TATA-binding protein-like protein (TLP/TRF2/TLF) negatively regulates cell cycle progression and is required for the stress-mediated G(2) checkpoint.
ABSTRACT: The TATA-binding protein (TBP) is a universal transcription factor required for all of the eukaryotic RNA polymerases. In addition to TBP, metazoans commonly express a distantly TBP-related protein referred to as TBP-like protein (TLP/TRF2/TLF). Although the function of TLP in transcriptional regulation is not clear, it is known that TLP is required for embryogenesis and spermiogenesis. In the present study, we investigated the cellular functions of TLP by using TLP knockout chicken DT40 cells. TLP was found to be dispensable for cell growth. Unexpectedly, TLP-null cells exhibited a 20% elevated cell cycle progression rate that was attributed to shortening of the G(2) phase. This indicates that TLP functions as a negative regulator of cell growth. Moreover, we found that TLP mainly existed in the cytoplasm and was translocated to the nucleus restrictedly at the G(2) phase. Ectopic expression of nuclear localization signal-carrying TLP resulted in an increase (1.5-fold) in the proportion of cells remaining in the G(2)/M phase and apoptotic state. Notably, TLP-null cells showed an insufficient G(2) checkpoint when the cells were exposed to stresses such as UV light and methyl methanesulfonate, and the population of apoptotic cells after stresses decreased to 40%. These phenomena in G(2) checkpoint regulation are suggested to be p53 independent because p53 does not function in DT40 cells. Moreover, TLP was transiently translocated to the nucleus shortly (15 min) after stress treatment. The expression of several stress response and cell cycle regulatory genes drifted in a both TLP- and stress-dependent manner. Nucleus-translocating TLP is therefore thought to work by checking cell integrity through its transcription regulatory ability. TLP is considered to be a signal-transducing transcription factor in cell cycle regulation and stress response.
Project description:We previously showed that reduced intracellular levels of the TATA binding protein (TBP), brought about by tbp heterozygosity in DT40 cells, resulted in a mitotic delay reflecting reduced expression of the mitotic regulator cdc25B but did not significantly affect overall transcription. Here we extend these findings in several ways. We first provide evidence that the decrease in cdc25B expression reflects reduced activity of the cdc25B core promoter in the heterozygous (TBP-het) cells. Strikingly, mutations in a previously described repressor element that overlaps the TATA box restored promoter activity in TBP-het cells, supporting the idea that the sensitivity of this promoter to TBP levels reflects a competition between TBP and the repressor for DNA binding. To determine whether cells might have mechanisms to compensate for fluctuations in TBP levels, we next examined expression of the two known vertebrate TBP homologues, TLP and TBP2. Significantly, mRNAs encoding both were significantly overexpressed relative to levels observed in wild-type cells. In the case of TLP, this was shown to reflect regulation of the core promoter by both TBP and TLP. Together, our results indicate that variations in TBP levels can affect the transcription of specific promoters in distinct ways, but overall transcription may be buffered by corresponding alterations in the expression of TBP homologues.
Project description:TATA-binding protein (TBP) is an essential factor for eukaryotic transcription. In this study, we demonstrated a mouse cDNA encoding a 21 kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of mouse TBP with 39% identity and 76% similarity. Northern blot analysis demonstrated that TLP mRNAs were expressed in various mammalian tissues ubiquitously and that their distribution pattern was analogous to that of TBP. By using anti-TLP antibody, we demonstrated the existence of TLP proteins in various mammalian cells and tissues. The Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor that binds to the TATA-box and activates transcription. TLP did not bind to the TATA-box nor direct transcription initiation. Multiple amino acids critical for TBP function were deleted or substituted in TLP, while amino acids in Drosophila TRF much resembled those in TBP. Similarity between Drosophila TRF and mouse TLP was considerably lower (alignment score 35) than that between Drosophila TBP and mouse TBP (alignment score 88). Identity of nucleotide sequences between mouse and putative human TLPs (94%) was higher than that between TBPs (91%) in these two animals. Expression of TLP was nearly constant throughout the P19 differentiation process. Accordingly, we suggest that, even if higher eukaryotes generally contain multiple tbp -related genes, TLP is not a bona fide mammalian counterpart of Drosophila TRF.
