Abstract 1477: The interactomes of H3.1 and H3.3 reveal novel interactions, and associations with histone chaperones
Notice bibliographique
Résumé
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.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,009 | 0,003 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».