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CHD8 Dosage Regulates Transcription in Pluripotency and Early Murine Neural Differentiation

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CHD8 Dosage Regulates Transcription in Pluripotency and Early Murine Neural Differentiation CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation Sabina Sooda,b,1, Christopher M. Webera,b,1, H. Courtney Hodgesc,d, Andrey Krokhotine, Aryaman Shalizia,b, and Gerald R. Crabtreea,b,e,2 aDepartment of Pathology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305; cDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; dCenter for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030; and eHoward Hughes Medical Institute, Chevy Chase, MD 20815 Contributed by Gerald R. Crabtree, July 7, 2020 (sent for review December 16, 2019; reviewed by Bradley E. Bernstein and Gordon L. Hager) The chromatin remodeler CHD8 is among the most frequently mu- Here, we utilized mouse embryonic stem cells (ESCs) and dif- tated genes in autism spectrum disorder (ASD). CHD8 has a dosage- ferentiation into neural progenitor cells (NPCs) as a model system sensitive role in ASD, but when and how it becomes critical to human to study the role of CHD8 in early embryonic gene regulation and social function is unclear. Here, we conducted genomic analyses of control of genome-wide accessibility. We created both homozygous heterozygous and homozygous Chd8 mouse embryonic stem cells and heterozygous Chd8 deletions, enabling us to characterize the and differentiated neural progenitors. We identify dosage-sensitive effect of gene dosage on these important processes. We demon- CHD8 transcriptional targets, sites of regulated accessibility, and an strate that CHD8 has a dosage-sensitive effect on transcriptional unexpected cooperation with SOX transcription factors. Collectively, regulation in ESCs and upon differentiation into NPCs that is our findings reveal that CHD8 negatively regulates expression of consistent with premature neuronal differentiation. Surprisingly, we neuronal genes to maintain pluripotency and also during differenti- find that CHD8 directly associates with SOX2 to maintain acces- ation. Thus, CHD8 is essential for both the maintenance of pluripo- sibility in support of the pluripotency gene network. Collectively, we tency and neural differentiation, providing mechanistic insight into demonstrate an essential role for CHD8 in regulating the accessible its function with potential implications for ASD. chromatin landscape and gene expression, providing insight into the function of CHD8 with potential implications for ASD. autism spectrum disorder (ASD) | chromatin remodeling | pluripotency | GENETICS neural progenitors | chromodomain helicase DNA-binding protein 8 (CHD8) Results Dosage-Sensitive Effects of CHD8 Deletion on Transcription in ESCs. ynamic regulation of chromatin structure is required to en- We were particularly motivated to study CHD8 gene dosage in Dsure rapid transcriptional responses during development and pluripotency and in neurogenesis because it is one of the most to maintain these programs in order to safeguard cellular identity. highly constrained genes in the human genome (19) and is hap- Central to these regulatory processes are chromatin-remodeling loinsufficient for human neurodevelopment and human social ′ enzymes, which hydrolyze adenosine 5 -triphosphate (ATP) to behavior, as indicated by its role in ASD. Consistent with these modify chromatin structure and DNA accessibility by reposition- observations, CHD8 has a very high probability of being intolerant ing, editing, or evicting nucleosomes (1). There are four major to the loss of function of a single allele in humans (Fig. 1A). subfamilies of ATP-dependent nucleosome remodelers classified Previous work has shown that CHD8 expression at embryonic day by phylogenetic relationships and distinct functional activities (2). 12.5 (E12.5) is ∼10-fold higher compared to the adult, and Chd8 Of these, the chromodomain helicase DNA-binding (CHD) class of remodelers is of particular interest because it is implicated in Significance human diseases. In mammals, there are nine CHD family mem- bers, each containing two tandemly arranged chromodomains The chromatin remodeler CHD8 is one of the most frequently upstream of their catalytic SNF2 helicase domain that are involved mutated genes in autism spectrum disorder (ASD), but the in nucleosome spacing and histone variant H3.3 incorporation (3, mechanistic basis remains unclear. Here, we identify dosage- 4). Individual CHD family members are frequently inactivated in sensitive roles for CHD8 in the regulation of transcription and ’ specific human diseases, including Hodgkin slymphoma(CHD3), define CHD8’s role in regulating genome-wide accessibility. neuroblastoma (CHD5), CHARGE