NIR, an inhibitor of histone acetyltransferases, regulates transcription factor TAp63 and is controlled by the cell cycle

Abstract

p63 is a sequence-specific transcription factor that regulates epithelial stem cell maintenance and epithelial differentiation. In addition, the TAp63 isoform with an N-terminal transactivation domain functions as an inducer of apoptosis during the development of sympathetic neurons. Previous work has indicated that the co-activator and histone acetyltransferase (HAT), p300, can bind to TAp63 and stimulate TAp63-dependent transcription of the p21Cip1 gene. Novel INHAT Repressor (NIR) is an inhibitor of HAT. Here, we report that the central portion of NIR binds to the transactivation domain and the C-terminal oligomerization domain of TAp63. NIR is highly expressed in G2/M phase of the cell cycle and only weakly expressed in G1/S. Furthermore, except during mitosis, NIR is predominantly localized in the nucleolus; only a small portion co-localizes with TAp63 in the nucleoplasm and at the p21 gene promoter. Consistent with NIR acting as a repressor, the induced translocation of NIR from the nucleolus into the nucleoplasm resulted in the inhibition of TAp63-dependent transactivation of p21. Conversely, knockdown of NIR by RNAi stimulated p21 transcription in the presence of TAp63. Thus, NIR is a cell-cycle-controlled, novel negative regulator of TAp63. The low levels of nucleoplasmic NIR might act as a buffer toward potentially toxic TAp63.

Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2

“…When human H1299 lung adenocarcinoma cells (p53-deficient, low MDM2 levels) were transfected with plasmids producing Flag-L11 or Flag-NIR for 24 h, both proteins were primarily localized in the nucleoli, consistent with previous findings (8,25). On treatment with low-dose ActD (10 nM) for 0, 1 or 7 h, the characteristic nucleolar staining produced by Flag-L11 vanished and a diffuse nucleoplasmic staining became visible as early as 1 h after treatment (Figure 1A, left panels).…”

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

“…Human Noc1p/CEBPζ/CBF was described to stimulate transcription from the hsp70 promoter (33–35). Rrp5p/NKBP was shown to interact with NF-κB (36,37) and Noc2p/NIR was described to act as an inhibitor of histone acetyl transferases and to modulate p53 and TAp63 activity (38,39), adding a possible supplementary link between ribosome biogenesis and the p53 stress response pathway in higher eukaryotes [reviewed in (40,41)].…”

Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2

“…Peripheral blood mononuclear cells (PBMC) were isolated from 20 ml of whole blood donated by healthy volunteers, using standard Ficoll gradient centrifugation.PBMC were cultured at 37°C, 5% CO 2 , for up to 48 h in RPMI medium (Sigma) containing 10% FCS and antibiotics. Human diploid fibroblasts (HDF) were grown and maintained as reported by us previously64 . For transient transfection of DNA, cells were seeded to reach 60-70% confluency at transfection and were then transfected with jetPEI reagent (Polyplus, Illkirch, France), following the manufacturer's instructions.…”

“…pcDNA3-HA-ubiquitin was purchased from Addgene. pcmdm2, pCMV-p53, p21-luc, MG15-luc and PG13-luc have been used by us before64,65 .Epoxomicin was purchased from Enzo (Farmingdale, USA), and MG132 from Sigma, as were the -actin monoclonal antibody AC-15, the peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies, and the monoclonal anti-glutathione-s-transferase antibody (clone GST-2). The secondary anti-rat antibody was provided by one of us (EK).…”