RESUMO
NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia are significantly associated with altered gene activation, transcription, and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labeling (BioID) combined with label-free quantitative MS to identify high confidence NSD2 interacting partners in MM cells. The top 24 proteins identified were involved in maintaining chromatin structure, transcriptional regulation, RNA pre-spliceosome assembly, and DNA damage. Among these, an important DNA damage regulator, poly(ADP-ribose) polymerase 1 (PARP1), was discovered. PARP1 and NSD2 have been found to be recruited to DNA double strand breaks upon damage and H3K36me2 marks are enriched at damage sites. We demonstrate that PARP1 regulates NSD2 via PARylation upon oxidative stress. In vitro assays suggest the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2-regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. This work provides clear evidence of cross-talk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation, and other pathways.
Assuntos
Cromatina/enzimologia , Histona-Lisina N-Metiltransferase/metabolismo , Mieloma Múltiplo/enzimologia , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Proteínas Repressoras/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/patologia , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/genética , Estresse Oxidativo/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Repressoras/genéticaRESUMO
The contributions of phosphorylation-mediated signaling networks to colon cancer metastasis are poorly defined. To interrogate constitutive signaling alterations in cancer progression, the global phosphoproteomes of patient-matched SW480 (primary colon tumor origin) and SW620 (lymph node metastasis) cell lines were compared with TiO2 and immobilized metal affinity chromatography phosphopeptide enrichment followed by liquid chromatography-tandem mass spectrometry. Network analysis of the significantly altered phosphosites revealed differential regulation in cellular adhesion, mitosis, and messenger RNA translational machinery. Messenger RNA biogenesis and splicing, transport through the nuclear pores, initiation of translation, and stability and degradation were also affected. Although alterations in these processes have been associated with oncogenic transformation, control of messenger RNA stability has typically not been associated with cancer progression. Notably, the single phosphosite with the greatest relative change in SW620 cells was Ser2 on eukaryotic translation initiation factor 2 subunit 2, suggesting that SW620 cells translate faster or with greater efficiency than SW480 cells. These broad changes in the regulation of translation also occur without overexpression of eukaryotic translation initiation factor 4E. The findings suggest that metastatic cells exhibit constitutive changes to the phosphoproteome, and that messenger RNA stability and translational efficiency may be important targets of deregulation during cancer progression.
