Difference between revisions of "Part:BBa K5317016"
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===Usage and Biology===
 
===Usage and Biology===
  
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ATF2 belongs to the ATF/CREB family and regulates genes involved in cell growth, stress responses and apoptosis. With the addition of the ATF2 gene, this plasmid enables the study of transcriptional regulation of ATF2 (Kirsch ''et al.'', 2020) and its phosphorylation by PknB, making it important for research into signaling pathways related to cell stress and survival. It could play also a role in modulating bacterial virulence by altering key regulatory proteins, affecting their role in infection or antibiotic resistance (Zhang'' et al.'', 2020).
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The cyclic AMP-dependent transcription factor ATF2 is a member of the basic leucine zipper domain (bZIP) DNA-binding protein family and is expressed by nearly all human cells (Miller ''et al.'', 2010). In these cells, ATF2 is phosphorylated by stress-activated protein kinase (SAPK) p38 and C-Jun N-terminal kinase (JNK) at amino acids Thr69 and Thr71 in response to a specific stimulus (Livingstone ''et al.'', 1995). This phosphorylation results in the formation of dimer structures that efficiently bind to the specific DNA consensus sequence 5′-TGACGTCA-3′ (Lin ''et al.'', 1988) in promoter regions of target genes. A short DNA fragment containing this consensus sequence element is called a cAMP response element (CRE) (Lin ''et al.'', 1988). This binding stimulates CRE-dependent transcription, thereby activating gene expression of genes involved in cell growth, stress responses, and apoptosis (Kawasaki ''et al.'', 2000; Miller ''et al.'', 2010).
  
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An interesting discovery by Miller and colleagues in 2010 shows that ATF2 is phosphorylated by the bacterial serine/threonine kinase PknB, specifically at Thr73.  This phosphorylation activates ATF2 and leads to transcription of genes.
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We used ATF2 as a transcription factor in our cell-based beta-lactam sensor, which in contrast to CcpA (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317014 K5317014]</span>) or GraR (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317015 K5317015]</span>) is derived from a eukaryotic background, to translate the PknB-detected signal into reporter gene expression.
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=Cloning=
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===Theoretical Part Design===
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The ATF2 gene was synthesized, and the gene sequence was explicity chose from its cDNA to exclude intons and shorten the gene sequence inserted into the final plasmid for characterization (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317021 K5317021]</span>).
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===Sequence and Features===
  
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K5317016 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5317016 SequenceAndFeatures</partinfo>
  
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===References===
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=Characterization=  
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The functionality of ATF2 in our cell-based sensor was assessed by analysing its general expression after transfection in HEK293T cells and assessing its localization by linking ATF2 with the reporter gene mRuby2 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317001 K5317001]</span>). For results please visit the registry entry <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317021 K5317021]</span>.
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=References=
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Kawasaki, H., Schiltz, L., Chiu, R., Itakura, K., Taira, K., Nakatani, Y., & Yokoyama, K. K. (2000). ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. 'Nature', 405(6783), 195–200. https://doi.org/10.1038/35012097
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Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. ''Nature Communications'', 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3
 
Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. ''Nature Communications'', 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3
  
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Lin, Y. S., & Green, M. R. (1988). Interaction of a common cellular transcription factor, ATF, with regulatory elements in both E1a- and cyclic AMP-inducible promoters. Proceedings of the National Academy of Sciences of the United States of America, 85(10), 3396–3400. https://doi.org/10.1073/pnas.85.10.3396
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Livingstone, C., Patel, G., & Jones, N. (1995). ATF-2 contains a phosphorylation-dependent transcriptional activation domain. The EMBO journal, 14(8), 1785–1797. https://doi.org/10.1002/j.1460-2075.1995.tb07167.x
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Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T. R., Diks, S. H., Dreisbach, A., Reilman, E., Gronau, K., Becher, D., Peppelenbosch, M. P., van Dijl, J. M., & Ohlsen, K. (2010). Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases. PloS one, 5(2), e9057. https://doi.org/10.1371/journal.pone.0009057
  
