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. 2023 Jul 15;150(14):dev201515.
doi: 10.1242/dev.201515. Epub 2023 Jul 18.

Pleiotropy of autism-associated chromatin regulators

Micaela Lasser  1   2 , Nawei Sun  1   2 , Yuxiao Xu  1   2 , Sheng Wang  1   2 , Sam Drake  1   2 , Karen Law  1   2 , Silvano Gonzalez  1   2 , Belinda Wang  1   2   3 , Vanessa Drury  1   2 , Octavio Castillo  1   2 , Yefim Zaltsman  1   2 , Jeanselle Dea  1   2 , Ethel Bader  1   2 , Kate E McCluskey  1   2 , Matthew W State  1   2   3   4 , A Jeremy Willsey  1   2   4 , Helen Rankin Willsey  1   2   5
Affiliations

Pleiotropy of autism-associated chromatin regulators

Micaela Lasser et al. Development. .

Abstract

Gene ontology analyses of high-confidence autism spectrum disorder (ASD) risk genes highlight chromatin regulation and synaptic function as major contributors to pathobiology. Our recent functional work in vivo has additionally implicated tubulin biology and cellular proliferation. As many chromatin regulators, including the ASD risk genes ADNP and CHD3, are known to directly regulate both tubulins and histones, we studied the five chromatin regulators most strongly associated with ASD (ADNP, CHD8, CHD2, POGZ and KMT5B) specifically with respect to tubulin biology. We observe that all five localize to microtubules of the mitotic spindle in vitro in human cells and in vivo in Xenopus. Investigation of CHD2 provides evidence that mutations present in individuals with ASD cause a range of microtubule-related phenotypes, including disrupted localization of the protein at mitotic spindles, cell cycle stalling, DNA damage and cell death. Lastly, we observe that ASD genetic risk is significantly enriched among tubulin-associated proteins, suggesting broader relevance. Together, these results provide additional evidence that the role of tubulin biology and cellular proliferation in ASD warrants further investigation and highlight the pitfalls of relying solely on annotated gene functions in the search for pathological mechanisms.

Keywords: Xenopus; Autism spectrum disorder; Cell cycle; Chromatin; Cilia; Human; Microtubules; Spindles; Tubulin.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
ASD-associated chromatin regulators localize to microtubules. (A) Human Strep-tagged constructs for ASD-associated chromatin regulators ADNP, CHD8, CHD2, POGZ and KMT5B (labeled by Strep, green) localize to the nucleus (labeled by DAPI, blue) during interphase when expressed in Xenopus. (B) However, these constructs localize to the mitotic spindle during mitosis. Negative controls do not show these localizations. Arrows indicate spindle poles. See also Figs S1-S3.
Fig. 2.
Fig. 2.
CHD2 is required for mitotic spindle organization, cell cycle progression, genome stability and cell survival. (A-D) CHD2 CRISPRi in human iPSC-derived NPCs causes an increase in mitotic spindle defects (arrows, multipolar spindle) (A), in cyclin B (G2/M marker) fluorescence per cell (B), in pH2AX (DNA damage marker) puncta per nucleus (C), and in the proportion of CCP3 (cell death marker) positive cells (D) compared with non-targeting CRISPRi. (E) Quantification of the data shown in A (χ2 test). (F) Quantification of the data shown in B. Box is interquartile range, line is median, and whiskers are maximum to minimum values. (G) Quantification of the data shown in C. Dot is at the mean and lines are 95% confidence intervals. (H) Quantification of the data shown in D. Box is interquartile range, line is median, and whiskers are maximum to minimum values. ****P<0.0001 (non-parametric rank sum test). See also Fig. S4.
Fig. 3.
Fig. 3.
A missense variant of CHD2 observed in an individual with ASD disrupts spindle localization. (A) Schematic of human CHD2 protein with functional domains and locations of likely pathogenic missense variants annotated. (B) Strep-tagged human CHD2 and ASD-associated variants CHD2D856G and CHD2G1174D expressed in Xenopus localize to the nucleus during interphase. (C) During mitosis, CHD2 and CHD2G1174D localize to spindles during mitosis, whereas CHD2D856G does not and instead remains localized to DNA (DAPI, blue). Arrows indicate spindle poles. (D) Quantification of the area of overlap between Strep and DAPI. Bars are mean and whiskers are interquartile range. **P<0.01 (one-way ANOVA). ns, not significant.
Fig. 4.
Fig. 4.
Enrichment of microtubule-related proteins in ASD. (A) hcASD risk proteins (taken from Fu et al., 2022) are over-represented in a microtubule-related centriolar satellite proteome (taken from Gheiratmand et al., 2019). A significant number of overlapping proteins (bold) are annotated as chromatin binding. (B) Genes encoding centriolar satellite-associated proteins (Gheiratmand et al., 2019) are more likely to carry protein-truncating variants (PTVs) in individuals with ASD (Fu et al., 2022) compared with non-network genes. (C) Model for pleiotropy of ASD-associated chromatin regulators. During interphase, they localize to nuclei, regulating gene expression. During mitosis, they regulate tubulin and organize the mitotic spindle (inspired by Yokoyama, 2016). In loss of function, the spindle is disorganized, leading to cell cycle defects, DNA damage and death. See also Table S2.
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