Date of Award

Spring 2022

Document Type

Open Access Thesis


Biological Sciences

First Advisor

Sofia B. Lizarraga


Autism Spectrum Disorders (ASD) are highly heritable neurodevelopmental disorders characterized by social and verbal impairments as well as repetitive behaviors. Large human genetic studies showed that genes encoding chromatin and transcriptional regulatory proteins are among the most commonly mutated genes in ASD. Absent, Small, or Homeotic 1-like (ASH1L) is a histone methyltransferase that has previously been identified as a major genetic risk factor for ASD. ASH1L di-methylates Histone H3 on Lysine 36 (H3K36me2) and tri-methylates Histone H3 on Lysine 4 (H3K4me3), which mainly coincides with transcriptional activation. ASH1L is a Trithorax protein that is proposed to counteract the activity of Polycomb Repressor Complex 2 (PRC2) by preventing the tri-methylation of Histone H3 on Lysine 27 (H3K27me3). Using co-expression correlation analysis, we identified an overrepresentation of genes associated with neuronal projection development or morphogenesis that are highly correlated with ASH1L in the developing human cerebral cortex. This suggests that ASH1L could have a role in regulating gene programs that are important for neuronal morphogenesis. Using two different models of ASH1L haploinsufficiency, we find that reduction in ASH1L leads to altered morphological phenotypes, including decreased neurite outgrowth and enlarged “growth cone-like” structures in human neurons. Additionally, we show that in an ASH1L shRNA knockdown neurons there is an increased percentage of DCX-positive neurons compared to control neurons. DCX is a marker of immature cortical neurons suggesting that ASH1L knockdown potentially leads to delayed maturation.

Using engineered iPSCs containing pathogenic mutations in ASH1L, we investigated the role of ASH1L in the maintenance of stem cell pluripotency and neuronal morphogenesis. Additionally, we analyzed whether ASH1L could be regulating FOXP1 a major genetic risk factor for ASD that has been previously implicated as a transcriptional regulator of stem cell pluripotency and neuronal morphogenesis. Previous studies have also implicated the Trithorax/Polycomb axis in regulation of FOXP1, but it is unknown how FOXP1 expression relates to the ASH1L/PRC2 axis. FOXP1 and the ASH1L/PRC2 axis independently have been previously implicated in the control of stem cell pluripotency. Therefore, using control and ASH1L mutant-iPSCs we characterized the expression of FOXP1 and of a stem cell specific isoform of FOXP1 (FOXP1 ES), which has previously been implicated in the regulation of pluripotency genes. We also present the characterization of the pluripotency markers NANOG and OCT4, as well as the levels of PRC2-mediated H3K27me3 in ASH1L-mutant lines. PRC2 is a major orchestrator of early embryonic development, and its function is carefully regulated to ensure maintenance of pluripotency. Our work will begin to determine the extent to which ASH1L contributes to the maintenance of stem cell pluripotency through counteracting the activity of PRC2-mediated H3K27me3 and through regulation of FOXP1 ES. We also investigated the extent to which ASH1L pathogenic mutations alter the expression of the transcription factor FOXP1 and its transcriptional regulator RUNX1 in human neurons. In summary, our work in human neurons demonstrates that ASH1L is an essential regulator of neuronal morphogenesis and maturation and begins to investigate how regulation of FOXP1 by ASH1L contributes to the mechanisms underlying ASH1L-related neuronal deficits. In addition, our work on iPSCs starts to uncover the role of the ASH1L/FOXP1 axis in the modulation of human pluripotency.

Available for download on Friday, May 31, 2024

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