scholarly journals Bigenomic transcriptional regulation of all thirteen cytochrome c oxidase subunit genes by specificity protein 1

Open Biology ◽  
2013 ◽  
Vol 3 (3) ◽  
pp. 120176 ◽  
Author(s):  
Shilpa S. Dhar ◽  
Kaid Johar ◽  
Margaret T. T. Wong-Riley
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Mobility Shift ◽  
Mitochondrial Transcription ◽  
Over Expression ◽  
Specificity Protein 1 ◽  
Nuclear Respiratory Factors ◽  
Specificity Protein

Cytochrome c oxidase (COX) is one of only four known bigenomic proteins, with three mitochondria-encoded subunits and 10 nucleus-encoded ones derived from nine different chromosomes. The mechanism of regulating this multi-subunit, bigenomic enzyme is not fully understood. We hypothesize that specificity protein 1 (Sp1) functionally regulates the 10 nucleus-encoded COX subunit genes directly and the three mitochondrial COX subunit genes indirectly by regulating mitochondrial transcription factors A and B ( TFAM , TFB1M and TFB2M ) in neurons. By means of in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, RNA interference and over-expression experiments, the present study documents that Sp1 is a critical regulator of all 13 COX subunit genes in neurons. This regulation is intimately associated with neuronal activity. Silencing of Sp1 prevented the upregulation of all COX subunits by KCl, and over-expressing Sp1 rescued all COX subunits from being downregulated by tetrodotoxin. Thus, Sp1 and our previously described nuclear respiratory factors 1 and 2 are the three key regulators of all 13 COX subunit genes in neurons. The binding sites for Sp1 on all 10 nucleus-encoded COX subunits, TFAM , TFB1M and TFB2M are highly conserved among mice, rats and humans.

NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene

2008 ◽  
Vol 86 (1) ◽  
pp. 46-56 ◽  
Author(s):  
José R. Blesa ◽  
Jesús A. Prieto-Ruiz ◽  
Beth A. Abraham ◽  
Bridget L. Harrison ◽  
Anita A. Hegde ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Colorectal Tumors ◽  
Methylation Analysis ◽  
Mobility Shift ◽  
Nuclear Respiratory Factors ◽  
Hela S3 ◽  
Electrophoretic Mobility Shift Assays

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear–mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5′ region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

scholarly journals Differential Cell-Specific Modulation of HOXA10 by Estrogen and Specificity Protein 1 Response Elements

2007 ◽  
Vol 92 (5) ◽  
pp. 1920-1926 ◽  
Author(s):  
Ryan Martin ◽  
Melissa B. Taylor ◽  
Graciela Krikun ◽  
Charles Lockwood ◽  
G. Edda Akbas ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Tissue Specificity ◽  
Academic Medical Center ◽  
Cell Type ◽  
Mobility Shift ◽  
Response Elements ◽  
Specificity Protein 1 ◽  
Cellular Expression ◽  
Cell Type Specific ◽  
Specificity Protein

Abstract Context: HOX genes are highly evolutionarily conserved regulators of embryonic development. HOXA10 also regulates differentiation of the adult reproductive tract and mammary gland in response to sex steroids. Objective: We recently identified two HOXA10 estrogen response elements (EREs). Here we demonstrate that estrogen-responsive HOXA10 expression is cell type specific. Design and Setting: We conducted an in vitro study at an academic medical center. Main Outcome Measure: Reporter assay, gel shift assays (electrophoretic mobility shift assay), and immunohistochemistry were done. Results: The HOXA10 EREs and a specificity protein 1 (Sp1) binding site differentially drive the cell-type-specific E2 response. In electrophoretic mobility shift assays, both estrogen receptor-α and -β bound both EREs but not the Sp1 site. In reporter assays, both EREs and the Sp1 site demonstrated estrogen responsiveness and tissue specificity; transiently transfected uterine Ishikawa cells or breast MCF-7 cells showed differential responses to E2 treatment. Each response element (Sp1, ERE1, and ERE2) drove distinct differential expression in each cell type. Sp1 protein was expressed in a menstrual-cycle stage-specific expression pattern in endometrium, first expressed in perivascular cells. Conclusions: Tissue specificity inherent to a regulatory element as well as differential cellular expression of transcription factors imparts differential tissue-specific estrogen responsiveness.

