To control gene expression, transcription factors (TFs) bind to DNA and recruit regulatory cofactors (COFs) that mediate diverse effects on chromatin, such as DNA and histone modifications. To better help understand TF and COF interactions, our lab has been developing the nuclear extract protein-binding microarray (nextPBM) approach that allows high-throughput characterization of COF recruitment to DNA. An extension of the nextPBM, the human TF array (hTF array), allows COF recruitment to be profiled to binding sites of hundreds of human TFs, providing a method to screen TF-COF complexes forming in cell nuclear extracts. DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) and (cytosine-5)-DNA-methyltransferase 3 beta (DNMT3B), two isoforms of the methyltransferase DNMT3, are COFs that are known to be implicated in B-cell lymphoma. hTF experiments for DNMT3A recruitment in B-cell lymphoma cells revealed a number of interactions with known B-cell TFs. In this work, I have attempted to validate the interactions seen on the hTF array by testing TF interactions with both the native DNMT3A/B proteins and the central TF-interacting ATRX-DNMT3-DNMT3L (ADD) domains of DNMT3A/B through the use of an immunoprecipitation (IP) assay in RC B-cell lymphoma cells. I successfully cloned and purified the ADD domains of DNMT3A and B as GST epitope-tagged constructs. As preliminary hTF experiments predicted interactions of DNMT3A with Interferon Regulatory Factor (IRF) proteins, I tested whether the DNMT3 ADD domains interacted with IRF3 present in B-cell lymphoma cell extracts, but was unable to identify an interaction. To examine whether native DNMT3A interacted with the IRF proteins, I performed a native IP using a DNMT3A native antibody. However, I was unable to confirm this interaction by native extract co-IP. Future work to validate DNMT3A/B-TF interactions, and TF-COF interactions identified by the hTF platform more generally, can help us understand at a molecular level what is facilitating transition between normal and cancerous states.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43365 |
| Date | 12 November 2021 |
| Creators | Wadden, Evan |
| Contributors | Siggers, Trevor |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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