Abnormal neuronal development can lead to a wide array of mental disorders. Genes important for neurodevelopment have been combed for coding mutations leading to psychiatric disease with limited success, suggesting that other regions in the genome could be causative. A variety of molecular and clinical data indicates that mutations associated with psychiatric disease can reside in gene regulatory sequences such as enhancers. However, only a few enhancers have been definitively linked with these disorders to date. This is primarily because regulatory mutations are challenging to functionally characterize and link to specific genes and phenotypes. To address this challenge, we will use functional genomics data, sequence motifs, and evolutionary signatures to train EnhancerFinder, software that we developed that predicts functional enhancers at high success rates, to now specifically identify active neurodevelopmental enhancers. Over 12,000 candidate neurodevelopmental enhancers will then be cloned and assayed en masse for their enhancer activity using massively parallel reporter assays (MPRAs) in three human embryonic stem cell (hESC) derived neuronal lines: early initiation, neural progenitor cell stage that produces only neurons upon further differentiation, and astrocytes. In addition, we will link enhancers to their target genes using a novel chromatin structure-based prediction approach, called TargetFinder, thereby establishing a network connecting regulatory regions to neurodevelopmental genes. By overlaying reproducible psychiatric disease associated loci with this network, we will identify and prioritize non-coding mutations that are likely to affect expression of neurodevelopmental genes with roles in psychiatric disease. These predictions will be validated using genome- editing techniques to knock out regulatory elements and then assay changes in chromatin interactions and gene expression in developing neurons. The key innovations of our approach are: (i) accurate, quantitative measurements of activity for thousands of psychiatric disease associated enhancer candidates in parallel, (ii) chromatin based inference of gene regulatory networks linking enhancer mutations to genes and pathways, and (iii) a well-characterized stem cell based system to apply these techniques in a high-throughput manner to developing human neurons. We will rapidly disseminate software, reagents, protocols, and datasets to enable follow-up functional studies in the labs of our mental health collaborators and many others. Our long-term aim is to pinpoint causative regulatory variants in the many genomic loci associated with psychiatric disease where an obvious coding mutation is lacking. This approach could easily be adapted to functionally characterize gene regulatory elements involved in other complex human diseases.

Public Health Relevance

Despite a wide variety of molecular and clinical data suggesting that psychiatric disease can be caused by mutations in gene regulatory elements such as enhancers that control when and where genes turn on and off during neuronal development, only a few such mutations have been identified thus far. In this project, we will use advanced computational tools to predict neurodevelopmental enhancers and their gene targets, plus a technique called massively parallel reporter assay (MPRA) to functionally test thousands of these predictions en masse in human embryonic stem cell derived neuronal cell lines. We will then identify and functionally characterize disease associated DNA variants in these validated enhancers that disrupt their normal gene regulatory functions in neuronal development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH109907-03
Application #
9433692
Study Section
Special Emphasis Panel (ZMH1)
Program Officer
Arguello, Alexander
Project Start
2016-05-01
Project End
2021-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
Inoue, Fumitaka; Kircher, Martin; Martin, Beth et al. (2017) A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity. Genome Res 27:38-52
Petit, Florence; Sears, Karen E; Ahituv, Nadav (2017) Limb development: a paradigm of gene regulation. Nat Rev Genet 18:245-258
Ryu, Ann H; Eckalbar, Walter L; Kreimer, Anat et al. (2017) Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation. Sci Rep 7:7533
Whalen, Sean; Truty, Rebecca M; Pollard, Katherine S (2016) Enhancer-promoter interactions are encoded by complex genomic signatures on looping chromatin. Nat Genet 48:488-96