Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System

Abstract

Ubiquitin is required under both normal and stress conditions. Under stress conditions, upregulation of the polyubiquitin gene UBC is essential to meet the requirement of increased ubiquitin levels to confer stress resistance. However, UBC upregulation is usually observed only under stress conditions and not under normal conditions. Therefore, it has not been possible to upregulate UBC under normal conditions to study the effect of excess ubiquitin on cellular machinery. Recently, the CRISPR/Cas9 system has been widely used in biological research as a useful tool to study gene disruption effects. In this study, using an inducible CRISPR/Cas9 variant, a dCas9–VP64 fusion protein, combined with a single guide RNA (sgRNA) containing MS2 aptamer loops and MS2-p65-HSF1, we developed a system to increase the ubiquitin pool via upregulation of UBC. Although it is challenging to upregulate the expression of a gene that is already expressed at high levels, the significance of our system is that UBC upregulation can be induced in an efficient, reversible manner that is compatible with cellular processes, even under normal conditions. This system can be used to study ubiquitin pool dynamics and it will be a useful tool in identifying the role of ubiquitin under normal and stress conditions.

Regulation of polyubiquitin genes to meet cellular ubiquitin requirement

“…In addition, regulation using this system was inducible using doxycycline and completely reversible ( 68 ). These findings suggest that even under normal conditions, it is possible to induce changes in the free Ub pools by upregulation of endogenous polyubiquitin genes ( 29 ).…”

“…Some known facts are that the intron region is important for the regulation of basal expression levels of UBC , and the promoter region is important for the upregulation of UBC under stress conditions ( 25 - 28 ). Recently, it has been reported that using the inducible CRISPR-activation system, a modified version of the CRISPR/Cas9 system, in which guide RNA was targeted to the intron region of UBC , upregulation of UBC could be achieved temporally under normal conditions ( 29 ).…”

“…VP16 is a transcription factor of herpes simplex virus type 1 and activates transcription by inducing chromatin remodeling through interaction with other transcription factors ( 67 ). The activation system consisting of a simple combination of guide RNA and dCas9-VP64 targeting the UBC intron region did not induce UBC upregulation ( 29 ). These results suggest that UBC , with high basal expression levels, may already have a relaxed chromatin structure for transcriptional activation ( 66 ).…”

“…Thus, modified guide RNA targeting the intron region of UBC , dCas9-VP64, and p65-HSF1 were required for the upregulation of UBC expression. Because UBB was not affected, this modified guide RNA-derived system is quite specific to UBC ( 29 ). In addition, regulation using this system was inducible using doxycycline and completely reversible ( 68 ).…”

A negative feedback mechanism links UBC gene expression to ubiquitin levels by affecting RNA splicing rather than transcription

“…In particular, elevated Ub levels are intrinsic features of a variety of pathophysiological conditions, that upregulate Ub 22–25 , but may also derive from exogenous manipulation of cellular Ub levels, leading to ectopic Ub overexpression 9,20 . In a very recent paper, Han and coworkers 26 developed a new system to increase the cellular Ub levels in a more physiological fashion; they used the CRISPR-Cas9 technology to induce upregulation of the endogenous UBC gene under normal conditions. The authors claim that this system may be useful to study the cellular response to an excess of Ub under normal conditions and to highlight if this prior upregulation of UBC may have a protective role towards incoming stress insults.…”