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.

Calcium (Ca2+) is an important ion in physiology and is found both outside and inside cells. The intracellular concentration of Ca2+ is tightly regulated as it is an intracellular signal molecule and can affect a variety of cellular processes. In immune cells Ca2+ has been shown to regulate e.g. gene transcription, cytokine secretion, proliferation and migration. Ca2+ can enter the cytoplasm either from intracellular stores or from outside the cells when Ca2+ permeable ion channels in the plasma membrane open. The Ca2+ release-activated (CRAC) channel is the most prominent Ca2+ ion channel in the plasma membrane. It is formed by ORAI1-3 and the channel is opened by the endoplasmic reticulum Ca2+ sensor proteins stromal interaction molecules (STIM) 1 and 2. Another group of Ca-2(+) channels in the plasma membrane are the voltage-gated Ca2+ (Ca-V) channels. We examined if a change in immunological tolerance is accompanied by altered ORAI, STIM and Ca-V gene expression in peripheral blood mononuclear cells (PBMCs) in pregnant women and in type 1 diabetic individuals. Our results show that in pregnancy and type 1 diabetes ORAI1-3 are up-regulated whereas STIM1 and 2 are down-regulated in pregnancy but only STIM2 in type 1 diabetes. Expression of L-, P/Q-, R- and T-type voltage-gated Ca2+ channels was detected in the PBMCs where the Ca(V)2.3 gene was up-regulated in pregnancy and type 1 diabetes whereas the Ca(V)2.1 and Ca(V)3.2 genes were up-regulated only in pregnancy and the Ca(V)1.3 gene in type 1 diabetes. The results are consistent with that expression of ORAI, STIM and Ca-V genes correlate with a shift in immunological status of the individual in health, as during pregnancy, and in the autoimmune disease type 1 diabetes. Whether the changes are in general protective or in type 1 diabetes include some pathogenic components remains to be clarified.

Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.

The gene for Drosophila POU/Oct transcription factor Nubbin (Nub)/Pdm1 encodes two independent isoforms, Nub-PB and Nub-PD, which act antagonistically in gut progenitor cells to maintain normal intestinal stem cell (ISC) proliferation. However, the role of Nub-PB in regulating midgut epithelium regeneration is not fully understood.  Here, we have created a viable nub mutant named nub^PB-3 and find that this mutant displays shortened adult lifespan in normal rearing conditions. Careful analysis of nub^PB-3 midgut morphology revealed an enlarged anterior midgut with increased ISC proliferation, enhanced enteroendocrine (EE) lineage differentiation and enforced delamination of enterocytes (ECs). Furthermore, we found that the increased ISC proliferation in the nub^PB-3 mutant is caused by the high activity of JAK/STAT signaling in the anterior midgut, indicating that Nub-PB normally represses the JAK/STAT activity in progenitor cells. A high rate of ISC proliferation caused by oral Ecc15 infection was, however, not dependent on Nub-PB.  This suggests a role for Nub-PB in suppressing the rate of ISC proliferation under normal conditions, a role which is lost during infection-induced proliferation. Interestingly, nub^PB-3 mutants were susceptible to Ecc15 infection and died shortly thereafter. While this was not due to gut epithelium leakage or impaired antimicrobial peptide gene expression, a more general failure in gut physiology, digestion and metabolism may be the underlying cause of death.