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ShafferSarah_06552200_2024.pdf
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- Homeobox (Hox) genes code for a group of transcription factors fundamentally involved in animal development with roles in organogenesis and responsible for patterning of the anterior-posterior axis. HOXA2 is critically involved in the segmentation of the rhombomeric territories of the hindbrain, specifically defining the boundary between rhombomeres 1 and 2 and expressed in neural crest cells emanating from rhombomere 4, which migrate to brachial arch 2. One aspect of HOX protein biology that remains largely neglected is the characterization of the post-translational modifications (PTMs) associated to HOX proteins and their influence on the activity regulation and functional specificity of these transcription factors. Previous studies from the lab of Professor René Rezsohazy highlighted that HOXA2 is ubiquitinated, that ubiquitinated HOXA2 is detected in the nucleus, and is found in the insoluble fraction of protein extracts containing histones and chromatin. Additionally, recent work has shown that the DNA binding domain, the homeodomain, is required for the nuclear localization of HOXA2 and that, in fact, the homeodomain is ubiquitinated. Hence, it is of interest to study the ubiquitination of HOXA2 and especially explore the modification of the homeodomain. Altogether, these data proposed a possible ubiquitination-mediated regulation mechanism influencing the DNA binding activity of HOXA2. In the context of all this, the aim of this thesis has been to determine whether the DNA binding activity of HOXA2 is influenced by ubiquitination using an Electrophoretic Mobility Shift Assay (EMSA). This will constitute a small part of a broader research project in the laboratory of Professor René Rezsohazy, led by postdoctoral fellow Laure Bridoux, which seeks to identify the modalities of HOXA2 activity regulation and how the functional specificity of this transcription factor is achieved. This question was addressed by extraction of the homeodomain of HOXA2 (HOXA2-HD) and the full length HOXA2 from cells. Bacterial proteins were produced as controls since it was known that bacterial purified proteins bind to an EMSA oligonucleotide probe that was derived from a target enhancer sequence. Then, purification of protein from eukaryotic HEK cells was utilized to produce protein bearing mammalian PTMs. Transfection and purification from HEK cells was also used as a method of separating ubiquitinated and non-ubiquitinated proteins to use in the EMSAs. These were the first purifications of HOXA2-HD and HOXA2 from HEK cells in our lab, necessitating optimization of the protocols for purification with Ni-NTA beads and their utilization in the EMSAs. Finally, Mass Spectrometry analysis was carried out in collaboration with UNamur which confirmed ubiquitination on several residues in the HOXA2-HD and HOXA2 sequence. It was discovered that binding in the EMSAs could be observed for both ubiquitinated and non-ubiquitinated HOXA2-HD. It can be concluded that the intrinsic DNA binding ability of HOXA2-HD is not impeded by ubiquitination while it is also not required for binding to occur. This correlates with previous findings that postulated a role for ubiquitinated HOXA2 in the nucleus, associated with chromatin. Thus, together with previous results obtained in the lab, a new role for ubiquitination can be proposed in the relation to HOXA2 activity, possibly regulating its nuclear localization and/or modulating its ability to bind DNA.