An organoid model to assay the role of CFTR in the human epididymis epithelium
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE148367 | GEO | 2021/01/16
REPOSITORIES: GEO
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Project description:The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were over-represented in epididymis-selective DHS identified epithelial transcription factors including ELF5 and ELF3, the androgen receptor, Pax2 and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium. HEE cells were cultured as described previously (Harris and Coleman 1989). RNA was isolated by Trizol extraction from three primary cultures of HEE cells. Total RNA was purified by Millipore Microcon YM-100 filter centrifugation and shipped to MoGene, MO, for gene expression analysis on Nimblegen 4 x 72K HG18 60mer arrays. This submission represents transcriptome component of study.
2013-09-06 | E-GEOD-50605 | biostudies-arrayexpress
Project description:Cystic fibrosis (CF)-related diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting ~35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of b cell CFTR deletion, and normal and CF human pancreas and islets. Specific deletion of CFTR from murine b cells did not affect b cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein/electrical activity was not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion with few changes in key islet-regulatory transcripts. Furthermore, ~65% of b cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is not caused by intrinsic islet dysfunction from CFTR mutation, but rather, by b cell loss and intra-islet inflammation in the setting of a complex pleiotropic disease
2018-04-25 | GSE110935 | GEO
Project description:Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here we use single cell RNA-sequencing to construct an atlas of the human proximal epididymis. We find that the cystic fibrosis transmembrane conductance regulator (CFTR), which is pivotal in normal epididymis fluid transport, is most abundant in surface epithelial cells in the efferent ducts and in rare clear cells in the caput epididymis, suggesting region-specific functional properties. We reveal transcriptional signatures for multiple cell clusters, which identify the individual roles of principal, apical, narrow, basal, clear, halo and stromal cells in the epididymis. A marked cell-type-specific distribution of function is seen along the duct with local specialization of individual cell types integrating processes of sperm maturation.
2020-08-25 | GSE148963 | GEO
Project description:Organotypic culture systems from disease-specific induced pluripotent stem cells (iPSCs) exhibit obvious advantages compared to immortalized cell lines and primary cell cultures but implementation of iPSC-based high throughput (HT) assays is still technically challenging. Here we demonstrate the development and conduction of an organotypic HT Cl-/I- exchange assay using Cystic Fibrosis (CF) disease-specific iPSCs. The introduction of a halide sensitive YFP variant enabled automated quantitative measurement of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in iPSC-derived intestinal epithelia. CFTR function was partially rescued by treatment with VX-770 and VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of CFTR function. The identification of a series of validated primary hits that improve the function of p.Phe508del CFTR from a library of ~ 42.500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture systems for disease modelling can also be utilized for drug screening at a true HT format.
2019-05-11 | GSE129168 | GEO
Project description:The differentiation of pluripotent stem cells has been broadly used in studying the disease mechanism and development process. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, to investigate whether the hPSCs-derived SGEPs are capable of modeling the characteristics of cystic fibrosis (CF), a disease impact SG function caused by mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene, the CFTR knockout hPSCs were differentiated into CF-SGEPs using the same protocol. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16-day of differentiation, the protein expression of CFTR was also decreased in SGEPs generated from CFTR knockout hPSCs. RNA-sequencing shows that multiple genes modulating SG development, function and were down-regulated and positive regulators of inflammation were up-regulated in CF-SGEPs, which was correlated with salivary phenotype in CF patients. These results demonstrated that suppression of CFTR disrupted the differentiation of hPSCs-derived SGEPs, which modeled the SG development of patients with CF. In summary, this study not only provided the proof that the hPSCs-derived SGEPs could serve as manipulatable and easily accessible models to study the SG developmental diseases, but also provided new avenues for the study of the CF mechanism.
2023-12-06 | GSE224547 | GEO
Project description:The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human fetal epididymis epithelial cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor binding sites (TFBS) that were over-represented in the HEE open chromatin, include the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which is well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase-seq and their open chromatin map overlapped that of primary HEE cells at ~ 65% of sites. Moreover, the PAX2-binding motif was again identified as an overrepresented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 in comparison to a non-targeting siRNA. In response to PAX2-represssion, 3142 transcripts were differentially expressed (1334 up-regulated and 1808 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with a predicted function in the epididymis epithelium. examination of open chromatin region in REP cells with 2 replicates
2015-01-07 | E-GEOD-59782 | biostudies-arrayexpress
Project description:The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human fetal epididymis epithelial cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor binding sites (TFBS) that were over-represented in the HEE open chromatin, include the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which is well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase-seq and their open chromatin map overlapped that of primary HEE cells at ~ 65% of sites. Moreover, the PAX2-binding motif was again identified as an overrepresented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 in comparison to a non-targeting siRNA. In response to PAX2-represssion, 3142 transcripts were differentially expressed (1334 up-regulated and 1808 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with a predicted function in the epididymis epithelium. mRNA profile of control and PAX2 knockdown REP cells
2015-01-07 | E-GEOD-59781 | biostudies-arrayexpress
Project description:The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human fetal epididymis epithelial cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor binding sites (TFBS) that were over-represented in the HEE open chromatin, include the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which is well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase-seq and their open chromatin map overlapped that of primary HEE cells at ~ 65% of sites. Moreover, the PAX2-binding motif was again identified as an overrepresented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 in comparison to a non-targeting siRNA. In response to PAX2-represssion, 3142 transcripts were differentially expressed (1334 up-regulated and 1808 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with a predicted function in the epididymis epithelium. Gene expression in REP cells was measured at 16 hours after exposure to 1nM R1881 or vehicle control.
2015-01-07 | E-GEOD-60381 | biostudies-arrayexpress
Project description:Viral infections of the epididymis are associated with epididymitis, which damages the epithelium and impairs fertility. We showed previously that innate immune response genes were differentially expressed in the corpus and cauda region of the human epididymis in comparison to the caput. Here we investigate the antiviral defense response mechanisms of human epididymis epithelial (HEE) cells. Toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I)- like receptors (RLRs) are enriched in HEE cells from the corpus and cauda region. Furthermore, corpus and cauda HEE cells show an enhanced response to antiviral ligands (poly(I:C) and HSV-60), as shown by increased IFN-β mRNA expression and secretion of IFN-β. We also show that paired box 2 (PAX2), which was implicated in regulating antiviral response pathways is required for basal expression of the DNA sensor, Z-DNA binding protein (ZBP1) and type I interferon, in caput but not in cauda cells.
2019-10-11 | GSE104944 | GEO
Project description:Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure. Examination of open chromatin region in Caco2 (colorectal adenocarcinoma cells), HBE (primary human bronchial epithelial cells), and primary adult human epididymis cells
2016-01-07 | E-GEOD-74709 | biostudies-arrayexpress