Biology subjects

Sadowski, I.

Publications and source records attributed to Sadowski, I..

4 recordsLinked to original sources

bioRxiv2022

Inhibition of the TRIM24 bromodomain reactivates latent HIV-1

Expression of the HIV-1 genome by RNA Polymerase II is regulated at multiple steps, as are most cellular genes, including recruitment of general transcription factors and control of transcriptional elongation from the core promoter. We recently discovered that tripartite motif protein TRIM24 is recruited to the HIV-1 Long Terminal Repeat (LTR) by interaction with TFII-I and causes transcriptional elongation by stimulating association of PTEF-b/ CDK9. Because TRIM24 is required for stimulation of transcription from the HIV-1 LTR, we were surprised to find that IACS-9571, a specific inhibitor of the TRIM24 C-terminal bromodomain, induces HIV-1 provirus expression in otherwise untreated cells. IACS-9571 reactivates HIV-1 in T cell lines bearing multiple different provirus models of HIV-1 latency. Additionally, treatment with this TRIM24 bromodomain inhibitor encourages productive HIV-1 expression in newly infected cells and inhibits formation of immediate latent transcriptionally repressed provirus. IACS-9571 synergizes with PMA, ionomycin, TNF- and PEP005 to activate HIV-1 expression. Furthermore, co-treatment of CD4+ T cells from individuals with HIV-1 on antiretroviral therapy (ART) with PEP005 and IACS-9571 caused robust provirus expression. Notably, IACS-9571 did not cause global activation of T cells; rather, it inhibited induction of IL2 and CD69 expression in human PBMCs and Jurkat T cells treated with PEP005 or PMA. These observations indicate the TRIM24 bromodomain inhibitor IACS-9571 represents a novel HIV-1 latency reversing agent (LRA), and unlike other compounds with this activity, causes partial suppression of T cell activation while inducing expression of latent provirus.

molecular biology↗

bioRxiv2021

TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation

The conserved HIV-1 LTR cis elements RBE1/3 bind the factor RBF2, consisting of USF1/2 and TFII-I, and are essential for reactivation of HIV-1 by T cell signaling. We determined that TFII-I recruits the tripartite motif protein TRIM24 to the LTR, and this interaction is required for efficient reactivation of HIV-1 expression in response to T cell signaling, similar to the effect of TFII-I depletion. Knockout of TRIM24 did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9 to the LTR. These results demonstrate that TFII-I promotes transcriptional elongation in response to T cell activation through recruitment of the co-factor TRIM24, which is necessary for efficient recruitment of the elongation factor P-TEFb.

molecular biology↗

bioRxiv2021

Cdk8 directly regulates glycolysis via phosphorylation of Gcr2

CDK8 encodes an evolutionarily conserved Mediator complex kinase subunit that functions in general and context-specific transcription regulation by phosphorylating core components of the transcription machinery and gene-specific transcription factors. To better understand the role Cdk8 in transcription regulation, we performed high-resolution gene expression time course analysis following nuclear depletion of Cdk8. Focusing on the earliest gene expression alterations revealed dysregulation of genes encoding glycolysis enzymes, suggesting a functional link to Gcr1 and Gcr2, key transcriptional activators of these genes. Consistently, we found that nuclear depletion of Cdk8 altered the mRNA levels of glycolysis genes as well as the promoter occupancy of Gcr2, but not Gcr1. Examination of the Gcr2 protein sequence revealed a putative Cdk8 phosphorylation site at serine 365, which we confirmed using in vitro and in vivo assays. Importantly, phospho-mutant GCR2 recapitulated the growth and gene expression defects of the GCR2 deletion mutant, effects not observed with a phospho mimetic mutant. As such, our work highlights Gcr2 as a new Cdk8 substrate, revealing that its phosphorylation is critical for the activation of genes encoding glycolysis enzymes.

molecular biology↗

bioRxiv2020

TOR signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Cdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth of gal3 yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

genetics↗