ABSTRACT
Growth signals, such as extracellular nutrients and growth factors, have substantial effects on genome integrity; however, the direct underlying link remains unclear. Here, we show that the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) pathway, a central regulator of growth signalling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis and impair its function in the DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumour suppressor liver kinase B1 (LKB1; also known as STK11) hyperactivates mTOR complex 1 (mTORC1)-S6K signalling and decreases RNF168 expression, resulting in defects in the DNA damage response. Expression of a phospho-deficient RNF168-S60A mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of mTORC1-S6K signalling in the DNA damage response and suggest a general mechanism that connects cell growth signalling to genome stability control.
Subject(s)
Cell Proliferation , DNA Repair , Neoplasms/enzymology , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , TOR Serine-Threonine Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , A549 Cells , AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases , Animals , DNA Breaks, Double-Stranded , Female , HCT116 Cells , HEK293 Cells , Humans , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Signal Transduction , TOR Serine-Threonine Kinases/genetics , Tumor Burden , Ubiquitin-Protein Ligases/geneticsABSTRACT
Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the first and rate-limiting step in the classical pathway of bile acids synthesis in liver and is crucial for maintaining lipid homeostasis. Hepatocyte nuclear factor 4α (HNF4α) and α1-fetoprotein transcription factor (FTF) are two major transcription factors driving CYP7A1 promoter activity in hepatocytes. Previous researches have shown that Prospero-related homeobox (Prox1) directly interacts with both HNF4α and FTF and potently co-represses CYP7A1 transcription and bile acid synthesis through unidentified mechanisms. In this work, mechanisms involved in Prox1-mediated co-repression were explored by identifying Prox1-associated proteins using immunoprecipitation followed by mass spectrometry (IP-MS) methodology. Multiple components of the epigenetically repressive lysine-specific demethylase 1 (LSD1)/nucleosome remodeling and histone deacetylase (NuRD) complex, most notably LSD1 and histone deacetylase 2 (HDAC2), were found to be associated with Prox1 and GST pulldown assay demonstrated that Prox1 directly interacts with LSD1. Sequential chromatin immunoprecipitation (ChIP) assays showed that Prox1 co-localizes with HNF4α, LSD1 and HDAC2 on CYP7A1 promoter in HepG2 cells. Furthermore, by using ChIP assay on HepG2 cells with endogenous Prox1 knocked down by RNA interference, Prox1 was shown to recruit LSD1 and HDAC2 onto CYP7A1 promoter and cause increased H3K4 demethylation. Finally, bile acids treatment of HepG2 cells, which significantly repressed CYP7A1 transcription, resulted in increased Prox1 and LSD1/NuRD complex occupancy on CYP7A1 promoter with a concurrent increase in H3K4 demethylation and H3/H4 deacetylation. These results showed that Prox1 interacts with LSD1 to recruit the repressive LSD1/NuRD complex to CYP7A1 promoter and co-represses transcription through epigenetic mechanisms. In addition, such Prox1-mediated epigenetic repression is involved in the physiologically essential negative feedback inhibition of CYP7A1 transcription by bile acids.
Subject(s)
Cholesterol 7-alpha-Hydroxylase/genetics , Epigenesis, Genetic , Histone Demethylases/metabolism , Homeodomain Proteins/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Animals , Bile Acids and Salts/biosynthesis , Cell Line , Gene Expression Regulation , Hep G2 Cells , Humans , Mice , Promoter Regions, Genetic , Protein BindingABSTRACT
SET8 (also known as PR-Set7/9, SETD8, KMT5A), a member of the SET domain containing methyltransferase family, which specifically catalyzes mono-methylation of K20 on histone H4 (H4K20me1), has been implicated in multiple biological processes, such as gene transcriptional regulation, cell cycle control, genomic integrity maintenance and development. In this study, we used GST-SET8 fusion protein as bait to search for SET8 interaction partners to elucidate physiological functions of SET8. In combination with mass spectrometry, we identified 40 proteins that potentially interact with SET8. DDX21, a nucleolar protein, was further confirmed to associate with SET8. Furthermore, we discovered a novel function of SET8 in the regulation of rRNA transcription.
Subject(s)
DEAD-box RNA Helicases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Protein Interaction Mapping/methods , Proteome/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , HEK293 Cells , HeLa Cells , Histone-Lysine N-Methyltransferase/genetics , Humans , Immunoblotting , Microscopy, Fluorescence , Protein Binding , Proteomics/methods , RNA, Ribosomal/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transcription, GeneticABSTRACT
Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1's corepressor activity.
Subject(s)
Homeodomain Proteins/metabolism , Protein Processing, Post-Translational , Repressor Proteins/metabolism , SUMO-1 Protein/metabolism , Tumor Suppressor Proteins/metabolism , Cell Line , Histone Deacetylases/metabolism , Homeodomain Proteins/genetics , Humans , Repressor Proteins/genetics , Transcription, Genetic , Tumor Suppressor Proteins/geneticsABSTRACT
Interferon (IFN) regulatory factors (IRFs) are a family of transcription mediators involved in the regulation of type I IFN (IFN-alpha/beta) transcription. Among the nine already identified IRFs, IRF-3 is a constitutively and ubiquitously expressed and plays a critical role in the transcriptional activation of type I IFN and IFN-stimulated genes including IFN-alpha1, IFN-beta and RANTES. In the present study, we report the identification of a novel alternatively spliced transcript of murine Irf-3 gene (mIrf-3) designated mIRF-3a. mIRF-3a is ubiquitously present in mouse tissues along with mIRF-3 and could be translocated into the nucleus in uninfected L929 cells. EMSA and reporter assay demonstrated that mIRF-3a binds to ISRE sequences in murine Ifn-beta promoter and represses Ifn-beta promoter activation induced by Newcastle disease virus infection. These results suggest that mIRF-3a may act as a modulator of mIRF-3 functions in vivo.
