scholarly journals Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Mingen Lin ◽  
Chenghao Huang ◽  
Wenfeng Ren ◽  
Jun Chen ◽  
Ningshao Xia ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colony Formation ◽  
Rna Polymerase Iii ◽  
Tumor Development ◽  
5S Rrna ◽  
Trna Genes ◽  
Related Factor ◽  
Mechanism Analysis ◽  
Primary Mouse ◽  
Pol Iii

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

scholarly journals Exploring the Role and Mechanism of pAMPKα-Mediated Dysregulation of Brf1 and RNA Pol III Genes

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Teng Wu ◽  
Dongkun Zhang ◽  
Mingen Lin ◽  
Lihong Yu ◽  
Ting Dai ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Colony Formation ◽  
Rna Polymerase Iii ◽  
Tumor Development ◽  
Related Factor ◽  
Survival Times ◽  
Lung Cancer Patients ◽  
High Level ◽  
Pol Iii

TF IIB-related factor 1 (Brf1) is a key transcription factor of RNA polymerase III (Pol III) genes. Our early studies have demonstrated that Brf1 and Pol III genes are epigenetically modulated by histone H3 phosphorylation. Here, we have further investigated the relationship of the abnormal expression of Brf1 with a high level of phosphorylated AMPKα (pAMPKα) and explored the role and molecular mechanism of pAMPKα-mediated dysregulation of Brf1 and Pol III genes in lung cancer. Brf1 is significantly overexpressed in lung cancer cases. The cases with high Brf1 expression display short overall survival times. Elevation of Brf1 expression is accompanied by a high level of pAMPKα. Brf1 and pAMPKα colocalize in nuclei. Further analysis indicates that the carcinogen MNNG induces pAMPKα to upregulate Brf1 expression, resulting in the enhancement of Pol III transcription. In contrast, inhibiting pAMPKα decreases cellular levels of Brf1, resulting in the reduction of Pol III gene transcription to attenuate the rates of cell proliferation and colony formation of lung cancer cells. These outcomes demonstrate that high Brf1 expression reveals a worse prognosis in lung cancer patients. pAMPKα-mediated dysregulation of Brf1 and Pol III genes plays important roles in cell proliferation, colony formation, and tumor development of lung cancer. Brf1 may be a biomarker for establishing the prognosis of lung cancer. It is a new mechanism that pAMPKα mediates dysregulation of Brf1 and Pol III genes to promote lung cancer development.

scholarly journals Alcohol Intake and Abnormal Expression of Brf1 in Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Chenghao Huang ◽  
Yanmei Zhang ◽  
Shuping Zhong
Keyword(s):  
Breast Cancer ◽  
Alcohol Consumption ◽  
Molecular Mechanism ◽  
Cell Transformation ◽  
Rna Polymerase Iii ◽  
Tumor Development ◽  
Epidemiological Studies ◽  
Tumor Formation ◽  
Related Factor ◽  
Pol Iii

Breast cancer is the most common malignant disease of females. Overall, one woman in every nine will get breast cancer at some time in her life. Epidemiological studies have indicated that alcohol consumption has most consistently been associated with breast cancer risk. However, the mechanism of alcohol-associated breast cancer remains to be addressed. Little is known about the effects of alcohol consumption on Brf1 (TFIIIB-related factor 1) expression and RNA Pol III gene (RNA polymerase III-dependent gene) transcription, which are responsible for protein synthesis and tightly linked to cell proliferation, cell transformation, and tumor development. Emerging evidences have indicated that alcohol induces deregulation of Brf1 and Pol III genes to cause the alterations of cell phenotypes and tumor formation. In this paper, we summarize the progresses regarding alcohol-caused increase in the expression of Brf1 and Pol III genes and analysis of its molecular mechanism of breast cancer. As the earlier and accurate diagnosis approach of breast cancer is not available yet, exploring the molecular mechanism and identifying the biomarker of alcohol-associated breast cancer are especially important. Recent studies have demonstrated that Brf1 is overexpressed in most ER+ (estrogen receptor positive) cases of breast cancer and the change in cellular levels of Brf1 reflects the therapeutic efficacy and prognosis of this disease. It suggests that Brf1 may be a potential diagnosis biomarker and a therapeutic target of alcohol-associated breast cancer.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

