Difference between revisions of "Part:BBa K1150003"
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<div style="text-align:left;">
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This part contains the catalytically active set domain of G9a on the murine <i>EHMT2</i> gene. G9a is able to transfer methyl groups to lysine 9 of histone 3 (H3K9), which is part of every nucleosome. In regions of open chromatin structures H3K9 methylation can lead to chromatin condensation and thereby inactivation of the concerned locus. This G9a part alone contains the histone methyltransferase domain needed for transcriptional repression and can be combined with dCas9 [[Part:BBa_K1150000]] to target different loci for transcriptional repression of gene expression.
+
This BioBrick is part of the Freiburg 2013 uniCAS toolkit for gene regulation and contains the histone methyltransferase G9a encoded by the murine <i>EHMT2</i> gene. G9a is able to induce heterochromatization by transferring methyl groups to lysine 9 of histone 3 (H3K9)4. This part only contains the catalytically active SET domain of G9a that is responsible for histone modification. By fusing an HMTase domain to tha DNA binding dCas9 device, specific endogenous gene targeting is possible. In regions of open chromatin structures H3K9 methylation can therefore lead to inactivation of the concerned locus. In combination with a customized [https://parts.igem.org/Part:BBa_K1150034 RNAimer plasmid] different loci can be targeted for transcriptional repression of gene expression.
  
 
</div>
 
</div>
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===Usage and Biology===
 
===Usage and Biology===
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This part belongs to the uniCAS toolkit of iGEM Team Freiburg 2013. In fusion with dCas9 [[Part:BBa_K1150024]] this part can be used to effectively repress target genes by di- or tri-methylation of H3K9. We offer four devices using this part:
+
The murine <i>EHMT2 (Euchromatic histone-lysine N-methyltransferase 2)</i> gene encodes a mammalian lysine-preferring histone methyltransferase called G9a [1]. The complete G9a protein contains a SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain, which catalyzes transfer of methyl groups to H3K9 and an ANK domain which has a role in <i>de novo</i> DNA methylation [2]. The G9a BioBrick only comprises the G9a-SET domain. To provide the possibility of stable endogenous repression in our uniCAS toolkit we constructed the following four devices using the G9a BioBrick.
 
<ol>
 
<ol>
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<li> the uniCAS Histone Modifier (CMV promoter) ([[Part:BBa_K1150024]]) </li>  
+
<li> the [https://parts.igem.org/Part:BBa_K1150024 uniCAS Histone Modifier (CMV promoter)] </li>  
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<li> the uniCAS Histone Modifier (SV40 promoter) ([[Part:BBa_K1150023]]) uses a medium strong SV40 promoter to optimize this device for means were a strong expression is suboptimal. </li>
+
<li> the [https://parts.igem.org/Part:BBa_K1150024 uniCAS Histone Modifier (SV40 promoter)]  
−
<li> the uniCAS Red Light Switch Part II - Histone Modifier ([[Part:BBa_K1150028]]) is a result of combining G9a with part of our red light switch, Phytochrome B [[Part:BBa_K1150004]]. PhyB will dimerize with PIF6 upon red light stimulus. PIF6 is fused to dCas9 in the uniCAS Red Light Switch Part I - Stimulator ([[Part:BBa_K1150025]]) . Using these two devices G9a is brought into proximity of the DNA targeting dCas9 and can therefore site specifically transfer methyl groups and repress gene expression.
+
<!-- uses a medium strong SV40 promoter to optimize this device for means were a strong expression is suboptimal.--> </li>
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<li> the uniCAS UV Light Switch Part II - Histone Modifier ([[Part:BBa_K1150032]]) </li>
+
<li> the [https://parts.igem.org/Part:BBa_K1150028 uniCAS Red Light Switch Part II - Histone Modifier] is a result of combining G9a with part of our red light switch, [https://parts.igem.org/Part:BBa_K1150004 PhytochromeB]. PhyB will dimerize with [https://parts.igem.org/Part:BBa_K1150005 PIF6] upon red light stimulus. PIF6 is fused to dCas9 in the [https://parts.igem.org/Part:BBa_K1150025 uniCAS Red Light Switch Part I - Stimulator]. Using these two devices G9a is brought into proximity of the DNA targeting dCas9 and should therefore be able to site specifically transfer methyl groups to histone H3 and to repress gene expression.
 +
<li> the [https://parts.igem.org/Part:BBa_K1150032 uniCAS UV Light Switch Part II - Histone Modifier] </li>
  
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===Functional tests===
+
===Expression tests===
−
==Expression==
+
The expression of G9a fusion constructs was determined via Western blotting. Therefore mammalian HEK293T cells were transfected with the [https://parts.igem.org/Part:BBa_K1150024 uniCAS Histone Modifier (CMV promoter)] in 6-well plates (150,000 cells/well). 48h after transfection the cells were harvested and the complete cell lysate was blotted with anti-HA antibody against the N-terminally HA-tagged dCas9 proteins. The fullength dCas9-G9a protein could be detected at the expected size of approximately 185kDa.  
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To ensure protein expression of the fusion protein HEK-293T cells were seeded into 6-wells at a density of 150,000 cells per well. 24 hours later they were transfected with this device. <br> 2 days post transfection the protein expression was evaluated by Western blot analysis of total cell lysates. Figure 2 demonstrates successful expression of the dCas9-G9a fusion protein by detection of the HA-tag.  
+
  
−
[[File:UniBAssblotfertig2.1-team_Freiburgforregistry.PNG|800px|]]
+
[[File:UniBAssblotfertig2.1-team_Freiburgforregistry.PNG|700px|]]
  
 
'''Figure 2.:''' HA-tag fused to different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.
 
'''Figure 2.:''' HA-tag fused to different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.
  
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==Proof of Function==
+
===Expression tests===
−
Targeting of dCas9-G9a to an open, endogenous locus should lead to changes in the epigenetic state and repression. To test this, the endogenous <b>VEGF-a locus </b> was chosen, as it is well characterized and open in HEK-293T cells. Additionally, VEGF production can easily be measured by ELISA analysis. <br> Cells were seeded into 24-well format at a density of 65,000 cells per well and were co-transfected with the desired crRNAs, based on the [https://parts.igem.org/Part:BBa_K1150034 RNAimer]. These plasmids are derivates of the RNAimer, the only difference was the insertion of the target sites. These were: [https://parts.igem.org/Part:BBa_K1150036 -8], [https://parts.igem.org/Part:BBa_K1150037 -573], [https://parts.igem.org/Part:BBa_K1150038 +434], [https://parts.igem.org/Part:BBa_K1150039 -475] and [https://parts.igem.org/Part:BBa_K1150043 -573/+434]. As an off-target control crRNA targeting [https://parts.igem.org/Part:BBa_K1150035 EMX] was used. Every site was targeted with [https://parts.igem.org/Part:BBa_K1150017 CMV:dCas9] wthout G9a, to differentiate the action of G9a from the CRISPRi effect. As an <b> internal standard </b> a constitutive SEAP reporter was used. 24 hours post transfection, VEGF levels and SEAP levels were measured and the ratio was calculated. Error bars represent standard deviations.
+
Targeting of dCas9-G9a to an open, endogenous locus should lead to changes in the epigenetic chromatin state and repression of gene expression. To test this, the endogenous <b>VEGF-A locus </b> was chosen, as it is well characterized and open in HEK-293T cells. Additionally, VEGF production can easily be measured by ELISA analysis. <br> Cells were seeded into 24-well format at a density of 65,000 cells per well and were co-transfected with the desired crRNAs, based on the [https://parts.igem.org/Part:BBa_K1150034 RNAimer]. These plasmids are derivates of the RNAimer, the only difference was the insertion of the target sites. These were: [https://parts.igem.org/Part:BBa_K1150036 -8], [https://parts.igem.org/Part:BBa_K1150037 -573], [https://parts.igem.org/Part:BBa_K1150038 +434], [https://parts.igem.org/Part:BBa_K1150039 -475] and [https://parts.igem.org/Part:BBa_K1150043 -573/+434]. As an off-target control crRNA targeting [https://parts.igem.org/Part:BBa_K1150035 EMX] was used. Every site was targeted with [https://parts.igem.org/Part:BBa_K1150017 CMV:dCas9] wthout G9a, to differentiate the action of G9a from the CRISPRi effect. As an <b> internal standard </b> a constitutive SEAP reporter was used. 24 hours post transfection, VEGF levels and SEAP levels were measured and the ratio was calculated. Error bars represent standard deviations.
  
