II) passes nucleosomes with the help of various elongation factors. Here, we show that RNA Pol II achieves efficient nucleosome passage when the human elongation factors DSIF, PAF1 complex (PAF), RTF1, SPT6, and TFIIS are present. The cryo-EM structure of an intermediate of the nucleosome passage shows a partially unraveled hexasome that lacks the proximal H2A-H2B dimer and interacts with the RNA Pol II jaw, DSIF, and the CTR9trestle helix. RNA Pol II adopts a backtracked state with the RNA 3' end dislodged from the active site and bound in the RNA Pol II pore. Additional structures and biochemical data show that human TFIIS enters the RNA Pol II pore and stimulates the cleavage of the backtracked RNA and nucleosome passage.
Asunto(s)
Nucleosomas , ARN Polimerasa II , Núcleo Celular/metabolismo , Humanos , Nucleosomas/genética , ARN , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A-H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA.
Asunto(s)
Proteínas Cromosómicas no Histona/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Proteínas del Grupo de Alta Movilidad/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Transcripción Genética , Factores de Elongación Transcripcional/ultraestructura , Cromatina/genética , Cromatina/ultraestructura , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Histonas/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Nucleosomas/genética , Nucleosomas/ultraestructura , ARN Polimerasa II/genética , ARN Polimerasa II/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Chromatin remodeling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodeling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here, we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 Å. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at superhelical location (SHL) +2, supporting the 'twist defect' model of chromatin remodeling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our structure also maps CHD4 mutations that are associated with human cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.
