Partial sequence identity in a 25-nucleotide long element is sufficient for transcriptional adaptation in the Caenorhabditis elegans act-5/act-3 model

Welker, Jordan M.; Serobyan, Vahan; Zaker Esfahani, Elhamalsadat; Stainier, Didier Y. R.

doi:10.1371/journal.pgen.1010806

Cited by 2 publications

(1 citation statement)

References 42 publications

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“…elegans act-5/act-3 TA model, where a 25 base pair-length sequence in the regulatory region of the act-3 locus, demonstrating a 60% identity to a sequence in the act-5 mRNA, was sufficient to induce TA (Welker, Serobyan, Zaker Esfahani, & Stainier, 2023).…”

Section: Crispr-cas13d-mediated Cytoplasmic Wild-type Mrna Degradatio...mentioning

confidence: 99%

Inducible mRNA degradation tools to study transcriptional adaptation in mammalian systems

Jakutis

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Discrepancies between knockdown and knockout animal model phenotypes have long stood as a perplexing phenomenon. Several mechanisms explaining such observations have been proposed, namely the toxicity or the off-target effects of the knockdown reagents, as well as, in certain cases, genetic robustness – an organism's ability to maintain its phenotype despite genetic perturbations. In addition to these explanations, transcriptional adaptation (TA), a phenomenon defined as an event whereby a mutation in one gene leads to transcriptional upregulation or downregulation of another, adapting, gene or genes expression, has been recently proposed as an alternative explanation for the conflicting knockdown and knockout phenotype paradox. Since its discovery in 2015, TA's precise mechanism remains a subject of ongoing research. Majority of evidence suggests that mutant mRNA degradation plays a central in TA. Epigenetic remodeling is also thought to play a role, as evidenced by an increase in active histone marks at the transcription start sites of the adapting genes. Whether mRNA degradation is indeed the key player in TA remains debated. Furthermore, it is still unknown how exactly TA develops, what adapting genes it targets, and whether genomic mutations that render mutant mRNA sensitive to degradation are required for TA to occur. Throughout the experiments described in this Dissertation, I have designed an inducible TA system where TA can be triggered on demand and its effects on the cell’s transcriptome followed through time. I have demonstrated that degradation-prone transgenes, once induced and expressed, can be efficiently degraded, resulting in the protein loss-independent upregulation of adapting genes via TA. Adapting genes with higher degree of sequence similarity become upregulated faster than genes with lower degree of sequence similarity. Further functionality of this approach to study TA is limited by the leakiness of the inducible gene expression system; however, constitutively expressed degradation-prone transgenes were used to demonstrate TA in human cells. In addition, I have developed an approach to target wild-type cytoplasmic mRNAs without altering the cell’s genome and reported a TA-like phenomenon, which manifested as adapting gene upregulation not relying on mutations in other genes. Cytoplasmic mRNA cleavage with CRISPR-Cas13d triggered a TA-like response in three different gene models: Actg1 knockdown, Ctnna1 knockdown, and Nckap1 knockdown. After comparing two different modes of triggering TA, CRISPR-Cas9 knockout versus CRISPR-Cas13d knockdown, I reported little overlap between the dysregulated genes and suggested that diverse mRNA degradation modes led to distinct TA responses. In addition, the transcriptional increase of Actg2 caused by CRISPR-Cas13d-mediated Actg1 mRNA cleavage did not require chromatin accessibility changes. Experiments and genetic tools described in this dissertation investigated how TA develops from its earliest onset, how it affects the global transcriptome of the cell, as well as provided compelling evidence for an mRNA degradation-central TA mechanism. I have created tools to study both direct and indirect TA gene targets and unveiled important insights into the temporal dynamics of TA. Genes with higher sequence similarity were found to be upregulated more rapidly than those with lower similarity. Furthermore, it was revealed that the epigenetic properties of TA responses vary depending on the triggering mechanism. Cas13d-mediated degradation of wild-type mRNAs led to immediate transcriptional enhancement independent of epigenetic changes, which stood in contrast to previously measured alterations in chromatin accessibility in CRISPR-Cas9 mutants. This research has thus significantly advanced our knowledge of TA and provided valuable tools and findings that contribute to the broader understanding of gene expression regulation in response to mRNA degradation.

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Section: Crispr-cas13d-mediated Cytoplasmic Wild-type Mrna Degradatio...mentioning

confidence: 99%

Inducible mRNA degradation tools to study transcriptional adaptation in mammalian systems

Jakutis

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Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells

Mellis,

Melzer,

Bodkin

et al. 2024

Genome Biol

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Background Cells and tissues have a remarkable ability to adapt to genetic perturbations via a variety of molecular mechanisms. Nonsense-induced transcriptional compensation, a form of transcriptional adaptation, has recently emerged as one such mechanism, in which nonsense mutations in a gene trigger upregulation of related genes, possibly conferring robustness at cellular and organismal levels. However, beyond a handful of developmental contexts and curated sets of genes, no comprehensive genome-wide investigation of this behavior has been undertaken for mammalian cell types and conditions. How the regulatory-level effects of inherently stochastic compensatory gene networks contribute to phenotypic penetrance in single cells remains unclear. Results We analyze existing bulk and single-cell transcriptomic datasets to uncover the prevalence of transcriptional adaptation in mammalian systems across diverse contexts and cell types. We perform regulon gene expression analyses of transcription factor target sets in both bulk and pooled single-cell genetic perturbation datasets. Our results reveal greater robustness in expression of regulons of transcription factors exhibiting transcriptional adaptation compared to those of transcription factors that do not. Stochastic mathematical modeling of minimal compensatory gene networks qualitatively recapitulates several aspects of transcriptional adaptation, including paralog upregulation and robustness to mutation. Combined with machine learning analysis of network features of interest, our framework offers potential explanations for which regulatory steps are most important for transcriptional adaptation. Conclusions Our integrative approach identifies several putative hits—genes demonstrating possible transcriptional adaptation—to follow-up on experimentally and provides a formal quantitative framework to test and refine models of transcriptional adaptation. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-024-03351-2.

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Partial sequence identity in a 25-nucleotide long element is sufficient for transcriptional adaptation in the Caenorhabditis elegans act-5/act-3 model

Cited by 2 publications

References 42 publications

Inducible mRNA degradation tools to study transcriptional adaptation in mammalian systems

Inducible mRNA degradation tools to study transcriptional adaptation in mammalian systems

Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells

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