Quantifying the impact of genetic mutations on enhancer dynamics

Area of Science:

• Genomics
• Molecular Biology
• Systems Biology

Background:

• Enhancers are key to transcriptional regulation, but their response to genetic perturbations is unclear.
• Understanding enhancer dynamics is crucial for deciphering gene expression control.

Purpose of the Study:

• To develop and apply a novel method for profiling enhancer activity and gene expression changes following genetic perturbations.
• To investigate the impact of transcription factor deletion on enhancer function and target gene regulation.

Main Methods:

• Developed UDI-UMI-STARR-seq, integrating dual indexes and unique molecular identifiers with RNA-seq.
• Assessed CRISPR/Cas9-mediated deletion of six transcription factors (TFs) across a large library of candidate enhancers.
• Utilized a deep learning model to analyze enhancer sequences and regulatory grammar.

Main Results:

• Identified TF-specific enhancer responses (repression or induction) and associated TF motifs.
• Revealed TF-specific regulatory programs, including impacts on Wnt/p53 targets, FIRRE locus, and RNA polymerase II regulators.
• Demonstrated enhancer dynamics linked to genes in axon guidance and synaptic plasticity in a neurodevelopmental disorder model.

Conclusions:

• UDI-UMI-STARR-seq provides a scalable method to study enhancer-gene regulatory dynamics.
• The findings elucidate TF-dependent enhancer grammar and its role in gene expression.
• This framework is generalizable to understanding the effects of genetic mutations on enhancer function.