This paper looks at selective inhibition of GR activity via a DNA-binding molecule called a polyamide that competes with and interrupts GR binding at consensus GREs. They confirm the function of the polyamide using GILZ (a well-characterized GR responsive gene with at least 2 known GREs) and further use this molecule to identify genes that physically interact with GR. Although they don't address any non-genomic affects of the polyamide on GR target genes they do control for specificity with a "mismatch" polyamide.
I think the most obvious goal of this type of regulation is to design drugs that selectively target GR regulated genes. This paper identifies a unique level of regulation different from antagonistic ligands. It would be nice to see more selective/specific polyamides screened, but at the least this paper opens the door for a new level of GR transcriptional regulation and hints at genes where GR seems to directly interact with a GRE to regulate expression.
I just found out this blogger has already been set up because the messages were sent to my junk mail folder without being noticed for some days till now. Nice job, Marcus!
In this paper, using a small programmable molecule, the researchers were able to distinguish the direct DNA-binding dependent activity of GR from the independent one.Since it is programmable, specific designs could be achieved for testing other nuclear receptors/transcription factors in a similar way.
Interestingly, the results showed that the mismatch control molecule did not affect the target gene expression even though it impacted the GR binding to a significant extent in ChIP assay. Such a phenomenon might impair the interpretation regarding those genes that were affected by both polyamide 1 and 2. In addition, assuming the overall activities of GR is coordinated accurately, I wonder whether interfering the DNA-binding dependent activity would affect the DNA-binding independent activity indirectly so that a readout might actually reflect such an indirect effect. Does anyone have an idea about this?
I do think this was an interesting paper and never knew that there were synthetic DNA binding compounds. Pretty cool. I do think this work opens the door for future research that may yield therapeutics to specifically target certain aspects of GR function, but it is going to take a lot of work and more controls.
What is lacking is in this type of research is a comprehensive read-out for indirect GR-binding. For the genes that displayed no sensitivity to polyamide treatment, there is no certainty that GR is not binding to those genes. GR could be binding to a site not inhibited by the compounds. Thus, genes regulated through indirect GR binding can only be determined through the process of elimination once genes that bind GR directly have been identified. Therefore, I agree with Marcus that it will be useful to develop more polyamide compounds, perhaps based on GR ChiP-CHIP data, which I am sure is available, in order to maximize inhibition of GR binding and characterize gene sets regulated by particular polyamides.
Chaowei, I do agree that the read out may reflect indirect effects, perhaps even independent of GR. For example, the authors never performed a control or addressed whether the polyamides overlap with other transcription factor binding sites. Since the W's can be As or Ts, that yields many different sequences that can be recognized by the polyamide.
Hey guys, this post is late, but I've been swamped with class work lately.
Definitely a fun read, and I believe this is the first time I've heard about a synthetic compound designed to bind a specific DNA sequence. I agree with what's been said already regarding specificity issues of the polyamide. As far as I understand this, the polyamide spans 4 common base-pairs within each GRE of the GILZ promotor. Could it be made more specific by simply making it longer? Perhaps two separate polyamides could be designed to span the entire GRE 1 and GRE 2 respectively, and then be administered simultaneously. Under these conditions, one could then do real-time RT-PCR as in Figure 5, and verify whether simultaneous administration of the two polyamides would have a greater inhibitory effect on dex-induced GILZ expression.
I don't know too much about what goes into making these polyamides. Do you think it would be easy to synthesize a longer fragment, and would this help with specificity issues?
This paper looks at selective inhibition of GR activity via a DNA-binding molecule called a polyamide that competes with and interrupts GR binding at consensus GREs. They confirm the function of the polyamide using GILZ (a well-characterized GR responsive gene with at least 2 known GREs) and further use this molecule to identify genes that physically interact with GR. Although they don't address any non-genomic affects of the polyamide on GR target genes they do control for specificity with a "mismatch" polyamide.
ReplyDeleteI think the most obvious goal of this type of regulation is to design drugs that selectively target GR regulated genes. This paper identifies a unique level of regulation different from antagonistic ligands. It would be nice to see more selective/specific polyamides screened, but at the least this paper opens the door for a new level of GR transcriptional regulation and hints at genes where GR seems to directly interact with a GRE to regulate expression.
I just found out this blogger has already been set up because the messages were sent to my junk mail folder without being noticed for some days till now. Nice job, Marcus!
ReplyDeleteIn this paper, using a small programmable molecule, the researchers were able to distinguish the direct DNA-binding dependent activity of GR from the independent one.Since it is programmable, specific designs could be achieved for testing other nuclear receptors/transcription factors in a similar way.
Interestingly, the results showed that the mismatch control molecule did not affect the target gene expression even though it impacted the GR binding to a significant extent in ChIP assay. Such a phenomenon might impair the interpretation regarding those genes that were affected by both polyamide 1 and 2. In addition, assuming the overall activities of GR is coordinated accurately, I wonder whether interfering the DNA-binding dependent activity would affect the DNA-binding independent activity indirectly so that a readout might actually reflect such an indirect effect. Does anyone have an idea about this?
I do think this was an interesting paper and never knew that there were synthetic DNA binding compounds. Pretty cool. I do think this work opens the door for future research that may yield therapeutics to specifically target certain aspects of GR function, but it is going to take a lot of work and more controls.
ReplyDeleteWhat is lacking is in this type of research is a comprehensive read-out for indirect GR-binding. For the genes that displayed no sensitivity to polyamide treatment, there is no certainty that GR is not binding to those genes. GR could be binding to a site not inhibited by the compounds. Thus, genes regulated through indirect GR binding can only be determined through the process of elimination once genes that bind GR directly have been identified. Therefore, I agree with Marcus that it will be useful to develop more polyamide compounds, perhaps based on GR ChiP-CHIP data, which I am sure is available, in order to maximize inhibition of GR binding and characterize gene sets regulated by particular polyamides.
Chaowei, I do agree that the read out may reflect indirect effects, perhaps even independent of GR. For example, the authors never performed a control or addressed whether the polyamides overlap with other transcription factor binding sites. Since the W's can be As or Ts, that yields many different sequences that can be recognized by the polyamide.
Thanks Marcus!
Hey guys, this post is late, but I've been swamped with class work lately.
ReplyDeleteDefinitely a fun read, and I believe this is the first time I've heard about a synthetic compound designed to bind a specific DNA sequence. I agree with what's been said already regarding specificity issues of the polyamide. As far as I understand this, the polyamide spans 4 common base-pairs within each GRE of the GILZ promotor. Could it be made more specific by simply making it longer? Perhaps two separate polyamides could be designed to span the entire GRE 1 and GRE 2 respectively, and then be administered simultaneously. Under these conditions, one could then do real-time RT-PCR as in Figure 5, and verify whether simultaneous administration of the two polyamides would have a greater inhibitory effect on dex-induced GILZ expression.
I don't know too much about what goes into making these polyamides. Do you think it would be easy to synthesize a longer fragment, and would this help with specificity issues?