  
  
  
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[[File:Freiburg2013_G9a_test.jpg|800px|]] <br>
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[[File:Freiburg2013_G9a_test_neu.jpg|800px|]] <br>
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'''Figure 3.:''' dCas9-G9a targeting of endogenous VEGF-A locus in HEK-293T cells.<br>ELISA values, normalized to internal SEAP standard. For some loci clear repressive effects up to 50% were detectable. Controls did not display any off-target effects. Combining two target sites did not show any stronger repression.<br><br>
+
'''Figure 3.:''' dCas9-G9a targeting of endogenous VEGF-A locus in HEK-293T cells.<br>ELISA values, normalized to internal SEAP standard. For some loci clear repressive effects up to 50% were detectable. Controls did not display any off-target effects. Combining two target sites did not show stronger repression than targeting one site at a time.<br><br>
 
<br>
 
<br>
−
Different VEGF target sites led to different repression efficiencies and simultaneous targeting of two sites did not further enhance the observed effects.  <br> In summary, the CMV:dCas9-G9a is an effective repressor of endogenous gene expression, by modulating the chromatin structure of the targeted locus. This offers application in fundamental research as well as in medical science.
+
Different VEGF target sites led to different repression efficiencies and simultaneous targeting of two sites did not further enhance the observed effects.  <br> In summary, the CMV:dCas9-G9a is an effective repressor of endogenous gene expression, by modulating the chromatin structure of the targeted locus. This offers application in fundamental research as well as in medical sciences.
  
  
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===References===
 
===References===
 
<small>
 
<small>
 +
<!--
 
[1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481. <br>
 
[1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481. <br>
 
[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166. <br>
 
[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166. <br>
−
[3]  Lee, D., et al. (2006). Histone 3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors. Journal of Biological Chemistry 281, 8476-8485.  <br>
+
[3]  Lee, D., et al. (2006). Histone 3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors. Journal of Biological Chemistry 281, 8476-8485.  <br>-->
 +
[1] Tachibana, M., et al. (2001). SET Domain-containing Protein, G9a, Is a Novel Lysine-preferring Mammalian Histone Methyltransferase with Hyperactivity and Specific Selectivity to Lysines 9 and 27 of Histone H3. The Journal of Biological Chemistry, 276, 25309-25317.<br>
 +
[2] Epsztejn-Litman, S., et al. (2008). De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes. Nat Struct Mol Biol. 15(11): 1176–1183. <br>
 
</small>
 
</small>
  

Revision as of 01:27, 4 October 2013

G9a

G9a
Function Histone Methyltransferase
Use in Mammalian cells
RFC standard RFC 25
Backbone pSB1C3
Organism Mus musculus
Source Albert Jeltsch, Stuttgart
Submitted by [http://2013.igem.org/Team:Freiburg Freiburg 2013]

This BioBrick is part of the Freiburg 2013 uniCAS toolkit for gene regulation and contains the histone methyltransferase G9a encoded by the murine EHMT2 gene. G9a is able to induce heterochromatization by transferring methyl groups to lysine 9 of histone 3 (H3K9)4. This part only contains the catalytically active SET domain of G9a that is responsible for histone modification. By fusing an HMTase domain to tha DNA binding dCas9 device, specific endogenous gene targeting is possible. In regions of open chromatin structures H3K9 methylation can therefore lead to inactivation of the concerned locus. In combination with a customized RNAimer plasmid different loci can be targeted for transcriptional repression of gene expression.

