Functional Interaction between the Mouse Notch1 Intracellular Region and Histone Acetyltransferases PCAF and GCN5

Hisanori Kurooka,Tasuku Honjo

doi:10.1074/jbc.m000909200

Abstract

The Notch receptor that plays an important role in cell fate determination is intracellularly cleaved by interaction with the ligand. The cleaved intracellular region (RAMIC) of Notch is translocated into the nucleus and interacts with a DNA-binding protein RBP-J to activate transcription of genes that regulate cell differentiation. Although RAMIC has been shown to facilitate the RBP-J-mediated transactivation by displacing the histone deacetylase corepressor complex from RBP-J, there is no evidence demonstrating the involvement of histone acetyltransferases (HATs) in the transactivation. Here we show that mouse Notch1 RAMIC interacts with two conserved HATs, mouse PCAF and GCN5, and recruits each of the HATs to RBP-J. The ankyrin repeats and the transactivation domain of RAMIC and the N-terminal regions of PCAF and GCN5, respectively, are required for the interaction. We also show that not only mouse Notch1 but also Drosophila Notch RAMIC interacts with mouse PCAF and GCN5 in mammalian cells. Furthermore, the RBP-J-mediated transactivation activity of RAMIC is repressed by two HAT inhibitor proteins, E1A and Twist. These results suggest that HATs including PCAF and GCN5 play an important role in the RBP-J-mediated transactivation by RAMIC.

Highlights

The Notch family consists of single transmembrane receptors that are involved in cell fate determination in multiple steps of development [1]
trichostatin A (TSA) Derepresses Transcactivation Activity of the Fusion Protein between RBP-J and the Mouse Notch1 transcriptional activation domain (TAD)—Previously we reported that the fusion protein between RBP-J and the C-terminal portion of mouse Notch1 RAMIC (RBP-J-IC⌬ANK, Fig. 1) failed to activate transcription of the pGa981-6 luciferase reporter gene carrying 12 canonical RBP-J-binding sites [23], the C terminus of mouse Notch1 activates transcription autonomously when fused to the yeast GAL4 DNAbinding domain (DBD) (Ref. 23 and see Fig. 5)
Since RBP-J interacts with histone deacetylase (HDAC) corepressor complexes [33, 35], we asked whether the fusion protein between RBP-J and the Cterminal portion of mouse Notch1 RAMIC is repressed by the HDAC activity

Summary

The abbreviations used are

Su(H), Suppressor of Hairless; bHLH, basic helix-loop-helix; RAM, RBP-J-associating molecule; ANK, ankyrin; NLS, nuclear localization signal; TAD, transcriptional activation domain; EBV, Epstein-Barr virus; EBNA2, EBV nuclear antigen 2; EBNA2RE, EBNA2-responsive element; HDAC, histone deacetylase; HAT, histone acetyltransferase; CBP, CREB-binding protein; PCAF, P300/CBP-associated factor; tk, thymidine kinase; CMV, cytomegalovirus; DBD, DNA-binding domain; IP, immunoprecipitation; PAGE, polyacrylamide gel electrophoresis; TSA, trichostatin A; GTF, general transcription factor; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay Kao et al [33] demonstrated that RBP-J interacts with a corepressor complex including the SMRT/NCoR family of nuclear receptor corepressors and histone deacetylase 1 (HDAC1) [34] They showed that SMRT/ NCoR competed with TAN-1 (human Notch RAMIC) for RBPJ-binding and antagonized the RBP-J-mediated transactivation activity of TAN-1. Our results suggest that HATs including PCAF and GCN5 may be involved in the RBP-J-mediated transactivation by Notch RAMIC

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION