Rohm, D. | Search bioRxiv

bioRxiv2024

Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing

SummaryEpigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

genomics↗

bioRxiv2019

Collateral fitness effects of mutations

The distribution of fitness effects (DFE) of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutations collateral fitness effects, which we define as effects that do not derive from changes in the proteins ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the E. coli TEM-1 {beta}-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins, collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper post-translational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly-expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against mutations with deleterious effects on protein activity and protein abundance. Significance StatementMutations provide the source of genetic variability upon which evolution acts. Deleterious protein mutations are commonly thought of in terms of how they compromise the proteins ability to perform its physiological function. However, mutations might also be deleterious if they cause negative effects on one of the countless other cellular processes. The frequency and magnitude of such collateral fitness effects is unknown. Our systematic study of mutations in a bacterial protein finds widespread collateral fitness effects that were associated with protein aggregation, improper protein processing, incomplete protein transport across membranes, incorrect disulfide-bond formation, induction of stress-response pathways, and unexpected changes in cell properties. Our results suggest that deleterious collateral fitness effects may be an important constraint on protein evolution.

evolutionary biology↗