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1.
Sci Adv ; 9(10): eadf2468, 2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36888719

RESUMO

The polymerase-associated factor 1 complex (PAF1C) is a key, post-initiation transcriptional regulator of both promoter-proximal pausing and productive elongation catalyzed by RNA Pol II and is also involved in transcriptional repression of viral gene expression during human immunodeficiency virus-1 (HIV-1) latency. Using a molecular docking-based compound screen in silico and global sequencing-based candidate evaluation in vivo, we identified a first-in-class, small-molecule inhibitor of PAF1C (iPAF1C) that disrupts PAF1 chromatin occupancy and induces global release of promoter-proximal paused RNA Pol II into gene bodies. Transcriptomic analysis revealed that iPAF1C treatment mimics acute PAF1 subunit depletion and impairs RNA Pol II pausing at heat shock-down-regulated genes. Furthermore, iPAF1C enhances the activity of diverse HIV-1 latency reversal agents both in cell line latency models and in primary cells from persons living with HIV-1. In sum, this study demonstrates that efficient disruption of PAF1C by a first-in-class, small-molecule inhibitor may have therapeutic potential for improving current HIV-1 latency reversal strategies.


Assuntos
HIV-1 , RNA Polimerase II , Humanos , RNA Polimerase II/metabolismo , HIV-1/genética , HIV-1/metabolismo , Simulação de Acoplamento Molecular , Linhagem Celular , Transcrição Gênica , Fatores de Transcrição/genética
2.
Mol Cell ; 82(18): 3412-3423.e5, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35973425

RESUMO

It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.


Assuntos
RNA Polimerase II , Fatores de Transcrição , Resposta ao Choque Térmico , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica
3.
Mol Cell ; 81(21): 4413-4424.e5, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34480849

RESUMO

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.


Assuntos
Proteínas Culina/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Sobrevivência Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Culina/química , Fibroblastos/metabolismo , Humanos , Ácidos Indolacéticos/química , Camundongos , Ubiquitina-Proteína Ligases Nedd4/química , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/química , Proteoma , Proteômica/métodos , Ubiquitina-Proteína Ligases/química , Proteína com Valosina/química , Proteína com Valosina/metabolismo
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