Difference between revisions of "Part:BBa K1150024"

Revision as of 19:32, 29 September 2013

uniCAS Histone Modifier (CMV promoter)

pCMV:HA-NLS-dCas9-G9a-NLS:tBGH
Function	DNA binding protein fused to a methyl histone transferase
Use in	Mammalian cells
RFC standard	RFC 25
Backbone	pSB1C3
Organism	Streptococcus pyogenes, Mus musculus
Source	Feng Zhang, Addgene Albert Jeltsch, University of Stuttgart
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

This device is combining the dCas9 protein, that enables multiple gene targeting with the set-domain of the murine G9a. dCas9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 Lysin 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a Set-Domain to an open locus leads to a transcriptionally inactive state. [1] The usage of the strong CMV promoter enables this device to be expressed in a strong manner. If weaker expression levels are needed, we recommend using our uniCAS Histone Modificator device with an SV40 promoter ([[1]]).

Usage and Biology

H3K9 methylation is a hallmark of repressed transcriptional states. [2] The murine G9a-Set domain is able to transfer methyl groups to H3K9 when targeting it to the DNA and repress transcription. G9a is also known to be involved in downstream signalling, but by targeting it to a specific locus we reduce the functionality to its histone modification ability. [3] The dCAS9 protein is able to be targeted to several loci at once as it interacts with small RNAs to build up a complex that will interact with complementary DNA strands. Its origin is the adaptive immune system of Streptococcus pyogenes called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting. The team Freiburg 2013 combined these two elements to create a transcriptional repressor that is able to repress by a specific mechanism the targeted locus. This approach offers new possibilities for fundamental epigenetic research, tissue engineering and cancer research. [1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481.
[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166.
[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.

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 576
Illegal BglII site found at 900
Illegal BglII site found at 5375
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 31: / Line 31: @@
 |}
-This device is combining the dCAS9 protein, that enables multiple gene targeting with the set-domain of the murine G9a. dCAS9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 Lysin 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a Set-Domain to an open locus leads to a transcriptionally inactive state.
+This device is combining the dCas9 protein, that enables multiple gene targeting with the set-domain of the murine G9a. dCas9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 Lysin 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a Set-Domain to an open locus leads to a transcriptionally inactive state. [1]
 The usage of the strong CMV promoter enables this device to be expressed in a strong manner. If weaker expression levels are needed, we recommend using our uniCAS Histone Modificator device with an SV40 promoter ([[https://parts.igem.org/Part:BBa_K1150023]]).
 ===Usage and Biology===
-H3K9 methylation is a hallmark of repressed transcriptional states. The murine G9a-Set domain is able to transfer methyl groups to H3K9 when targeting it to the DNA (see Snowden et.al., 2003) and repress transcription. G9a is also known to be involved in downstream signalling, but by targeting it to a specific locus we reduce the functionality to its histone modification ability.
+H3K9 methylation is a hallmark of repressed transcriptional states. [2] The murine G9a-Set domain is able to transfer methyl groups to H3K9 when targeting it to the DNA and repress transcription. G9a is also known to be involved in downstream signalling, but by targeting it to a specific locus we reduce the functionality to its histone modification ability. [3]
 The dCAS9 protein is able to be targeted to several loci at once as it interacts with small RNAs to build up a complex that will interact with complementary DNA strands. Its origin is the adaptive immune system of <i> Streptococcus pyogenes </i> called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting.
 The team Freiburg 2013 combined these two elements to create a transcriptional repressor that is able to repress by a specific mechanism the targeted locus.
 This approach offers new possibilities for fundamental epigenetic research, tissue engineering and cancer research.
+[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>
+[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>