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Scholarly Works (3 results)

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  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2021)

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor with the average survival being only a year. To date the only treatment is radiotherapy, but it is not very effective leaving a need to better understand DIPG biology to develop more effective treatment methods. About 80% of DIPGs have a point mutation in one allele of H3F3A, one of two genes that codes for histone variant H3.3, resulting in lysine 27 being mutated to methionine. The H3.3K27M mutation is thought to be one of the first mutations to take place in DIPG tumorigenesis and results in the global depletion of the repressive mark H3K27me3 and an increase in the activating mark H3K27ac. In order to further investigate the effects of H3.3K27M and its subsequent abrogation on normal histone post-translational modifications (PTMs) patterns, we used CRISPR-Cas9 to revert H3.3K27M back to wild-type H3.3 in two patient DIPG cell lines. Here I used ATAC-seq in these isogenic cell lines to study the unique chromatin structure and function at work in DIPG tumors, including specifically what features are due to the H3.3K27M mutation as opposed to other mutations DIPGs are known to have. H3.3K27M results in increased chromatin accessibility surrounding genes that regulate neurogenesis and neuronal development, processes that have been found to be key in DIPG tumor formation and maintenance. The binding sites of several transcription factors including NOTCH signaling factor ASCL1 are enriched in unique open chromatin regions in K27M cells. This provides further support for drug treatments targeting NOTCH signaling and ASCL1. Overall, these studies indicate that both wildtype and mutant H3.3 are key regulators of chromatin dynamics and that K27M likely drives aberrant transcription in DIPG in part through altered chromatin structure.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2022)

    Background

    The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences.

    Results

    Recently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1.

    Conclusions

    Altogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type.
      Cover page: Histone H3.3 K27M chromatin functions implicate a network of neurodevelopmental factors including ASCL1 and NEUROD1 in DIPG
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2020)
      Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered H3K27me3 activity including within super-enhancers, which exhibited perturbed transcriptional function. This was largely independent of H3.3 DNA binding. The K27M and G34R mutations induced several of the same pathways suggesting key shared oncogenic mechanisms including activation of neurogenesis and NOTCH pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH pathway gene knockdown and drug inhibition, reducing their viability in culture. Reciprocal editing of cells generally produced reciprocal effects on tumorgenicity in xenograft assays. Overall, our findings define common and distinct K27M and G34R oncogenic mechanisms, including potentially targetable pathways.
        Cover page: Reciprocal H3.3 gene editing identifies K27M and G34R mechanisms in pediatric glioma including NOTCH signaling
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