Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. METHODS: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). RESULTS: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. CONCLUSION: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

Reengineering a tRNA Methyltransferase To Covalently Capture New RNA Substrates.

Covalent RNA modifications can alter the function and metabolism of RNA transcripts. Altering the RNA substrate specificities of the enzymes that install these modifications can provide powerful tools to study and manipulate RNA. To develop new tools and probe the plasticity of the substrate specificity of one of these enzymes, we examined the engineerability of the uridine-54 tRNA methyltransferase, TrmA. Starting from a mutant that remains covalently bound to its substrate RNA (E358Q, TrmA*), we were able to use both rational design and a high-throughput sequencing assay to examine the RNA substrates of TrmA*. Although rational engineering substantially changed TrmA* specificity, the rationally designed substrate mutants we developed still retained activity with the wild-type protein. Using high-throughput substrate screening of additional TrmA* mutants, we identified a triple mutant of the substrate RNA (C56A;A58G;C60U) that is efficiently trapped by a TrmA* double mutant (E49R;R51E) but not by the wild-type TrmA*. This work establishes a foundation for using protein engineering to reconfigure substrate specificities of RNA-modifying enzymes and covalently trap RNAs with engineered proteins.