Rahmatollahi, Mahdieh
(2020).
Transcriptional Regulation in Podocytes.
PhD thesis, Universität zu Köln.
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Abstract
Prolonged and progressive podocyte injury leads to extensive proteinuria, scarred glomeruli and a decline in renal function. When left untreated, it develops chronic kidney disease (CKD) which often has poor prognosis. Disturbed gene regulation serves as both cause and/or consequence in the context of podocyte injury. Unravelling the gene regulatory circuits within podocytes is therefore a key step to introduce new curative and or palliative strategies for chronic kidney disease. Wt1 is a master transcription factor (TF) in podocytes and previous work has determined Wt1dependent gene regulatory networks in healthy adult podocytes. In this thesis, analysis of Wt1directed gene regulation in a genetic mouse model of podocyte damage demonstrated that Wt1 executes a differential binding tactic on genomic elements during early phases of podocyte damage. This differential binding on the genome is reflected as differential pathway regulation in the course of podocyte injury. Key podocyte pathways such as collagen biosynthesis, collagen metabolism as well as Eph/Ephrin signaling are markedly compromised. Perturbation of these critical functions entails glomerular basement membrane thickening, collapse of foot processes and podocyte detachment from the basement membrane. Next, a genome-wide analysis of Tead1, another master transcription factor in podocytes, indicated the cooperative function of Wt1 and Tead1 on gene regulatory elements. Both Wt1 and Tead1 are members of the podocyte-specific transcription factor network which regulates homeostasis and cell survival in podocytes. Interestingly, analysis of Tead1-dependent functions showed that Tead1 mainly regulates actin cytoskeleton dynamics and cell adhesion in podocytes. Tead1-dependent functions are chiefly directed via distal regulatory elements. Previously, enhancer-based gene regulation by Wt1 has been shown in podocytes. In line with this, examining the peak distribution of both Wt1 and Tead1 across the genome accentuates the highimpact role of enhancers in gene regulation in podocytes. Next, De novo motif enrichment analysis and motif conservation scores around the Tead1 peaks revealed enrichment not only for Tead1 motif but also for Wt1 and other partners of podocyte-specific TF network. Co-binding analysis of the Tead1 ChIPseq with our previous Wt1 ChIPseq dataset suggested that some of the Tead1-dependent functions are co-directed by Wt1 and vice versa. Finally, another set of complementary analysis was done in this thesis which unveils a different aspect of gene regulation in podocytes. Here, a highly parallel transcriptional profiling of more than 13,000 cells obtained from isolated mouse glomeruli was performed which identified the three main glomerular cell types i.e. podocytes, endothelial cells and mesangial cells. The comprehensive gene expression profile of all identified cell types is gathered in an interactive online atlas which can be queried based on gene names. In addition, novel markers for podocytes, endothelial cells and mesangial cells were introduced and validated by crossreferencing with glomerular expression of such markers in Human Protein Atlas. Furthermore, cell heterogeneity assessment for podocytes and endothelial cells was done. Endothelial cells showed four subpopulations relevant to different phases of their activation and proliferation. Podocytes, however, exhibited a more subtle trace for cellular heterogeneity. Thus, a new immunofluorescent staining approach was adopted to address the identified podocyte subpopulations. The results validated identification of subpopulations within podocytes based on the differential immunofluorescence signal for subpopulation markers observed in different podocytes within a glomerulus. Altogether, the novel findings throughout this thesis casts some light on tens of thousands of questions surrounding gene regulation in podocytes.
| Item Type: | Thesis (PhD thesis) | ||||||||
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| URN: | urn:nbn:de:hbz:38-116048 | ||||||||
| Date: | 2020 | ||||||||
| Language: | English | ||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||
| Divisions: | CECAD - Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases | ||||||||
| Subjects: | Natural sciences and mathematics Life sciences |
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| Date of oral exam: | 13 May 2020 | ||||||||
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| Refereed: | Yes | ||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/11604 |
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