ROS Signaling-Mediated Novel Biological Targets: Brf1 and RNA Pol III Genes

Biomolecule metabolism produces ROS (reactive oxygen species) under physiological and pathophysiological conditions. Dietary factors (alcohol) and carcinogens (EGF, DEN, and MNNG) also induce the release of ROS. ROS often causes cell stress and tissue injury, eventually resulting in disorders or diseases of the body through different signaling pathways. Normal metabolism of protein is critically important to maintain cellular function and body health. Brf1 (transcript factor II B-related factor 1) and its target genes, RNA Pol III genes (RNA polymerase III-dependent genes), control the process of protein synthesis. Studies have demonstrated that the deregulation of Brf1 and its target genes is tightly linked to cell proliferation, cell transformation, tumor development, and human cancers, while alcohol, EGF, DEN, and MNNG are able to induce the deregulation of these genes through different signaling pathways. Therefore, it is very important to emphasize the roles of these signaling events mediating the processes of Brf1 and RNA Pol III gene transcription. In the present paper, we mainly summarize our studies on signaling events which mediate the deregulation of these genes in the past dozen years. These studies indicate that Brf1 and RNA Pol III genes are novel biological targets of ROS.

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

Parasitology ◽

10.1017/s0031182015001122 ◽

2015 ◽

Vol 142 (13) ◽

pp. 1563-1573 ◽

Cited By ~ 6

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.22.17.6131-6141.2002 ◽

2002 ◽

Vol 22 (17) ◽

pp. 6131-6141 ◽

Cited By ~ 10

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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Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major

Genes ◽

10.3390/genes12020280 ◽

2021 ◽

Vol 12 (2) ◽

pp. 280

Author(s):

Luis E. Florencio-Martínez ◽

Andrés Cano-Santiago ◽

Fabiola Mondragón-Rosas ◽

Maricarmen Gómez-García ◽

Carlos Flores-Pérez ◽

...

Keyword(s):

Leishmania Major ◽

Affinity Purification ◽

Rna Polymerase Iii ◽

Rrna Genes ◽

Related Factor ◽

Messenger Rnas ◽

Small Nuclear Rna ◽

Rna Molecules ◽

18S Rrna Genes ◽

Pol Iii

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

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Intracellular Communication among Morphogen Signaling Pathways during Vertebrate Body Plan Formation

Genes ◽

10.3390/genes11030341 ◽

2020 ◽

Vol 11 (3) ◽

pp. 341

Author(s):

Kimiko Takebayashi-Suzuki ◽

Atsushi Suzuki

Keyword(s):

Growth Factor ◽

Signaling Pathways ◽

Cell Fate ◽

Target Genes ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Body Plan ◽

The Body ◽

Fine Tuning ◽

Axis Formation

During embryonic development in vertebrates, morphogens play an important role in cell fate determination and morphogenesis. Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor-β (TGF-β) family control the dorsal–ventral (DV) patterning of embryos, whereas other morphogens such as fibroblast growth factor (FGF), Wnt family members, and retinoic acid (RA) regulate the formation of the anterior–posterior (AP) axis. Activation of morphogen signaling results in changes in the expression of target genes including transcription factors that direct cell fate along the body axes. To ensure the correct establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated by a fine-tuning of morphogen signaling. In this review, we focus on the interplay of various intracellular regulatory mechanisms and discuss how communication among morphogen signaling pathways modulates body axis formation in vertebrate embryos.

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

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/5554932 ◽

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.

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Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.25.21.9406-9418.2005 ◽

2005 ◽

Vol 25 (21) ◽

pp. 9406-9418 ◽

Cited By ~ 17

Author(s):

Ashish Saxena ◽

Beicong Ma ◽

Laura Schramm ◽

Nouria Hernandez

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Function Analysis ◽

Rna Polymerase Iii ◽

Related Factor ◽

Pol Ii ◽

Terminal Domain ◽

U6 Promoter ◽

Pol Iii ◽

Type 3

ABSTRACT The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAPc, a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

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Comparative analysis of T-cell costimulation and CD43 activation reveals novel signaling pathways and target genes

Blood ◽

10.1182/blood-2004-04-1536 ◽

2004 ◽

Vol 104 (10) ◽

pp. 3302-3304 ◽

Cited By ~ 21

Author(s):

Ivan Mattioli ◽

Oliver Dittrich-Breiholz ◽

Mark Livingstone ◽

Michael Kracht ◽

M. Lienhard Schmitz

Keyword(s):

T Cell ◽

Signaling Pathways ◽

Target Genes ◽

Phosphorylation Site ◽

Cell Receptor ◽

Nuclear Factor Κb ◽

Lymphocyte Adhesion ◽

Peripheral T Cells ◽

Signaling Events ◽

Surface Receptor

Abstract The CD43 lymphocyte surface receptor is involved in the regulation of lymphocyte adhesion and activation. Many CD43 functions remain controversial or unclear, and it is not known to which extent CD43 signaling pathways are shared with or distinct from those used by the T-cell receptor (TCR). Here, we systematically compared signaling events and target gene expression induced by CD43 or T-cell costimulation in primary human peripheral T cells. These studies identify nuclear factor-κB (NF-κB) p65 serine 468 as a novel inducible phosphorylation site strongly induced by T-cell costimulation and only weakly triggered by CD43 ligation. We also identified CD43 as a novel Jun N-terminal kinase (JNK) activator and a comprehensive analysis of further signaling events suggests that both stimuli use overlapping but also distinct signaling pathways. Microarray analysis of inflammatory genes shows 1 group of genes coregulated by both stimuli and 2 further groups of target genes affected solely by costimulation or primarily by CD43. (Blood. 2004;104:3302-3304)

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

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/2067959 ◽

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.

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Alcohol Intake and Abnormal Expression of Brf1 in Breast Cancer

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/4818106 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 2

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.

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Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922821117 ◽

2020 ◽

Vol 117 (27) ◽

pp. 15702-15711

Author(s):

Xiaoling Wang ◽

Alan Gerber ◽

Wei-Yi Chen ◽

Robert G. Roeder

Keyword(s):

Rna Polymerase ◽

Knockout Mice ◽

Mammalian Cells ◽

Target Genes ◽

Rna Polymerase Iii ◽

Embryonic Stem ◽

Mouse Development ◽

Polymerase Iii ◽

Pol Iii

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIβ). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

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