Investigating the role of CHASERR in CHD2 regulation, chromatin architecture, and gene expression during neurodevelopment - PROJECT SUMMARY Haploinsufficiency of chromatin remodeler CHD2 causes a neurodevelopmental disorder (NDD) characterized by developmental delay, intellectual disability, and epilepsy. Adjacent and upstream of CHD2 is a conserved long non-coding RNA (lncRNA) CHASERR. Deletion of CHASERR causes CHD2 overexpression and a more severe, early onset developmental disorder with significant motor and language delay, intellectual disability, and structural brain defects in humans. RNA-seq and western blot analysis of patient-specific induced pluripotent stem cells (iPSCs) and CRISPR-generated HAP1 cells have shown that CHASERR deletion increases CHD2 expression and protein levels in cis. While there is growing evidence of the role of lncRNAs in gene regulation, the mechanism of how CHASERR regulates CHD2, and the downstream consequences of too much CHD2 on global chromatin dynamics and neurodevelopment, is not well understood. Prior studies suggest that CHASERR is concentrated within its locus and binds to SPEN, a protein known to recruit HDAC3 and other chromatin remodeling proteins to repress transcription. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in HAP1s showed loss of HDAC3 occupancy at CHD2 locus in CHASERR knockout (KO) but not wildtype (WT), suggesting that the CHASERR-SPEN complex is essential to recruit HDAC3 and repress CHD2 expression. Taken together, I hypothesize that CHASERR deletion results in loss of recruitment of SPEN and other repressive proteins at the CHD2 locus, leading to a more open chromatin state permissive of increased CHD2 expression, resulting in CHD2 overproduction and global changes in chromatin dynamics. Using CRISPR-generated CHASERR KO and antisense oligonucleotide (ASO) to knockdown CHASERR in WT HAP1 cells, as well as patient-specific iPSCs and neural progenitor cells (NPCs), I will address my hypothesis with two aims. In Aim 1, I will conduct CUT&RUN on chromatin remodeling proteins (HDAC3, CHD2, EZH2) and histone modifications (H3K27ac, H3K27me3) to determine which proteins CHASERR and SPEN recruit to change local chromatin structure and repress CHD2 expression. In Aim 2, I will use a multi- omics approach with WT, ASO knockdown, and patient-derived CHASERR+/- NPCs, to understand how CHD2 overexpression affects global chromatin dynamics and transcription in a neural model. Because treatment for CHD2-related NDDs requires precise dosage control of functional CHD2, the proposed studies will help understand CHASERR’s regulation of CHD2 and its potential as a therapeutic target for CHD2 patients. Furthermore, this study will contribute to broader understandings of lncRNA biology and the biological underpinnings of childhood developmental disorders. The diverse team of mentors and the premier facilities and equipment at Northwestern will be available throughout the award period to support rigorous training to carve a successful physician-scientist career uncovering genetic mechanisms of pediatric neurological disorders.
