Innate immune reactions protect organisms against a variety of infections.  In metazoans, these reactions involve both cellular and humoral responses. The immune responses have to be well-tuned, as excessive immune activation is associated with tissue-specific pathologies. However, the transcriptional regulatory mechanisms underlying how immune responses are balanced are still not well understood. The aim of this study was to investigate the role of the Drosophila POU/Oct transcription factor Nubbin (Nub) in regulating Drosophila innate immunity, with a special focus on intestinal immune and epithelium homeostasis.

In Paper I, we show that the nub gene encodes two independent transcription factor isoforms, Nub-PB and Nub-PD. The short isoform, Nub-PD, acts as a repressor of NF-κB/Relish-dependent antimicrobial peptide (AMP) gene expression in healthy flies. Furthermore, we demonstrate that Nub-PD directly binds to Oct sequence motifs located in the distal promoter region of several AMP genes, thereby inhibiting gene transcription. In addition, loss of Nub-PD diminishes the number of cultivatable gut bacteria, possibly due to high expression levels of AMP genes. In Paper II, we show that the large isoform, Nub-PB, in a sharp contrast to Nub-PD, activates AMP gene expression, both independently of and together with Relish. Importantly, Nub-PB and Nub-PD regulated the same target AMP gene expression antagonistically. In addition, Nub-PB expression in gut enterocytes (ECs) negatively correlated with gut microbial loads and host lifespan. Finally, we found that enforced Nub-PB expression in ECs promotes a pro-inflammatory signature and stimulated epithelium renewal. In Paper III, we show that Nub-PB and Nub-PD are not only expressed in differentiated gut ECs, but also present in midgut progenitor cells. Depletion of Nub-PB in gut progenitor cells results in hyperproliferation of intestinal stem cells (ISCs), via direct or indirect de-repression of Escargot expression. Furthermore, enforced Nub-PB expression in ISCs and enteroblasts (EBs) inhibited Notch RNAi-induced tumor formation. In addition, Nub-PD was necessary for both basal and infection-induced ISC proliferation. Strikingly, Nub-PB and Nub-PD regulated ISC proliferation in antagonistic manners. In Paper IV, we created a Nub-PB-specific mutant and found that this mutant impairs normal gut development, giving rise to short and wide anterior midguts. Furthermore, loss of Nub-PB promoted rapid ISC proliferation, increased EC delamination, and increased numbers of enteroendocrine cells in the anterior midgut.

Taken together, we have characterized a novel isoform-specific regulatory mechanism, involved in maintaining Drosophila intestinal immune homeostasis and epithelial regeneration. 

Dysregulation of physiological and cellular processes underlies various pathological conditions, including cancer and inflammatory disorders. Unraveling the molecular mechanisms driving these processes is crucial. The aim of this thesis was to investigate the roles of evolutionarily conserved POU/Oct transcription factors using Drosophila melanogaster as a model organism. The thesis highlights the functions of Nubbin (Nub) protein isoforms (Nub-PB and Nub-PD) in the regulation of cellular proliferation and mitosis, epithelial regeneration, and innate immune responses.

In paper I, we demonstrate that Nub-PB acts as a potent transcriptional activator of immune and stress response genes, while Nub-PD represses their expression, indicating transcriptional antagonism by these Nub isoforms. Overexpression of Nub-PB in midgut cells effectively cleared local infections. However, prolonged Nub-PB overexpression caused a hyperactive immune response, leading to pro-inflammatory reactions, apoptosis, and reduced adult lifespan. These findings emphasize the importance of Nub protein isoforms in fine-tuning immune responses. 

In Paper II, we generated and phenotypically characterized a Nub-PB-specific mutant revealing impaired gut morphology, disorganized visceral muscles, and aberrant lineage specification in the midgut. In addition, it displays impaired immune gene activation, shortened lifespan, and enhanced reactive oxygen species (ROS) expression, which correlates with increased numbers of gut microbiota, featuring an important role of Nub-PB in intestinal epithelium homeostasis. 

In Paper III, we show that Nub-PD is necessary for proper nuclear divisions in transcriptionally silent pre-blastoderm embryos. The Nub-PD protein is enriched around the mitotic spindles in metaphase, requiring intact spindle microtubules. Live imaging of mitotic divisions revealed that Nub-PD is involved in the maintenance of spindle organization and its dynamics. We also infer similar mitotic roles for Nub-PD in S2 cells and for Oct1/POU2F1 in human cell culture. Our findings unveil a direct role of POU/Oct factors in proper mitotic progression, which may be evolutionarily preserved among insects and mammals.

In Paper IV, we study how the loss of Nub and Pdm2 proteins affects wing growth and development. We found that Nub-PD is specifically required for cell proliferation, while balanced Nub-PB and Nub-PD expression levels at the dorso-ventral boundary are essential for correct wing margin formation. 

Overall, this thesis elucidates crucial roles of Drosophila POU proteins in maintaining immune and tissue homeostasis and aditionally uncovers mitotic roles of POU/Oct factors, suggesting new functions in regulation of cell proliferation and development.