Downregulation of Sulfiredoxin (Srx) Suppressed Cell Proliferation Migration and Invasion Through Inhibiting Extracellular Signal-Regulated Kinase/Nrf2 Pathway in Hepatocellular Carcinoma

2021 ◽  
Vol 11 (11) ◽  
pp. 2137-2145
Author(s):  
Xuejuan Zhu ◽  
Danqian Lu
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Colony Formation ◽  
Migration And Invasion ◽  
Related Factor ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Nrf2 Signaling Pathway

Background: Sulfiredoxin (Srx) has been identified to play important roles in the development of various cancers. However, the precise effects and underlying mechanism of Srx on the progression of HCC are far from being fully understood. Materials and Methods: The abundances of Srx in THLE-2 cell and HCC cell lines were determined by western blot and RT-qPCR. Next, SK-Hep-1 cells were transfected with shRNA-Srx or shRNA-NC and treated with TBHQ (an extracellular signal-regulated kinase (ERK) activator) for functional experiments. Then, CCK8 and colony formation assays were used to determine cell proliferation and clone-forming abilities in vitro. Cell migration and invasion were assessed via wound healing and transwell assays. The expression of MMP2, MMP9 and key members in ERK/nuclear factor E2 related factor (Nrf2) signaling pathway was detected by performing western blot analysis. Results: We reported evidence that Srx was frequently up-regulated in HCC cell lines. Srx interference constrained cell proliferation, colony formation rate, migration and invasion of SK-Hep-1 cells. Moreover, mechanistic investigations indicated that Srx interference significantly inhibited the activation of ERK/Nrf2 signaling pathway, and ERK activator TBHQ can reverse the functions of Srx interference in SK-Hep-1 cells. Conclusion: Overall, Downregulation of Srx might impede HCC progression by suppressing ERK/Nrf2 signaling pathway. Findings in the current study reported the functional involvement and molecular mechanism of Srx in HCC, suggesting that Srx might have a potential therapeutic value in HCC treatment.

scholarly journals Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome

2006 ◽  
Vol 396 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Akhila Parthasarthy ◽  
Karumathil P. Gopinathan
Keyword(s):  
Transcriptional Activation ◽  
Multigene Family ◽  
Rna Polymerase Iii ◽  
Upstream Region ◽  
Trna Genes ◽  
Naked Dna ◽  
Gene Copies ◽  
Direct Binding ◽  
Pol Iii ◽  
Mulberry Silkworm

All of the members of a tRNA1Gly multigene family from the mulberry silkworm, Bombyx mori, have identical coding regions and consequently identical internal promoter elements, but are transcribed at different levels. A moderately expressed copy, tRNA1Gly-4 from within this multigene family, which was transcribed to 30–50% of the highly transcribed gene copies harboured two typical TATAA box sequences in the 5′ upstream region at positions −27 nt and −154 nt with respect to the +1 nt of mature tRNA. Deletion of the distal TATAA sequence at −154 nt brought down the transcription more than 70%, whereas mutation of the proximal element did not affect transcription. tRNA1Gly-4 could be readily assembled into chromatin, with a positioned nucleosome in the upstream region, and the assembled nucleosome formed stable complexes with the transcription factors TFIIIC and TFIIIB. Organization of the gene into nucleosomes also enhanced transcription significantly above that of the naked DNA, reaching transcription levels comparable with those of the highly transcribed copies. This nucleosome-mediated enhancement in transcription was absent when the distal TATAA sequences were deleted, whereas mutation of the proximal TATAA element showed no effect. In the absence of the distal TATAA sequences, assembly into the nucleosome inhibited transcription of tRNA1Gly-4. TFIIIB bound directly through the distal TATAA sequence at −154 nt and the positioned nucleosome facilitated its interaction with TFIIIC. The direct binding of TFIIIB to the DNA provided anchoring of the factor to the template DNA which conferred a higher stability on the TFIIIB–TFIIIC–DNA complex. We have proposed a novel mechanism for the nucleosome-mediated stimulation of pol III (RNA polymerase III) transcription of tRNA genes, a model not presented previously.

