Transcriptome profile of MafB-cKO of podocyte
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE96044 | GEO | 2021/08/03
REPOSITORIES: GEO
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Project description:We identified binding sites of the Wilms' tumor suppressor protein WT1 in the mouse podocyte genome in vivo by ChIP-seq. Furthermore, we provide a podocyte transcriptome derived from primary podocytes that were isolated by FACS on mouse glomeruli. In short, we show that WT1 activates a highly specific podocyte transcriptome by binding to putative podocyte-specific enhancers and TSS of target genes. Genes bound by WT1 in podocytes include the majority of genes mutated in hereditary podocytopathies as well as components of the slit diaphragm, actin cytoskeleton, extracellular matrix, and within endocytosis pathways. Furthermore, we infer a podocyte TF network from DNA-binding motifs enriched at WT1-bound loci that includes Tead, Lmx1b, Mafb, Tcf21, and Fox-class transcription factors. Examination of transcription factor binding sites for WT1 by ChIP-seq. Transcriptome analysis of podocytes by RNA-seq.
2014-12-11 | E-GEOD-64063 | biostudies-arrayexpress
Project description:We identified binding sites of the Wilms' tumor suppressor protein WT1 in the mouse podocyte genome in vivo by ChIP-seq. Furthermore, we provide a podocyte transcriptome derived from primary podocytes that were isolated by FACS on mouse glomeruli. In short, we show that WT1 activates a highly specific podocyte transcriptome by binding to putative podocyte-specific enhancers and TSS of target genes. Genes bound by WT1 in podocytes include the majority of genes mutated in hereditary podocytopathies as well as components of the slit diaphragm, actin cytoskeleton, extracellular matrix, and within endocytosis pathways. Furthermore, we infer a podocyte TF network from DNA-binding motifs enriched at WT1-bound loci that includes Tead, Lmx1b, Mafb, Tcf21, and Fox-class transcription factors.
2014-12-11 | GSE64063 | GEO
Project description:The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the lipid-binding protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore now analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and RNA levels of podocinwildtype and podocinΔexon. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology. Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 hours after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas RNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.
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Project description:The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remains to be elucidated. Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed ‘cell junctions’, ‘actin binding’, and ‘cytoskeleton organization’ as top terms. Known foot process components were identified and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and supports the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.
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Project description:The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remains to be elucidated. Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed ‘cell junctions’, ‘actin binding’, and ‘cytoskeleton organization’ as top terms. Known foot process components were identified and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and supports the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.
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Project description:Podocytes are cells of the visceral epithelium in the kidneys and form a crucial component of the glomerular filtration barrier, contributing to size selectivity and maintaining a massive filtration surface. We are interested in pursuing a microarray analysis to identify the glycosylation-related genes that are modulated in podocytes during insulin and glucose stimulation. As such, we propose to isolate RNA from cultured AB 8/13 human podocyte cell line in their normal state and treated with different concentrations of insulin and glucose. Initially, we would like to analyze 12 samples (Normal control, 2 Starved controls, Insulin experiments 1-4, and glucose experiments 1-5). These experiments would give us information on the gene regulation changes in different healthy and diabetic conditions. In addition to providing possible identification of the unknown proteins involved with diabetic nephropathy, such analysis would provide novel information about early responses by podocytes in response to insulin and glucose level changes in vitro. The results from this analysis will be utilized to focus our research with regard to glycosylation of the proteins in the podocyte slit diaphragm.
2011-10-02 | E-GEOD-32553 | biostudies-arrayexpress
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2014-05-20 | GSE47606 | GEO
Project description:Glomerular podocyte cells are critical for the function of the renal ultrafiltration barrier. The highly specialized cell-cell junction of podocytes, the slit diaphragm, has a central role in the filtration barrier. Dendrin is a poorly characterized cytosolic component of the slit diaphragm in where it interacts with nephrin and Cd2ap. In this study, we have generated a dendrin knockout mouse line and explored the molecular interactions of dendrin. Dendrin-deficient mice were viable, fertile and had normal life span. To reveal the glomerular gene expression changes in the dendrin knockout mouse, Affymetrix Mouse Genome 430 2.0 Array were used to profiling the dendrin knockout and control glomeruli. Three dendrin knockout and three littermate control mice at age of 13 months were used to profile glomerular transcriptomes. Total glomerular RNA was extracted and hyrbridized on the Mouse Genome 430 2.0 Array according to standard procedures.
2014-05-20 | E-GEOD-47606 | biostudies-arrayexpress
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