Developing optogenetic control of NF-κB dynamics to elucidate their influence on the transcriptional output

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The Nuclear Factor-kappa B (NF-κB) pathway is a crucial and extensively studied signalling cascade, implicated in a plethora of biological processes, including immune responses, cell proliferation, apoptosis, cancer, and aging. Members of the NF-κB transcription factor (TF) family form dimers, the most prominent of which is the p65/p50 heterodimer, commonly known as NF-κB. There are more than 300 target genes controlled by NF-κB. Albeit in the case of NF-κB the existence of several different heterodimers explains in part how such multiplexing is achieved, many years of research on gene expression and TFs in eukaryotic cells have established that, beyond TF identity, post-translational modifications and chromatin accessibility play a role in controlling which promoters are activated. For many TFs, including NF-κB, it was also observed that, upon pathway activation by different ligands, these signalling molecules exhibit different dynamics––defined as the shape of the curve describing the change over time of the TF activity/nuclear localization. Despite extensive efforts to elucidate how NF-κB dynamics influence cellular responses, it remains unclear whether dynamics alone are necessary and sufficient to activate specific promoters. Traditional stimulation methods based on ligands, such as TNFα or LPS, activate multiple pathways alongside NF-κB. This leads to crosstalk and substantial cellular heterogeneity, both of which obscure the direct role of NF-κB dynamics in gene regulation. This thesis addresses this gap by employing a novel optogenetic strategy for precise, light-controlled manipulation of NF-κB dynamics. I chose to control the localization of p65, rather than p50, because p65 contains transactivation domains and can elicit gene expression independently. Unlike conventional approaches that trigger the pathway at the receptor level, this innovative method directly controls p65, allowing for specific interrogation of the protein's dynamics without perturbing other components of the signalling cascade and without triggering the activity of other proteins. I compared three different strategies to control p65 dynamics: (1) C-terminal fusion of p65 to the light-inducible nuclear localization tool LINuS; (2) N-terminal fusion of p65 to LINuS; and (3) fusion of p65 to GFP combined with the concomitant expression of a nanobody (Nb) against GFP fused to LINuS. Different nuclear translocation patterns of these fusion proteins were elicited by imposing specific light regimes. Of the three designs evaluated, the one based on the Nb and GFP-p65 proved to be more effective, yielding enhanced nuclear localization and greater transcriptional activity. This work was conducted using transiently transfected cells. This presented its own set of problems, especially in regard to the reproducibility of the experiments, which hindered me from drawing more robust conclusions about whether different illumination patterns (e.g., continuous versus pulsed light) yield distinct transcriptional outcomes. It is clear, though, that the optogenetic constructs do not fully replicate the behaviour of the NF-kB pathway activated by ligands: in my experiments, an illumination of twenty-four hours was required for a small increase in target gene expression, whereas the ligand-activated pathway achieves higher fold changes in less than one hour. I speculate that the absence of post-translational modifications and, consequently, cofactor recruitment likely limited the transcriptional activity of the p65-containing optogenetic constructs, thereby obscuring clear and reliable differences between illuminated and non-illuminated samples. Despite these challenges, the optogenetic platform I developed offers an unprecedented opportunity to dissect the information encoded within p65 dynamics. Future investigations leveraging this system could enable detailed analyses of the full transcriptome in response to defined p65 dynamics. It will also facilitate exploring the interplay between NF-κB dynamics and post-translational modifications. Ultimately, this work establishes a powerful methodology to unravel fundamental aspects of NF-κB signalling, advancing our understanding of cellular decisions in health and disease.

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