Molecular and regulatory interactions of HOXA5 with SMAD1, SOX2 and TSHZ3

Details

Supervisors

Gofflot, Françoise

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Hox genes are homeogenes encoding transcription factors essential for establishing the anterior-posterior axis during embryonic development. Recent studies have revealed that 24 Hox genes remain expressed in the postnatal and adult mouse brain, including Hoxa5 gene, whose protein has been detected in the adult brain and is enriched in precerebellar nuclei within the pons and medulla oblongata. RNA-seq analyses have shown that postnatal functional loss of HOXA5 alters the expression of several genes involved in the development, maturation, and synaptic plasticity of precerebellar circuits. While new postnatal functions of HOXA5 are being explored, its mechanisms of action remain unclear. To deepen the understanding of the transcription factor HOXA5's activity in the postnatal development of the central nervous system, Pre. Gofflot’s laboratory investigates its mechanisms of action in postmitotic neurons. This master thesis aims to characterize the functional interactions of HOXA5 with its candidate partners SMAD1, SOX2, and TSHZ3, selected based on literature and database analyses. The first part of this master thesis aims to assess the impact of potential interactants SMAD1, SOX2, and TSHZ3 on HOXA5 transcriptional activity in the presence of the cofactors PREP1 and PBX1A. This study is conducted through a luciferase assay normalized by a ß-galactosidase assay, using the uce2.12 enhancer, a sequence containing a HOX-PBX Response Element to which HOXA5 is able to bind. The results show that SOX2 may act as an antagonist to the transcriptional activities of HOXA5 and PREP1 and PBX1A by reducing the luciferase signal. Additionally, the results also suggest that SOX2 can activate the uce2.12 enhancer on its own, despite the absence of any known binding site for SOX2 in this sequence. The use of mutated enhancers (uce2.12* and uce2.12**), targeting potential HOXA5 binding sites, reveals that SOX2 likely interacts with other binding sites on the uce2.12 enhancer, as these mutations do not affect its transcriptional activity. Results regarding SMAD1 indicate that the protein has no impact on HOXA5 transcriptional activity, either with the uce2.12 enhancer or its mutated versions. This lack of effect may be explained by the potential absence of a SMAD1 binding site on the uce2.12 enhancer. Finally, experiments involving TSHZ3 could not be performed due to time constraints. The second part of this master thesis aims to study the nature of the potential dimer formed by SOX2 and HOXA5 when HOXA5 binds to the uc.212 probe, using Electrophoresis Mobility Shift Assay (EMSA) and Western blot experiments. Since the DNA binding motifs of HOXA5 were not yet known at the beginning of this master thesis, the uc.212 probe, studied by Lampe et al. (2008), was selected for these experiments. The binding of SOX2 to the uc.212 probe in monomeric or homodimeric form had previously been observed in Mr. Ruelle’s master thesis, as well as the possible heterodimerization of SOX2 and HOXA5 by BiFC, and the competitive relationship between the two proteins to form homo- or heterodimers with SOX2. In this thesis, EMSA experiments using proteins purified from the soluble cell fraction revealed several closely spaced shifts, potentially resulting from the interaction of the complex formed by SOX2 and HOXA5, or by SOX2 alone in homodimeric form, with the probe. Since the molecular weights of these two proteins are similar, distinguishing the shifts corresponding to homo- and heterodimers is challenging. Nevertheless, these results suggest the possible formation of several multimers between SOX2 and HOXA5, resulting from potential dynamic complexes that can rapidly form and dissociate, reflecting transient binding states to the uc.212 probe. However, the identity of the shifts could not be confirmed, neither using antibodies in the EMSA experiments nor through Western blot attempts. Finally, EMSA experiments using proteins purified from the insoluble fraction revealed no shifts. This absence of results suggests that the loss of protein function may be due to their purification under denaturing conditions or to their structure in the insoluble fraction, which may prevent them from binding to DNA in the same way as proteins from the soluble fraction. These results provide new insights into the functional interactions of HOXA5 with SOX2, SMAD1, and TSHZ3, as well as their potential implications in HOXA5 transcriptional activity and DNA binding selectivity. They also contribute to a better understanding of HOXA5 functions in the postnatal brain.