Team:Penn/MethylaseCharacterization
From 2013.igem.org
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<center><b>Bisulfite Sequencing Confirms TALE Binding Blocks Methylation | <center><b>Bisulfite Sequencing Confirms TALE Binding Blocks Methylation | ||
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- | <div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/23/TaleIndOn102513-1.png" alt="Workflow" width=" | + | <div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/23/TaleIndOn102513-1.png" alt="Workflow" width="500px"><figcaption><i>Figure 5: MaGellin standard bisulfite sequencing primers were used to bisulfite sequence 500 bp including the target site. Rows are individual clones, circles are CpG sites, and distances reflect their distance in bp. Filled in circles are methylated CpGs. </i></figcaption></figure></div> |
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The next step for characterizing functional constructs with MaGellin is to use the standard bisulfite sequencing primers (LINK) for a higher resolution look at individual CpG methylation. We bisulfite sequenced the target site of the induced TALE-M.SssI with the TALE binding site present 4 nucleotides upstream of the target AvaI cut site. The data further validated the enzymatic activity of our TALE-M.SssI in the region of the TALE binding site. It was also the first demonstration that the MaGellin assay and standard primers can easily be used for bisulfite sequencing with a TOPO cloning kit with ~7 day turnaround. Interestingly, no clones were methylated at the CpG site that is within the AvaI site itself (Figure 5). | The next step for characterizing functional constructs with MaGellin is to use the standard bisulfite sequencing primers (LINK) for a higher resolution look at individual CpG methylation. We bisulfite sequenced the target site of the induced TALE-M.SssI with the TALE binding site present 4 nucleotides upstream of the target AvaI cut site. The data further validated the enzymatic activity of our TALE-M.SssI in the region of the TALE binding site. It was also the first demonstration that the MaGellin assay and standard primers can easily be used for bisulfite sequencing with a TOPO cloning kit with ~7 day turnaround. Interestingly, no clones were methylated at the CpG site that is within the AvaI site itself (Figure 5). | ||
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This data, in combination with the varied induction conditions and COBRA results, led us to hypothesize a new model for the TALE’s mechanism that successfully explains each result. Although 4 nucleotides between the zinc finger binding site and the target cut site was optimal for a published zinc finger fusion with a short linker. That distance is too short for use with a TALE fusion with our linker length. The TALE is at least three times the size of the zinc finger and so TALE binding occludes that CpG from interacting with the methylase.</br></br> | This data, in combination with the varied induction conditions and COBRA results, led us to hypothesize a new model for the TALE’s mechanism that successfully explains each result. Although 4 nucleotides between the zinc finger binding site and the target cut site was optimal for a published zinc finger fusion with a short linker. That distance is too short for use with a TALE fusion with our linker length. The TALE is at least three times the size of the zinc finger and so TALE binding occludes that CpG from interacting with the methylase.</br></br> | ||
We have now shown that the novel TALE-M.SssI binds to its binding site strongly, as it almost fully protected that site from the methylase for 24 hours (Figure 4). We have shown that it exhibits methylating activity (Figure 2). Perhaps most interestingly, we have demonstrated that the TALE is large enough to physically occlude neighboring nucleotides from access to its linked effector, which has significant consequences for the recently published slew of TALE fusions – including the TALE-histone methylases, TALE-histone demethylases, and TALE-DNA demethylases for epigenetic engineering. We expect the same result will hold for Cas9-effector fusions, and are in the process of validating that hypothesis. We have already constructed the first dCas9-methylase fusion and demonstrated its enzymatic methylase activity in vivo (Figure 6). | We have now shown that the novel TALE-M.SssI binds to its binding site strongly, as it almost fully protected that site from the methylase for 24 hours (Figure 4). We have shown that it exhibits methylating activity (Figure 2). Perhaps most interestingly, we have demonstrated that the TALE is large enough to physically occlude neighboring nucleotides from access to its linked effector, which has significant consequences for the recently published slew of TALE fusions – including the TALE-histone methylases, TALE-histone demethylases, and TALE-DNA demethylases for epigenetic engineering. We expect the same result will hold for Cas9-effector fusions, and are in the process of validating that hypothesis. We have already constructed the first dCas9-methylase fusion and demonstrated its enzymatic methylase activity in vivo (Figure 6). | ||
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+ | </br><b><center>Novel dCas9-M.SssI Methylation Activity Reported by MaGellin</b></center> | ||
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Revision as of 12:04, 27 October 2013
Methylase Characterization
The software package calculated for us that the largest band we were seeing on the TALE gel was the result of simultaneous target site and off target site methylation while the second largest band was only off target site methylation. We used this information to formulate the Targeting Score to reflect increased site-specificity. We varied induction conditions, expecting one might be more optimal than our previous inductions. As desired, the negative control produced a baseline Targeting Score of almost exactly 1 (1.06). However, no induction condition increased Targeting Score, rather there was a steady decline (Figure 4). This indicated the TALE could be giving negative feedback to the site-specific methylation.