In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.

Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes1,2. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy whilst maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants3,4 (4-fold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantial improved specificity and observed no off-target sites for 4 of the 8 sgRNAs tested. Finally, we showed that following long-term expression (40 days), evoCas9 strongly limited the unspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-targets.

Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.DOI: http://dx.doi.org/10.7554/eLife.15161.001

Sustaining energy homeostasis is of pivotal importance for all living organisms. In , evolutionarily conserved SnRK1 kinases (Snf1-RELATED KINASE1) control metabolic adaptation during low energy stress. To unravel starvation-induced transcriptional mechanisms, we performed transcriptome studies of inducible knockdown lines and found that S-basic leucine zipper transcription factors (S-bZIPs) control a defined subset of genes downstream of SnRK1. For example, S-bZIPs coordinate the expression of genes involved in branched-chain amino acid catabolism, which constitutes an alternative mitochondrial respiratory pathway that is crucial for plant survival during starvation. Molecular analyses defined S-bZIPs as SnRK1-dependent regulators that directly control transcription via binding to G-box promoter elements. Moreover, SnRK1 triggers phosphorylation of group C-bZIPs and the formation of C/S-heterodimers and, thus, the recruitment of SnRK1 directly to target promoters. Subsequently, the C/S-bZIP-SnRK1 complex interacts with the histone acetylation machinery to remodel chromatin and facilitate transcription. Taken together, this work reveals molecular mechanisms underlying how energy deprivation is transduced to reprogram gene expression, leading to metabolic adaptation upon stress.

In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.