Project description:TLP (TBP-like protein), which is a new protein dis-covered by us, has a structure similar to that of the C-terminal conserved domain (CCD) of TBP, although its function has not yet been elucidated. We isolated cDNA and genomic DNA that encode chicken TLP (cTLP) and determined their structures. The predicted amino acid sequence of cTLP was 98 and 91% identical to that of its mammalian and Xenopus counterparts, respectively, and its translation product was ubiquitously observed in chicken tissues. FISH detection showed that chicken tlp and tbp genes were mapped at 3q2.6-2.8 and 3q2.4-2.6 of the same chromosome, respectively. Genome analysis revealed that the chicken tlp gene was spliced with five introns. Interestingly, the vertebrate tbp genes were also found to be split by five introns when we focused on the CCDs, and their splicing points were similar to those of tlp. On the contrary, another TBP-resembling gene of Drosophila, trf1, is split by only one intron, as is the Drosophila 's tbp gene. These results support our earlier assumption that vertebrate TLPs did not directly descend from Drosophila TRF1. On the basis of these results together with phylogenetical exam-ination, we speculate that tlp diverged from an ancestral tbp gene through a process of gene duplication and point mutations.
Project description:The lack of direct targets for TATA-binding protein (TBP)-like factors (TLFs) confounds the understanding of their role in gene expression. Here we report that human TLF (also called TBP-related factor 2 [TRF2]) activates a number of different genes, including the neurofibromatosis type 1 (NF1) gene. The overexpression of TLF increases the amount of NF1 mRNA in cells. In vivo, TLF binds to and upregulates transcription from a fragment of the NF1 promoter. In vitro, purified TLF-TFIIA binds directly to the same NF1 promoter fragment that is required for TLF responsiveness in cells. Furthermore, targeted deletion of TLF in mice reduces NF1 levels. In contrast, TLF inhibits transcription driven by a fragment from the TATA-containing c-fos promoter by sequestering TFIIA. TBP affects the NF1 and c-fos promoters in a manner reciprocal to that of TLF, stimulating the c-fos promoter and inhibiting NF1 transcription. We conclude that TLF is a functional regulator of transcription with targets distinct from those of TBP.
Project description:Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors ?-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.
Project description:Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multiple physiological functions in cells and its bioavailability is negatively controlled through active-site binding to a specific thioredoxin-binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx and the effect of overexpression of TBP-2 in human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a sevenfold increase in TBP-2 and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day 7. Cell cycle analysis showed that TBP-2 OE cells arrested at the G2/M stage and that they displayed low expression of the cell cycle elements P-cdc2(Y15), cdc2, cdc25A, and cdc25C. Furthermore, TBP-2 OE cells were more sensitive to oxidation. Under H2O2 (200?M, 24h) treatment, these cells lost 80% viability and became highly apoptotic. Brief oxidative stress (200?M, 30min) to TBP-2 OE cells disrupted the Trx antiapoptotic function by dissociating the cytosolic and mitochondrial Trx-ASK binding complexes. The same H2O2-treated cells also showed activated ASK (P-ASK), increased Bax, lowered Bcl-2, cytochrome c release, and elevated caspase 3/7 activity. We conclude from these studies that high cellular levels of TBP-2 can potentially suppress Trx bioavailability and increase oxidation sensitivity. Overexpression of TBP-2 also causes slow growth by mitotic arrest and apoptosis by activating the ASK death pathway.
Project description:TATA-binding protein (TBP)-related factor 2 (TRF2) is one of four closely related RNA polymerase II transcription factors. We compared the intracellular localizations of TBP and TRF2 during the cell cycle and mitosis in HeLa cells. We show that during interphase, endogenous or exogenously expressed TRF2 is located almost exclusively in the nucleolus in HeLa or Cos cells. TRF2 localization is not affected by stress or mitotic stimuli, but TRF2 is rapidly released from the nucleolus upon inhibition of pol I transcription or treatment by RNase. These results suggest that localization of HeLa TRF2 requires a nucleolar-associated RNA species. In contrast, in 3T3 fibroblast cells, exogenously expressed TRF2 localizes to the nucleoplasm. Constitutive expression of ectopic TRF2 in 3T3 cells leads to a prolonged S phase of the cell cycle and reduced proliferation. Together with previous data, our results highlight the cell-specific localization and functions of TRF2. Furthermore, we show that during cell division, HeLa TRF2 and TBP are localized in the mitotic cytoplasm and TRF2 relocalizes into the nascent nucleoli immediately after mitosis, whereas TBP reassociates with the chromatin. Although partially contradictory results have been reported, our data are consistent with a model where only small proportion of the cellular TBP remains associated with specific promoter loci during mitosis.
