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S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression.

ABSTRACT: Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription.

SUBMITTER: Cesari M

PROVIDER: S-EPMC1219912 | biostudies-other | 1998 Dec

REPOSITORIES: biostudies-other

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Project description:Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.

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S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression.

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