Tuesday, May 7, 2024
5/7/2024
Project Summary The goal of this proposal is to uncover the mechanistic connection between R-loops and gene expression through effects on genome architecture to understand how R-loop deregulation can contribute to neurodevelopmental disorders. R-loops are poorly understood RNA-containing chromatin structures that accumulate in, and contribute to the etiology of, several neurodevelopmental disorders. This includes Activity-dependent neuroprotective protein (ADNP) syndrome, also known as Helsmoortel-Van Der Aa syndrome, which is a rare condition in children that exhibit signs of autism. At present, how R-loops contribute to neurological disorders like ADNP syndrome is unclear. Accumulated R loops lead to an increased DNA damage response and can also alter transcription of neighboring genes. However, the mechanistic basis for R loops-mediated changes in gene expression are unknown and the functional relevance of this process to disorders like ADNP syndrome is unexplored. We have made the surprising finding that R-loops are highly enriched at a subset of binding sites for CTCF. Preliminary data show that R-loops strengthen CTCF interactions with chromatin. We found that conditions leading to loss or gain of R-loops can decrease or increase CTCF recruitment, respectively, and affect long- range genome interactions. We recently demonstrated that the activity-dependent neuroprotective protein (ADNP), a critical protein for brain development, is a site-specific R-loop resolver. ADNP heterozygous missense or frameshift mutations cause ADNP syndrome, a severe neurodevelopmental disorder. Human induced pluripotent stem cells (hiPSCs) derived from patients with ADNP syndrome show increased R-loops and CTCF accumulation at ADNP binding sites. We find that ADNP binding sites are enriched for sequences that are recognized by the genome architectural protein YY1, which has important functions in regulating enhancer- promoter interactions especially in the neural lineage. ADNP and YY1 co-localizes at active enhancers. Our preliminary data also identified changes in DNA methylation in ADNP syndrome hiPSCs that can potentially impact CTCF and YY1 binding to cause pathogenic genome misfolding and aberrant neural gene expression in ADNP syndrome. We hypothesize that R-loops have a regulatory function, and that they target CTCF and YY1 to specific genomic sites during neurodifferentiation. We posit that they may be critical for long-range genome interactions that reinforce neural lineage specific gene expression programs. We propose to decipher the impact of R-loop deregulation on genome organization and gene expression in the neural lineage through the lens of ADNP syndrome. In Aim 1, we will evaluate R-loop mediated regulation of CTCF and YY1 localization during neurodifferentiation. In Aim 2, we will examine the epigenetic consequences of distinct ADNP syndrome mutations and their impact on genome regulatory interactions.
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