Using a Massively-Parallel Reporter Assay to Evaluate Synthetic Enhancer Sequences Acting with Cell Type Specificity


Natalia Fuentes

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In: Student Reports, Summer 2019, The Jackson Laboratory


Enhancer elements arc regions of DNA which are able to recruit transcription factors that promote and control gene expression. Understanding the fundamental architecture of enhancer regions, especially their ability to fine-tune expression across different cell types, will aid in efforts such as gene therapy and estimating the heritability of complex diseases. One approach involves using machine-learning approaches to score an input on its likelihood of acting with enhancer activity, we can work with a neural network as a prediction model for enhancer elements. To develop and evaluate improved models and of gene regulation, we need a tool that is able to test the activity of thousands of individual regulatory elements in parallel across multiple cellular states.

A massively parallel reporter assay (MPRA) reports the expression levels of enhancer sequences spliced into episomal reporter vectors. We can use :MPRA to understand regulatory elements by designing an assay that compares the expression levels of thousands of proposed enhancer elements within a single library. By testing the library across multiple cell types and comparing activity measurements we can identify enhancer elements acting Vvith cell-type specificity. To accomplish this, we developed an ,oligo library of 30k sequence elements selected for having cell specific regulatory characteristics (marked by open chromatin or H3K27ac). We transfected the reporter library into 4 different cell types (K562, HepG2, GM12878 and SK-NSH), harvested the reporter rnRNA and generated Illumina sequencing libraries to measure the enhancer activity for all 30k elements in parallel.

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