Novel siRNA Expressing Lymphoma Model Identify Distinct Molecular Mechanisms Regulating NFkB1 and NFkB2 Induced Cell Proliferation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2372-2372
Author(s):  
Leon Bernal-Mizrachi ◽  
John Harding ◽  
Ratner Lee
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Dna Synthesis ◽  
Cell Line ◽  
Cell Lines ◽  
Thymidine Incorporation ◽  
Tumor Development ◽  
Nfkb Pathway ◽  
Pol Iii

Abstract The NFkB pathway has been implicated in tumorigenesis of several lymphoid malignancies. Most aggressive lymphomas have a constitutively active NFkB. Activation of NFkB results in processing of p105 and p100 to produce p50 and p52, respectively, and degradation of IkB. As a result, heterodimers of p65 and p50 or p52 and REL-B are formed and translocated to the nucleus where they induce transcription. Previously, it has been suggested that NFkB regulation of cyclin D and Myc expression is important for cell proliferation. However, these studies fail to explain all the NFkB regulatory effects, as well as the role of each individual pathway. To address these questions we tested the effect of NFkB on proliferation in two virally mediated murine lymphoma cell lines over-expressing Tax/HTLV-1 oncoprotein (SC and BLA) after knocking down (KD) p105 or p100, using a lentivirus expressing siRNAs. After synchronization, cells were released for 24 hrs and the rate of proliferation and cell cycle analysis were measured by thymidine incorporation and PI staining (4,8,16,24 hrs), respectively. As it is seen below, KD p100 or p105 cell lines had a reduced rate of thymidine incorporation compare to controls (Luciferase-siRNA). However, minimal changes were observed between cell lines in cell cycle analysis. To confirm these findings, xenograft experiments with siRNA expressing cell lines were developed. These experiments demonstrated that KD p100 or p105 in xenografts, prevented (in some) or delayed tumor formation, reduced tumor size and prolonged disease free survival. We then investigated NFkB regulation of cell proliferation by assessing gene expression in each cell line. Our results revealed a distinct proliferation-related gene profile between pathways: KD p105 dependent genes include: Cell cycle: Cyclin D2, B2 and G, CDK-8, P27, RB6, Transcription factor Dp2 and AKT-substrate-1, Growth factors: Platelet derived growth factor and RhoG, RNA synthesis: RNA Pol III. On the other hand KD p100 dependent genes include: Cell cycle: Cyclin D1,B2, retinoblastoma-like 1 (p107), p16, ring box-1 and histone deacetylase 2, Growth factors: HRAS and RhoG, RNA/DNA synthesis: RNA pol III and DNA pol (p17), histone 1 and 2, and thymidine kinases. Interestingly, both KD cell lines share many over-expressed genes such as TGFB, p21, p53, RB7 and BRCA1. In conclusion, our novel experimental model demonstrates: the main effect of both NFkB pathways involves DNA replication and early S phase. KD of either p100 or p105 reduces tumor development. The mechanism of cell proliferation regulated by each NFkB pathway is more complex than what was previously suspected; we suggest that RNA, DNA synthesis and regulation of p21, BRCA-1 and other cell cycle regulatory proteins also play an important role. Effect of Knocking Down p100 or p105 in Tumor Development Luciferase siRNA P100 siRNA P105 siRNA NR=Non reached. *Total 58 days f/u. Ten animals per cell line. H3 Thymidine SC 1 ± 0.09 0.38 ± 0.16 0.68 ± 0.37 H3 Thymidine BLA 1 ± 0.09 0.53 ± 0.07 0.69 ± 0.21 Tumor weight (mg) 617.18 ± 251 257.1± 285.1 258.6 ± 195.7 Tumor initiation (day) 32 ± 11 51 ± 11 43 ± 10 Tumor (+) animals (%) 100% 45% 78% Median DFS (day)* 30 NR 37

BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei

Parasitology ◽  
2015 ◽  
Vol 142 (13) ◽  
pp. 1563-1573 ◽  
Author(s):  
D. E. VÉLEZ-RAMÍREZ ◽  
L. E. FLORENCIO-MARTÍNEZ ◽  
G. ROMERO-MEZA ◽  
S. ROJAS-SÁNCHEZ ◽  
R. MORENO-CAMPOS ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Homology Modelling ◽  
Rna Polymerase Iii ◽  
Related Factor ◽  
Characteristic Structure ◽  
Rna Molecules ◽  
Polymerase Iii ◽  
Pol Iii

SUMMARYRNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

scholarly journals Inhibition of tRNA Gene Transcription by the Immunosuppressant Mycophenolic Acid

2019 ◽  
Vol 40 (1) ◽  
Author(s):  
Aneta Jurkiewicz ◽  
Ewa Leśniewska ◽  
Małgorzata Cieśla ◽  
Neuton Gorjão ◽  
Theodoros Kantidakis ◽  
...  
Keyword(s):  
Mycophenolic Acid ◽  
Mammalian Cells ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Sharp Decrease ◽  
Trna Genes ◽  
Anticancer Chemotherapy ◽  
Transcriptional Inhibition ◽  
Cycle Arrest ◽  
Pol Iii

ABSTRACT Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling.

scholarly journals Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes.

1994 ◽  
Vol 14 (7) ◽  
pp. 4704-4711 ◽  
Author(s):  
V J Wolf ◽  
T Dang ◽  
P Hartl ◽  
J M Gottesfeld
Keyword(s):  
Rna Polymerase Iii ◽  
Cyclin B ◽  
Trna Genes ◽  
5S Rna ◽  
Transcription System ◽  
Transcription Complexes ◽  
Pol Iii ◽  
Differential Transcription ◽  
5S Rna Genes ◽  
Rna Genes

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.

scholarly journals A Gain-of-Function Mutation in the Second Tetratricopeptide Repeat of TFIIIC131 Relieves Autoinhibition of Brf1 Binding

2002 ◽  
Vol 22 (17) ◽  
pp. 6131-6141 ◽  
Author(s):  
Robyn D. Moir ◽  
Karen V. Puglia ◽  
Ian M. Willis
Keyword(s):  
Binding Affinity ◽  
Rna Polymerase Iii ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Tetratricopeptide Repeat ◽  
Related Factor ◽  
Back Side ◽  
Binding Studies ◽  
Gain Of Function ◽  
Pol Iii

ABSTRACT The interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC, TFIIIC131, and the TFIIB-related factor Brf1 represents a limiting step in the assembly of the RNA polymerase III (pol III) initiation factor TFIIIB. This assembly reaction is facilitated by dominant mutations that map in and around TPR2. Structural modeling of TPR1 to TPR3 from TFIIIC131 shows that one such mutation, PCF1-2, alters a residue in the ligand-binding groove of the TPR superhelix whereas another mutation, PCF1-1, changes a surface-accessible residue on the back side of the TPR superhelix. In this work, we show that the PCF1-1 mutation (H190Y) increases the binding affinity for Brf1, but does not affect the binding affinity for Bdp1, in the TFIIIC-dependent assembly of TFIIIB. Interestingly, binding studies with TFIIIC131 fragments indicate that Brf1 does not interact directly at the site of the PCF1-1 mutation. Rather, the data suggest that the mutation overcomes the previously documented autoinhibition of Brf1 binding. These findings together with the results from site-directed mutagenesis support the hypothesis that gain-of-function mutations at amino acid 190 in TPR2 stabilize an alternative conformation of TFIIIC131 that promotes its interaction with Brf1.

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