# Early Epigenetic Footprint of Neurological Diseases

> **NIH NIH R00** · RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL · 2021 · $249,000

## Abstract

PROJECT SUMMARY:
Abnormal neuronal development can lead to a wide array of mental disorders. This is thought to be caused, amongst others, by aberrant gene regulation during early neural development. While we know the role of several key regulators, no study has thoroughly investigated the regulatory landscape during early time points of this process. The primary goal of this project is to comprehensively map and investigate the function of gene regulatory elements associated with early neural differentiation
and understand their role in neural disease. During the K99 phase of the award, I created a model system that extensively characterizes neural induction (Aim K1). Using neural induction from human pluripotent stem cells (hPSCs) as a paradigm, we interrogated these dynamics by performing RNA-seq, ChIP-seq and ATAC-seq at seven time points during early neural differentiation. We found that changes
in DNA accessibility precede H3K27ac, which is followed by gene expression changes. Using massively parallel reporter assays (MPRAs) to test the activity of 2,464 candidate regulatory sequences at all seven time points, we show that many of these sequences have temporal activity patterns that correlate with their respective cell-endogenous gene expression and chromatin changes. A prioritization method incorporating all genomic and MPRA data further identified key transcription factors (TFs) involved in driving neural fate. These results provide a comprehensive resource of genes and regulatory elements that orchestrate neural induction and illuminate temporal frameworks during differentiation (Cell Stem Cell 2019). I extended this work to identify functional TF binding in early neural differentiation (Aim K2). To that end, I designed a MPRA experiment to test the effect of perturbing hundreds of TF binding sites across hundreds of regulatory regions relevant to neural differentiation. These perturbed regions along with wild type regions and control sequences were tested in similar temporal points. Computational analysis highlights: Temporal TF binding effects; Different types of effects; Interactions between TFs (Manuscript in preparation).
In the R00 phase of the award, I will explore regions and factors that can trigger neural differentiation from hESC without environmental signal by (i) designing a single-cell RNA profiling of pooled overexpression for top TF candidates and (ii) targeting selected regulatory elements using CRISPR/Cas9 based activation. This analysis will elucidate determinants of neural induction (Aim R1). Finally, I will map variants that are associated with neural phenotype and reside in candidate enhancer regions, to perform a comparative reference/alternative MPRA that will specifically highlight variants that alter the
regulatory potential of a region and indicate their functionality in early neural development (Aim R2). Overall, this framework will identify key enhancers linked to neural differentiation, and provide hypothese...

## Key facts

- **NIH application ID:** 10240738
- **Project number:** 5R00MH117393-04
- **Recipient organization:** RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
- **Principal Investigator:** Anat Kreimer
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $249,000
- **Award type:** 5
- **Project period:** 2018-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10240738

## Citation

> US National Institutes of Health, RePORTER application 10240738, Early Epigenetic Footprint of Neurological Diseases (5R00MH117393-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10240738. Licensed CC0.

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