Abstract 1477: The interactomes of H3.1 and H3.3 reveal novel interactions, and associations with histone chaperones
Bibliographic record
Abstract
Abstract Pediatric high grade gliomas are incurable brain tumors with very high mortality rates. Recent genomic studies have uncovered unique driver mutations involving histone genes encoding either H3.1 or H3.3. Histone H3 interacts with diverse variety of cellular machinery which regulates chromatin structure and function, transcription, and DNA replication. Previous efforts using IP-mass spectroscopy have revealed a great deal about H3.1 and H3.3 biology and the different interaction networks between them, including the chaperones that transport histones around the cell and incorporate them into chromatin. For example HIRA specifically deposits H3.3 into active chromatin, while CAF-1 has been shown to deposit H3.1 during DNA replication. However, these studies typically use high salt extraction, which disrupts all but the most stable protein-protein interactions. In order to more fully characterise the interactome of histone H3.1 and H3.3 in an unbiased fashion and provide insight into their potentially different roles in pediatric brain tumors, we employed proximity dependent biotinylation (BioID). We generated Flp-In HEK293 cells expressing FLAG-BirA*, alone or fused to H3.1 or H3.3, under the control of a doxycycline-inducible promoter. BirA* is a highly promiscuous biotin ligase that biotinylates proteins within a 20 nm radius. Cells were induced with doxycycline and biotin for 24 hours. SDS-based lysis and streptavidin pulldowns followed by mass-spectrometry analysis were used to identify proteins interacting with FLAG-BirA*-H3.1/H3.3 but not FLAG-BirA* alone. We validated our results by affinity purification followed by western blotting, and proximity ligation assays. FLAG-BirA*-H3 displays normal cellular localisation and is incorporated into DNA in nucleosomes of the same stability as endogenous H3-containing nucleosomes. Furthermore, chromatin stability is not changed in the presence of FLAG-BirA*-H3, showing that FLAG-BirA* fusion does not affect normal histone functions. Comparison with mass spectroscopy data indentified many previously described interactors, as well as 465 interactors not previously identified by affinity purification-mass spectroscopy, suggesting novel histone functions. The interactomes of H3.1 and H3.3 were strikingly similar, however key difference were identified. As expected, the histone chaperones NASP and ASF1 were similarly enriched in both H3 proteins, while HIRA was enriched with H3.3 vs. H3.1. Surprisingly, however, CAF-1 was not enriched in H3.1 vs. H3.3 suggesting CAF-1 may also serve as a chaperone for H3.3. Our results suggest BioID is a useful tool for unbiased interactome characterization, including proteins such as histones that require harsh extraction methods. Identification and understanding of histone H3.1 and H3.3 interactors will further aid in uncovering what role histone mutations may play in cancer initiation. Citation Format: Scott Milos, Robert Siddaway, Sanja Pajovic, Eric Campos, Brian Raught, Cynthia Hawkins. The interactomes of H3.1 and H3.3 reveal novel interactions, and associations with histone chaperones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1477.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.009 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".