Effects of H3.3G34V mutation on genomic H3K36 and H3K27 methylation patterns in isogenic pediatric glioma cells

Tina Yi-Ting Huang,Marc Morgan,Andrea Piunti,Elizabeth Bartom,Jin Qi,Ali Shilatifard,Amanda M Saratsis

doi:10.1186/s40478-020-01092-4

Tina Yi-Ting Huang, Marc Morgan + Show 5 more

Open Access

https://doi.org/10.1186/s40478-020-01092-4

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Abstract

Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. Taken together, our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines.

Highlights

Pediatric high-grade glioma is the number one cause of cancer death in children, with a 5-year survival of less than 20%
Western blot analysis of whole-cell extracts confirmed successful knockdown of H3.3G34V mutant protein in glioma cells treated with doxycycline, compared to those not treated with doxycycline and those transduced with H3F3A for wild-type H3.3 expression, with no significant reduction in H3K36 trimethylation across conditions (Fig. 1b)
We observed DOX-induced expression of H3.3G34V mutant protein in cDNA transduced astrocytes, with no mutant protein detected in untreated cells, or in cells transduced with H3F3A (Fig. 1c)

Summary

Introduction

Pediatric high-grade glioma (pHGG) is the number one cause of cancer death in children, with a 5-year survival of less than 20%. Several studies comparing H3.3G34V mutant and wild type cell lines suggest distinct epigenetic changes at H3K27 and H3K36 in association with H3.3G34V/R mutation, as well as alteration to DNA repair pathways leading to transcriptional upregulation, increased mutational burden and genomic instability [2, 10, 16]. We demonstrate changes in genomic enrichment of multiple chromatin marks after DOX-inducible knockdown of H3F3A in an H3.3G34V mutant pediatric glioma cell line, and H3.3G34V mutation transduction in wildtype astrocytes, providing insight on epigenetic effects of this mutation that promote tumorigenesis

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