Large-scale mapping and mutagenesis of human transcriptional effector domains

DelRosso, Nicole; Tycko, Josh; Suzuki, Peter; Andrews, Cecelia; Aradhana,; Mukund, Adi; Liongson, Ivan; Ludwig, Connor; Spees, Kaitlyn; Fordyce, Polly M.; Beer, M; Bintu, Lacramioara

doi:10.1038/s41586-023-05906-y

Cited by 62 publications

(65 citation statements)

References 60 publications

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“…Interestingly, P HI fused to Gal4 DB acted as a transcriptional activation domain even in the absence of dNS3-VPR cotransfection, while Gal4 DB -P MED displayed no observable activation on its own (Figure S1). The P HI sequence contains features that are consistent with that of many known ADs, including an enrichment of acidic, aliphatic, and aromatic side chains (see Table S1 for sequence). Consequently, only P MED was used for the transcriptional effector work while P HI was reserved for future, nontranscriptional applications.…”

Section: Resultsmentioning

confidence: 86%

Controlled Protein Activities with Viral Proteases, Antiviral Peptides, and Antiviral Drugs

et al. 2023

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Chemical control of protein activity is a powerful tool for scientific study, synthetic biology, and cell therapy; however, for broad use, effective chemical inducer systems must minimally crosstalk with endogenous processes and exhibit desirable drug delivery properties. Accordingly, the drug-controllable proteolytic activity of hepatitis C cisprotease NS3 and its associated antiviral drugs have been used to regulate protein activity and gene modulation. These tools advantageously exploit non-eukaryotic and non-prokaryotic proteins and clinically approved inhibitors. Here, we expand the toolkit by utilizing catalytically inactive NS3 protease as a high affinity binder to genetically encoded, antiviral peptides. Through our approach, we create NS3peptide complexes that can be displaced by FDA-approved drugs to modulate transcription, cell signaling, and split-protein complementation. With our developed system, we invented a new mechanism to allosterically regulate Cre recombinase. Allosteric Cre regulation with NS3 ligands enables orthogonal recombination tools in eukaryotic cells and functions in divergent organisms to control prokaryotic recombinase activity.

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Section: Resultsmentioning

confidence: 86%

Controlled Protein Activities with Viral Proteases, Antiviral Peptides, and Antiviral Drugs

et al. 2023

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“…CRX and other homeodomain transcription factors have been shown to be capable of acting as both activators and repressors at different target sequences (White et al 2016;Shepherdson et al 2024;DelRosso et al 2023). Further characterization is required, but balancing activation and repression in the transcriptional effector domain may necessitate the "intermediate" activation strength residue composition observed for CRX.…”

Section: Discussionmentioning

confidence: 99%

Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Shepherdson,

Granas,

et al. 2024

Preprint

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Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of over 1,800 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.

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“…Additionally, the SAM and Suntag systems recruit TADs in trans via motifs appended to the tracrRNA and dCas9, respectively 8,9 . Heterologous combinations of TADs have also been developed, such as VPR, which combines VP64, p65, and Rta 10 domains, and recent studies have explored the landscape of potential TADs in high throughput [11][12][13] . CRISPRa technology has been deployed for genome-wide genetic screens across a diversity of phenotypes.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Griffith

Zheng

McGee

et al. 2023

Preprint

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Cas12a CRISPR technology, unlike Cas9, allows for multiplexing guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and we provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

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Large-scale mapping and mutagenesis of human transcriptional effector domains

Cited by 62 publications

References 60 publications

Controlled Protein Activities with Viral Proteases, Antiviral Peptides, and Antiviral Drugs

Controlled Protein Activities with Viral Proteases, Antiviral Peptides, and Antiviral Drugs

Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

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