C-MYB and C/EBP Family Transcription Factors Regulate the Gfi-1 Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4214-4214
Author(s):  
Richard Dahl ◽  
Kristin S. Owens
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Myeloid Cells ◽  
Transient Transfection ◽  
Mobility Shift ◽  
Reporter Assays ◽  
Immature Myeloid Cells ◽  
Factor C

Abstract Gfi-1 −/− mice generate abnormal immature myeloid cells exhibiting characteristics of both monocytes and granulocytes. One of Gfi-1’s critical functions is to downregulate monocyte specific genes in order for granulocytes to develop properly. Since the transcription factors C/EBP alpha and C/EBP epsilon are needed for granulocyte development we hypothesized that these factors may regulate Gfi-1 expression. The Gfi-1 promoter contains several putative C/EBP binding sites and we show by electrophoretic mobility shift and chromatin immunoprecipitation that C/EBP family members can bind to some of these sites. However we were unable to see activation of the Gfi-1 promoter by C/EBP proteins in transient transfection reporter assays. Other groups have shown that C/EBP proteins can synergize with the transcription factor c-myb. We observed that the Gfi-1 promoter contains sites for the hematopoietic transcription factor c-myb. Sevral of these c-myb binding sites are adjacent to C/EBP binding sites. In reporter assays in non-hematopoietic cells c-myb activated the Gfi-1 promoter by itself and this activity was enhanced when we included either C/EBP alpha or epsilon in the transfection. Our data suggests that C/EBP proteins and c-myb regulate the transcription of Gfi-1 in myeloid cells.

scholarly journals NF-κB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-κB site in the ICAM-1 gene

2009 ◽  
Vol 38 (1) ◽  
pp. 42-53 ◽  
Author(s):  
Jiaping Xue ◽  
Prabhakar B. Thippegowda ◽  
Guochang Hu ◽  
Kurt Bachmaier ◽  
John W. Christman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Leukocyte Adhesion ◽  
Reporter Expression ◽  
Mobility Shift ◽  
Intron 1 ◽  
Tnf Α ◽  
Protease Activated Receptor 1

Activation of NF-κB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-κB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-κB binding sites in intron-1 (+70, NF-κB site 1; +611, NF-κB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-κB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-κB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (−1,385 to +234) construct ∼25-fold and mutation of intronic NF-κB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-α-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-κB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

scholarly journals The binding sites of Zn^ and Cd^ in bovine heart cytochrome c oxidase.

2001 ◽  
Vol 41 (supplement) ◽  
pp. S115
Author(s):  
K. Hirata ◽  
E. Yamashita ◽  
T. Tsukihara ◽  
K. Muramoto ◽  
S. Aonami ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Binding Sites ◽  
Bovine Heart

scholarly journals AP-2αInhibits c-MYC Induced Oxidative Stress and Apoptosis in HaCaT Human Keratinocytes

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Lei Yu ◽  
Michael J. Hitchler ◽  
Wenqing Sun ◽  
Ehab H. Sarsour ◽  
Prabhat C. Goswami ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Regulatory Region ◽  
Human Keratinocytes ◽  
Epidermal Keratinocytes ◽  
Over Expression ◽  
Cellular Processes ◽  
Ros Accumulation ◽  
Proliferation And Apoptosis ◽  
Steady State Levels

AP-2αand c-MYC are important transcription factors involved in multiple cellular processes. They each display the paradoxical capacities to stimulate both cell proliferation and apoptosis under different conditions. In the present study we found that over expression of c-MYC was associated with accumulation of reactive oxygen species (ROS) and apoptosis in human keratinocytes, both of which were significantly inhibited by co-expression of AP-2. The effects of AP-2 on c-MYC were active at several levels. First, AP-2 and c-MYC were confirmed to interact at the protein level as previously described. In addition, forced expression of AP-2 significantly decreased steady state levels of c-MYC mRNA and protein. These findings suggested that AP-2 may have a direct effect on thec-mycgene. Chromatin immunoprecipitation assays demonstrated that AP-2 proteins bound to a cluster of AP-2 binding sites located within a 2 kb upstream regulatory region ofc-mycThese results suggest that the negative regulation of AP-2 on c-MYC activity was achieved through binding of AP-2 protein to thec-mycgene. The effects of AP-2 on c-MYC induced ROS accumulation and apoptosis in epidermal keratinocytes are likely to play an important role in cell growth, differentiation and carcinogenesis of the skin.

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