syndrome (CHD7), and au- Importantly, we present new results that help to define the tism spectrum disorder (ASD) (CHD8) (5). CHD8 is of particular molecular function of CHD8 both in the context of pluripotency interest because it is one of the most commonly mutated genes in and in neural differentiation with implications for its role in ASD (6), but the mechanistic basis remains unclear. ASD. By determining the execution point at which mutations in CHD8 regulates important developmental pathways and is Chd8 might contribute to the disease, we hope to discover the essential during embryogenesis. For example, CHD8 inhibits potential for therapeutic approaches. β-catenin and Wnt-signaling pathways as well as p53-dependent transactivation, preventing widespread apoptosis (7–9). Consis- Author contributions: S.S., C.M.W., and G.R.C. designed research; S.S., C.M.W., and A.S. tent with a specific role in neurodevelopmental disorders, CHD8 performed research; S.S. and C.M.W. contributed new reagents/analytic tools; S.S., C.M.W., H.C.H., A.K., and A.S. analyzed data; and S.S., C.M.W., and G.R.C. wrote targets pathways associated with ASD and intellectual disability the paper. – +/− (10 13). Additionally, Chd8 and CHD8 knockdown mouse Reviewers: B.E.B., Harvard Medical School and Broad Institute; and G.L.H., National models recapitulate some ASD-like behavioral phenotypes, sug- Cancer Institute. gesting a causal role in promoting ASD etiology (14–17). While The authors declare no competing interest. these studies have demonstrated that CHD8 is a critical tran- Published under the PNAS license. scriptional regulator during neurogenesis, there are discrepancies 1S.S. and C.M.W. contributed equally to this work. regarding CHD8’s role in cell cycle exit (18); moreover, it remains 2To whom correspondence may be addressed. Email: [email protected]. unclear how CHD8 is targeted to its chromatin sites, the down- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ stream effect of its recruitment on accessibility, and how these doi:10.1073/pnas.1921963117/-/DCSupplemental. biochemical activities regulate transcription. First published August 24, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1921963117 PNAS | September 8, 2020 | vol. 117 | no. 36 | 22331–22340 Downloaded by guest on September 24, 2021 knockout mice arrest at E7.5, implying an essential role for CHD8 sequencing (RNA-seq) to characterize the effect of CHD8 de- during the neural progenitor stage (20). Thus, we generated single letion on gene expression changes over the approximately five or double allelic Chd8 deletions in mouse ESCs, a well-studied passages required to generate the mutant ESC lines (SI Appen- and context-relevant model system. Using a single guide RNA dix, Fig. S1B). As expected, there were significantly more differ- (sgRNA), we targeted CRISPR-Cas9 to exon 12, which includes entially expressed genes (DEGs) upon homozygous CHD8 deletion the ATPase/helicase domain (Fig. 1B) (21). Single or double de- (709 up-regulated and 673 down-regulated) compared to hetero- letion clones were validated by Sanger sequencing and Western zygous deletion (162 up-regulated and 110 down-regulated genes) − blotting, which confirmed that CHD8 protein level was reduced to (Fig. 1E). Interestingly, the Chd8+/ DEGs showed substantial − − − − − ∼50% in the Chd8+/ line and was undetectable in the Chd8 / overlap with Chd8 / for both increased (77.8% overlap, 126 of cell line, enabling investigation into CHD8 dosage. 162) and decreased (78.2% overlap, 86 of 110) genes, confirming Consistent with previous results (22), CHD8 knockout cells that CHD8 activity is dosage sensitive (haploinsufficient) in ESCs. − − proliferated at a significantly slower rate, with a doubling time Consistent with this finding, many DEGs in the Chd8 / ESCs − that was twice as long as wild-type (WT) and Chd8+/ cells exhibited moderate dosage-sensitive expression changes when a (Fig. 1C; n = 3, P < 0.05). The decreased proliferation rate was single allele of CHD8 was deleted (Fig. 1F). not due to cell cycle arrest (SI Appendix, Fig. S1A). To assay the To identify the key regulatory pathways controlled by CHD8, effect of CHD8 deletion on pluripotency, we stained cells for we conducted gene ontology (GO) term analysis of biological alkaline phosphatase (Fig. 1D). While all genotypes stained with processes on DEGs (Fig. 1G). Genes that were up-regulated in − − − − similar intensity, the Chd8 / cells did not form colonies like Chd8 / were enriched for processes associated with nervous − the WT and Chd8+/ cells (2i growth conditions), indicative of system development, neurogenesis, and cell cycle (P < 0.01 for all − compromised pluripotency. To explore this result, we used RNA terms), whereas Chd8+/ did
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