Research Article: Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens

Date Published: January 29, 2019

Publisher: Public Library of Science

Author(s): Bhaven B. Patel, Andres M. Lebensohn, Ganesh V. Pusapati, Jan E. Carette, Julia Salzman, Rajat Rohatgi, Bart O. Williams.

http://doi.org/10.1371/journal.pone.0198463

Abstract

The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

Partial Text

An outstanding challenge in genomics is the identification of functional regulatory elements that control spatial and temporal expression of protein-coding genes and non-coding RNAs. The Encyclopedia of DNA Elements (ENCODE) project has the ambitious goal of generating a candidate list of all functional elements in the human genome using sequence features, such as evolutionary conservation, and biochemical features, such as chromatin accessibility and chromatin modifications [1]. Functional approaches to identify regulatory elements have thus far focused on specific regions of the genome and include massively parallel reporter assays or dense clustered regularly-interspaced short palindromic repeats (CRISPR)-mediated mutagenesis of <1 megabase pair segments around a locus of interest (reviewed in [2]). We developed a new bioinformatics tool to analyze haploid genetic screens with the explicit goal of uncovering regulatory elements. We analyzed screen data in a way that discerned GT insertion patterns distinct from those predicted to disrupt the coding sequence of genes, and found that atypical insertions in introns and regions upstream of the TSS can cause down-regulation of genes, leading to the phenotype selected for during the screens. When we applied this new analysis to haploid genetic screens interrogating the WNT pathway, we found that antisense GT insertions in the first intron of TFAP4 and upstream of the LRP6 promoter resulted in marked changes in the expression of these genes. These types of insertions had not been accounted for in previous analyses of haploid genetic screens.   Source: http://doi.org/10.1371/journal.pone.0198463

 

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