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Isoform-specific regulation of Drosophila gut immunity and regeneration by the POU/Oct transcription factor Nub/Pdm1
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0001-9875-8829
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2018. , p. 62
Keywords [en]
POU, Nubbin, Drosophila, intestinal stem cell, epithelium regeneration, midgut, mitosis, Antimicrobial peptides, innate immunity, NF-κB, bacterial infection, transcriptional regulation, homeostasis
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-155393ISBN: 978-91-7797-276-1 (print)ISBN: 978-91-7797-277-8 (electronic)OAI: oai:DiVA.org:su-155393DiVA, id: diva2:1199238
Public defence
2018-06-07, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrehnius väg 20, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2018-05-15 Created: 2018-04-19 Last updated: 2020-05-08Bibliographically approved
List of papers
1. The Oct1 homolog Nubbin is a repressor of NF-kappa B-dependent immune gene expression that increases the tolerance to gut microbiota
Open this publication in new window or tab >>The Oct1 homolog Nubbin is a repressor of NF-kappa B-dependent immune gene expression that increases the tolerance to gut microbiota
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2013 (English)In: BMC Biology, ISSN 1741-7007, E-ISSN 1741-7007, Vol. 11, article id 99Article in journal (Refereed) Published
Abstract [en]

Background: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-kappa B transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood. Results: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-kappa B/Relish-driven antimicrobial peptide gene expression in flies. In nub(1) mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism. Conclusions: This study demonstrates that a large number of genes that are activated by NF-kappa B/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.

Keywords
Antimicrobial peptides, Drosophila, Gene regulation, Host-pathogen interaction, Immune signaling, Innate immunity, NF-kappaB, Oct /POU transcription factors, Stress response
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-95772 (URN)10.1186/1741-7007-11-99 (DOI)000325347400001 ()
Note

AuthorCount:8;

Available from: 2013-11-04 Created: 2013-11-04 Last updated: 2019-12-04Bibliographically approved
2. Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis
Open this publication in new window or tab >>Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis
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2018 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 14, no 3, article id e1006936Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Immunology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-155384 (URN)10.1371/journal.ppat.1006936 (DOI)000430192300029 ()
Available from: 2018-04-19 Created: 2018-04-19 Last updated: 2019-12-04Bibliographically approved
3. The POU/Oct Transcription Factor Nubbin Controls the Balance of Intestinal Stem Cell Maintenance and Differentiation by Isoform-Specific Regulation
Open this publication in new window or tab >>The POU/Oct Transcription Factor Nubbin Controls the Balance of Intestinal Stem Cell Maintenance and Differentiation by Isoform-Specific Regulation
2018 (English)In: Stem Cell Reports, ISSN 2213-6711, Vol. 10, no 5, p. 1565-1578Article in journal (Refereed) Published
Abstract [en]

Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. Here, we show that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. We demonstrate that the nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. We suggest that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.

Keywords
Pdm1, POU, homeodomain, Drosophila, stem cell, epithelium regeneration, Escargot, Notch, mitosis, midbody
National Category
Developmental Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-155389 (URN)10.1016/j.stemcr.2018.03.014 (DOI)000432931400015 ()
Available from: 2018-04-19 Created: 2018-04-19 Last updated: 2018-06-25Bibliographically approved
4. Characterization of the role of Nub-PB in gut epithelium regeneration
Open this publication in new window or tab >>Characterization of the role of Nub-PB in gut epithelium regeneration
(English)Manuscript (preprint) (Other academic)
Abstract [en]

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 nubPB-3 and find that this mutant displays shortened adult lifespan in normal rearing conditions. Careful analysis of nubPB-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 nubPB-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, nubPB-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.

National Category
Developmental Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-155386 (URN)
Available from: 2018-04-19 Created: 2018-04-19 Last updated: 2018-04-23Bibliographically approved

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