Project description:Spaceflight is a special abiotic stress condition. In recent years, it has been confirmed that the spaceflight caused the stress response of rice seeds, and the protein level, transcription level, and methylation level will change during the planting process after returning to the ground. However, the changes at the metabolome level are not very clear. In this study, two kinds of rice seeds, Dongnong423 (DN3) and Dongnong416 (DN6), were carried on the ShiJian-10 retractable satellite (SJ-10) for 12.5 days in orbit, returned to the ground and planted in the field until the three-leaf (TLP) and tillering stage (TS). The results of antioxidant enzyme activity, soluble sugar, and electron leakage rate revealed that the spaceflight caused the stress response of rice. The TLP and TS of DN3 identified 110 and 57 different metabolites, respectively, while the TLP and TS of DN6 identified 104 and 74 different metabolites, respectively. These metabolites included amino acids, sugars, fatty acids, organic acids and secondary metabolites. We used qRT-PCR technology to explore the changes of enzyme genes in the tricarboxylic acid cycle (TCA) and amino acid metabolism pathway. Combined with the results of metabolomics, we determined that during the TLP, the TCA cycle rate of DN3 was inhibited and amino acid metabolism was activated, while the TCA cycle rate of DN6 was activated and amino acid metabolism was inhibited. In TS, the TCA cycle rate of DN3 was inhibited, and amino acid metabolism was not significantly changed, while the TCA cycle rate of DN6 was activated and amino acid metabolism was inhibited. These results suggested that the response mechanisms of the two different rice strains to spaceflight stress are different, and these differences may be reflected in energy consumption and compound biosynthesis of rice in different growth and development stages. This study provided new insights for further exploring the effects of spaceflight.
Project description:It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin ?2/?1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.
Project description:Background:In the recent past, many studies have been focused on extracts of BF and multiple biologically active factors and their effects on humoral immune system in chickens and birds. However, the mechanism of those immunomodulatory peptides on the B lineage cells proliferation and antibody production in chicken is fairly unknown. DT40 cell line, an avian leucosis virus-induced chicken pre-B cell line, expresses immunoglobulin M (IgM) isotype B cell reporter in the plasma membrane. There are many evidences suggesting that DT40 cells are best characterized as a bursal stem cell line. Because of the unique characteristics of DT40 cell line, it has been widely used to observe biological processes of pre-B lymphocyte cell within living cells. Methods:The chicken B cell line DT40 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and cytotoxicity was studied. Also, effect of BP5 on cell proliferation and cell cycle distribution of DT40 cells was studied. Also, the effect of BP5 on sIgM mRNA expression was studied by using real-time PCR. Objectives:To investigat the effects of Bursopentin (Cys-Lys-Arg-Val-Tyr, BP5) on a chicken promyelocyte cell line DT40, assays of cell proliferation, cell cycle distribution, detection of surface immunoglobulin G (sIgM) mRNA expression and gene microarray analysis were performed. Results:The results showed that BP5 displayed concentration-dependent effects on the proliferation, cell cycle, and sIgM mRNA expression in DT40 cells. And the analysis of expression profiles identified a signature set of 3022 genes (1254 up regulated genes, 1762 down regulated genes), which clearly discriminated the BP5-treated DT40 cells from control with high certainty (P?0.02). The results of microarray analysis were confirmed by quantitative reverse transcription-polymerase chain reaction for 12 of the differentially expressed genes. Conclusion:Theses findings showed the immuno-activity effect of BP5 on B lymphocyte and indicated that BP5 treatment regulated eight signaling pathways, in which Toll-like signaling pathway was the most significant enrichment pathway.