Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response

Feb 23, 2026Nucleic acids research

Single-cell analysis shows different roles of alternative gene starters in drug response

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Abstract

Over 51.6% of targeted genes exhibit distinct transcriptional programs driven by alternative promoters.

screens show significant promoter specificity, with compensatory upregulation observed at untargeted promoters.
The development of Isoform-Specific single-cell Perturb-Seq enables systematic analysis of alternative promoter function.
Promoter selection is suggested to be a key mechanism for generating cellular diversity rather than just redundancy.
In breast cancer models, different estrogen receptor (ESR1) promoters have opposing effects on tamoxifen sensitivity and patient survival.
Promoter-level analysis is highlighted as essential in functional genomics for potential therapeutic strategies.

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(CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed Isoform-Specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 51.6% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.

Key numbers

51.6%

Distinct Transcriptional Programs

Percentage of targeted genes exhibiting unique transcriptional programs

2.3×

Increased P1 Promoter Usage

Fold increase in P1 promoter usage in tamoxifen-resistant cells compared to parental cells

1.9

Survival Hazard Ratio

Hazard ratio for survival based on P1 promoter expression

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Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response

Abstract

Key numbers

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