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Zhang, C., Zhang, R., Dai, X., Cao, X., Wang, K., Huang, X., & Ren, Q. (2020). Activating transcription factor 2 (ATF2) negatively regulates the expression of antimicrobial peptide genes through tumor necrosis factor (TNF) in Macrobrachium nipponense. ''Fish & Shellfish Immunology'', 107, 26–35. https://doi.org/10.1016/j.fsi.2020.09.043
 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 20:25, 1 October 2024


ATF2

Usage and Biology

The cyclic AMP-dependent transcription factor ATF2 is a member of the basic leucine zipper domain (bZIP) DNA-binding protein family and is expressed by nearly all human cells (Miller et al., 2010). In these cells, ATF2 is phosphorylated by stress-activated protein kinase (SAPK) p38 and C-Jun N-terminal kinase (JNK) at amino acids Thr69 and Thr71 in response to a specific stimulus (Livingstone et al., 1995). This phosphorylation results in the formation of dimer structures that efficiently bind to the specific DNA consensus sequence 5′-TGACGTCA-3′ (Lin et al., 1988) in promoter regions of target genes. A short DNA fragment containing this consensus sequence element is called a cAMP response element (CRE) (Lin et al., 1988). This binding stimulates CRE-dependent transcription, thereby activating gene expression of genes involved in cell growth, stress responses, and apoptosis (Kawasaki et al., 2000; Miller et al., 2010).

An interesting discovery by Miller and colleagues in 2010 shows that ATF2 is phosphorylated by the bacterial serine/threonine kinase PknB, specifically at Thr73. This phosphorylation activates ATF2 and leads to transcription of genes.

We used ATF2 as a transcription factor in our cell-based beta-lactam sensor, which in contrast to CcpA (K5317014) or GraR (K5317015) is derived from a eukaryotic background, to translate the PknB-detected signal into reporter gene expression.

Cloning

Theoretical Part Design

The ATF2 gene was synthesized, and the gene sequence was explicity chose from its cDNA to exclude intons and shorten the gene sequence inserted into the final plasmid for characterization (K5317021).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal XbaI site found at 324
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal XbaI site found at 324
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 21
    Illegal EcoRI site found at 283
    Illegal XbaI site found at 324
    Illegal PstI site found at 731
    Illegal PstI site found at 1180
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1031

Characterization

The functionality of ATF2 in our cell-based sensor was assessed by analysing its general expression after transfection in HEK293T cells and assessing its localization by linking ATF2 with the reporter gene mRuby2 (K5317001). For results please visit the registry entry K5317021.

References

Kawasaki, H., Schiltz, L., Chiu, R., Itakura, K., Taira, K., Nakatani, Y., & Yokoyama, K. K. (2000). ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. 'Nature', 405(6783), 195–200. https://doi.org/10.1038/35012097

Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. Nature Communications, 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3

Lin, Y. S., & Green, M. R. (1988). Interaction of a common cellular transcription factor, ATF, with regulatory elements in both E1a- and cyclic AMP-inducible promoters. Proceedings of the National Academy of Sciences of the United States of America, 85(10), 3396–3400. https://doi.org/10.1073/pnas.85.10.3396

Livingstone, C., Patel, G., & Jones, N. (1995). ATF-2 contains a phosphorylation-dependent transcriptional activation domain. The EMBO journal, 14(8), 1785–1797. https://doi.org/10.1002/j.1460-2075.1995.tb07167.x

Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T. R., Diks, S. H., Dreisbach, A., Reilman, E., Gronau, K., Becher, D., Peppelenbosch, M. P., van Dijl, J. M., & Ohlsen, K. (2010). Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases. PloS one, 5(2), e9057. https://doi.org/10.1371/journal.pone.0009057