Usage and Biology

The murine EHMT2 (Euchromatic histone-lysine N-methyltransferase 2) gene encodes a mammalian lysine-preferring histone methyltransferase called G9a [1]. The complete G9a protein contains a SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain, which catalyzes transfer of methyl groups to H3K9 and an ANK domain which has a role in de novo DNA methylation [2]. The G9a BioBrick only comprises the G9a-SET domain. To provide the possibility of stable endogenous repression in our uniCAS toolkit we constructed the following four devices using the G9a BioBrick.

  1. the uniCAS Histone Modifier (CMV promoter)
  2. the uniCAS Histone Modifier (SV40 promoter)
  3. the uniCAS Red Light Switch Part II - Histone Modifier is a result of combining G9a with part of our red light switch, PhytochromeB. PhyB will dimerize with PIF6 upon red light stimulus. PIF6 is fused to dCas9 in the uniCAS Red Light Switch Part I - Stimulator. Using these two devices G9a is brought into proximity of the DNA targeting dCas9 and should therefore be able to site specifically transfer methyl groups to histone H3 and to repress gene expression.
  4. the uniCAS UV Light Switch Part II - Histone Modifier

    5. Expression tests

    The expression of G9a fusion constructs was determined via Western blotting. Therefore mammalian HEK293T cells were transfected with the uniCAS Histone Modifier (CMV promoter) in 6-well plates (150,000 cells/well). 48h after transfection the cells were harvested and the complete cell lysate was blotted with anti-HA antibody against the N-terminally HA-tagged dCas9 proteins. The fullength dCas9-G9a protein could be detected at the expected size of approximately 185kDa.

    UniBAssblotfertig2.1-team Freiburgforregistry.PNG

    Figure 2.: HA-tag fused to different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.

    Expression tests

    Targeting of dCas9-G9a to an open, endogenous locus should lead to changes in the epigenetic chromatin state and repression of gene expression. To test this, the endogenous VEGF-A locus was chosen, as it is well characterized and open in HEK-293T cells. Additionally, VEGF production can easily be measured by ELISA analysis.
    Cells were seeded into 24-well format at a density of 65,000 cells per well and were co-transfected with the desired crRNAs, based on the RNAimer. These plasmids are derivates of the RNAimer, the only difference was the insertion of the target sites. These were: -8, -573, +434, -475 and -573/+434. As an off-target control crRNA targeting EMX was used. Every site was targeted with CMV:dCas9 wthout G9a, to differentiate the action of G9a from the CRISPRi effect. As an internal standard a constitutive SEAP reporter was used. 24 hours post transfection, VEGF levels and SEAP levels were measured and the ratio was calculated. Error bars represent standard deviations.



    Freiburg2013 G9a test neu.jpg
    Figure 3.: dCas9-G9a targeting of endogenous VEGF-A locus in HEK-293T cells.
    ELISA values, normalized to internal SEAP standard. For some loci clear repressive effects up to 50% were detectable. Controls did not display any off-target effects. Combining two target sites did not show stronger repression than targeting one site at a time.


    Different VEGF target sites led to different repression efficiencies and simultaneous targeting of two sites did not further enhance the observed effects.
    In summary, the CMV:dCas9-G9a is an effective repressor of endogenous gene expression, by modulating the chromatin structure of the targeted locus. This offers application in fundamental research as well as in medical sciences.


    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 597
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      COMPATIBLE WITH RFC[1000]


    References

    [1] Tachibana, M., et al. (2001). SET Domain-containing Protein, G9a, Is a Novel Lysine-preferring Mammalian Histone Methyltransferase with Hyperactivity and Specific Selectivity to Lysines 9 and 27 of Histone H3. The Journal of Biological Chemistry, 276, 25309-25317.
    [2] Epsztejn-Litman, S., et al. (2008). De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes. Nat Struct Mol Biol. 15(11): 1176–1183.