PROJECT SUMMARY
The abundance of mutations in chromatin regulatory proteins in autism spectrum disorders (ASD) highlights the
significance of chromatin structure in normal brain development. A relatively new and little understood chromatin
structure that has been implicated in several neurodevelopmental disorders is the R-loop. R-loops are three-
stranded, nucleic acid structures containing a DNA:RNA hybrid and a displaced single stranded DNA, that form
and resolve continually. However, when R-loops accumulate, they can impede gene transcription, promote DNA
damage, and result in disease. R-loops are implicated in several neurodegenerative disorders, such as
frontotemporal dementia and amyotrophic lateral sclerosis, and in neurodevelopmental disorders such as Fragile
X Syndrome. Whether R-loop deregulation contributes to the etiology of ASD is not clear. The central challenge
in linking R-loop deregulation to ASDs has been pinpointing factors that are mutated in ASD that are also R-loop
regulators. We have identified factors that localize to R-loops using a proximity labeling proteomic approach.
Our approach yielded chromatin remodelers, homeobox transcription factors, and helicases. A number of these
proteins had also been reported to be mutated in ASD, e.g. ADNP, POGZ, CHD2, and DHX30. These
preliminary results provide the first indication that R-loop dysfunction may contribute to ASD. We focus our study
on the analysis of ADNP, an R-loop regulator that is frequently mutated in ASD and is causal in ADNP syndrome.
Our preliminary studies show that ADNP loss in mouse embryonic stem cells (mESCs) results in R-loop
accumulation specifically at ADNP binding sites. Importantly, our data show that the homeodomain of ADNP is
critical for R-loop suppression and that deletion of the homeodomain compromises the ability of mESCs to
differentiate into neural progenitor cells (NPCs). We found that human induced pluripotent stem cells (hiPSCs)
with an ADNP tyrosine 719* mutation (ADNP Tyr719*) also show R-loop deregulation. Based on these data, we
hypothesize that accumulation of R-loops at specific genomic regions changes normal gene expression
programs leading to improper neuronal differentiation, which can ultimately contribute to ASD. Here we will
determine how R-loop accumulation in ADNP Tyr719* hiPSCs affects gene expression in the pluripotent state
and upon neuronal differentiation. We will evaluate the neurodifferentiation potential of ADNP Tyr719* using a
cerebral organoid model and determine if R-loop attenuation can ameliorate the neurodifferentiation defects
observed in ADNP mutants.