APOBEC3A Mediated Genome Targeting of the Thyroid Hormone Response Element
Start Date
15-4-2021 9:30 AM
End Date
15-4-2021 10:30 AM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Allison Land
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
APOBEC3A (A3A) is a hydrolase enzyme that deaminates cytosine in DNA sequences to produce uracil. A3A is specific for the nucleotide sequence of nnnTCA and is one of the most potent human DNA cytosine deaminases. My goal is to use A3A’s specificity as a genome targeting tool, specifically targeting the thyroid hormone response element (TRE), which is a DNA sequence important for the thyroid hormone receptor transcription cascade. The DNA sequence of the TRE contains the A3A specific nucleotide sequence in two palindromic nnnTCAnnnnnnnTCA sites. I hypothesized that A3A would mutate this cytosine in the TRE sequence, obliterating the TRE and halting its transcriptional function. Thus far, I have shown that A3A, transfected into 293T cells, successfully mutates an oligonucleotide sequence with the target AGGTCA to AGGTUA. This was shown through subsequent uracil removal by Uracil DNA Glycosylase and cleavage by NaOH/heat treatment visualized on a 15% TBE-Urea electrophoresis gel. In order to observe deamination by A3A in vivo, both A3A and the TRE sequence must be present in live cells. For future in vitro experiments, a plasmid containing the TRE sequence will be transfected into two cell populations. One population (experimental) will also be transfected with an A3A expression plasmid. The second population (control) will be transfected with plasmid encoding a catalytically inactive version of the enzyme. Both cell groups will be observed and analyzed using genome sequencing. Successful A3A deamination will be shown by mutations of the cytosine in the TRE sequence when active A3A plasmid is present. The ability to perform genome targeting and editing of the TRE, using A3A, will allow for observation of metabolic changes produced in response to a lack of a functional TRE and greater understanding of the thyroid hormone receptor transcription cascade.
APOBEC3A Mediated Genome Targeting of the Thyroid Hormone Response Element
APOBEC3A (A3A) is a hydrolase enzyme that deaminates cytosine in DNA sequences to produce uracil. A3A is specific for the nucleotide sequence of nnnTCA and is one of the most potent human DNA cytosine deaminases. My goal is to use A3A’s specificity as a genome targeting tool, specifically targeting the thyroid hormone response element (TRE), which is a DNA sequence important for the thyroid hormone receptor transcription cascade. The DNA sequence of the TRE contains the A3A specific nucleotide sequence in two palindromic nnnTCAnnnnnnnTCA sites. I hypothesized that A3A would mutate this cytosine in the TRE sequence, obliterating the TRE and halting its transcriptional function. Thus far, I have shown that A3A, transfected into 293T cells, successfully mutates an oligonucleotide sequence with the target AGGTCA to AGGTUA. This was shown through subsequent uracil removal by Uracil DNA Glycosylase and cleavage by NaOH/heat treatment visualized on a 15% TBE-Urea electrophoresis gel. In order to observe deamination by A3A in vivo, both A3A and the TRE sequence must be present in live cells. For future in vitro experiments, a plasmid containing the TRE sequence will be transfected into two cell populations. One population (experimental) will also be transfected with an A3A expression plasmid. The second population (control) will be transfected with plasmid encoding a catalytically inactive version of the enzyme. Both cell groups will be observed and analyzed using genome sequencing. Successful A3A deamination will be shown by mutations of the cytosine in the TRE sequence when active A3A plasmid is present. The ability to perform genome targeting and editing of the TRE, using A3A, will allow for observation of metabolic changes produced in response to a lack of a functional TRE and greater understanding of the thyroid hormone receptor transcription cascade.
