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BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.
Assuntos
Biomarcadores , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Modelos Animais de Doenças , Células Estreladas do Pâncreas , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Animais , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Ovinos , Pâncreas/metabolismo , Pâncreas/patologia , Gravidez , Pancreatopatias/genética , Pancreatopatias/metabolismo , Pancreatopatias/patologia , Transcriptoma , Humanos , Perfilação da Expressão GênicaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5 kb 5' to the CFTR gene promoter. Activation of the -9.5 kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5 kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone. This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5 kb site and in which the locus is normally inactive. However, the -9.5 kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays. These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Regiões Promotoras Genéticas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Masculino , Animais , Regulação da Expressão Gênica , Epididimo/metabolismo , Cromatina/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , CamundongosRESUMO
A "universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF), regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in â¼0.01% of alleles primarily occurring at a nononcogenic off-target site and large indels in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes, except in the region corresponding to the CFTR cDNA and tCD19 cassette, indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the universal strategy showed minor undesirable genomic changes.
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We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Regulação da Expressão Gênica , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células CACO-2 , Elementos Facilitadores Genéticos/genética , Genômica , CromatinaRESUMO
The airway epithelial cell line, 16HBE14o- , is an important cell model for studying airway disease. 16HBE14o- cells were originally generated from primary human bronchial epithelial cells by SV40-mediated immortalization, a process that is associated with genomic instability through long-term culture. Here, we explore the heterogeneity of these cells, with respect to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) transcript and protein. We isolate clones of 16HBE14o- with stably higher and lower levels of CFTR in comparison to bulk 16HBE14o- , designated CFTRhigh and CFTRlow . Detailed characterization of the CFTR locus in these clones by ATAC-seq and 4C-seq showed open chromatin profiles and higher order chromatin structure that correlate with CFTR expression levels. Transcriptomic profiling of CFTRhigh and CFTRlow cells showed that the CFTRhigh cells had an elevated inflammatory/innate immune response phenotype. These results encourage caution in interpreting functional data from clonal lines of 16HBE14o- cells, generated after genomic or other manipulations.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Sistema Respiratório , Células Epiteliais/metabolismo , Cromatina/metabolismoRESUMO
The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Pulmão , Animais , Fibrose Cística/genética , Fibrose Cística/patologia , Fibrose Cística/veterinária , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/uso terapêutico , Perfilação da Expressão Gênica , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Ovinos/genética , Transcriptoma , Inflamação/genética , Inflamação/patologiaRESUMO
Highly effective modulator therapies for cystic fibrosis (CF) make it a treatable condition for many people. However, although CF respiratory illness occurs after birth, other organ systems particularly in the digestive tract are damaged before birth. We use an ovine model of CF to investigate the in utero origins of CF disease since the sheep closely mirrors critical aspects of human development. Wildtype (WT) and CFTR -/- sheep tissues were collected at 50, 65, 80, 100, and 120 days of gestation and term (147 days) and used for histological, electrophysiological, and molecular analysis. Histological abnormalities are evident in CFTR-/- -/- animals by 80 days of gestation, equivalent to 21 weeks in humans. Acinar and ductal dilation, mucus obstruction, and fibrosis are observed in the pancreas; biliary fibrosis, cholestasis, and gallbladder hypoplasia in the liver; and intestinal meconium obstruction, as seen at birth in all large animal models of CF. Concurrently, cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short circuit current is present in WT tracheal epithelium by 80 days gestation and is absent from CFTR -/- tissues. Transcriptomic profiles of tracheal tissues confirm the early expression of CFTR and suggest that its loss does not globally impair tracheal differentiation.
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Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-ß signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.
Assuntos
Epididimo , Células Epiteliais , Masculino , Humanos , Epididimo/metabolismo , Células Epiteliais/metabolismo , Redes Reguladoras de Genes , Diferenciação Celular , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by both ubiquitous and cell-type selective cis-regulatory elements (CREs). These CREs include extragenic and intronic enhancers that bind lineage-specific transcription factors, and architectural protein-marked structural elements. Deletion of the airway-selective -35 kb enhancer in 16HBE14o- lung epithelial cells was shown earlier to disrupt normal enhancer-promoter looping at the CFTR locus and nearly abolish its expression. Using a 16HBE14o- clone that lacks the endogenous -35 kb CRE, we explore the impact of relocating the functional core of this element to an ectopic site in intron 1. The -35 kb sequence establishes a de novo enhancer signature in chromatin at the insertion site, and augments CFTR expression, albeit not fully restoring WT levels. The relocated -35 kb enhancer also initiates de novo chromatin contacts with the CFTR promoter and other known CFTR CREs. These results are broadly relevant to therapeutic gene editing.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Elementos Facilitadores Genéticos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fatores de Transcrição/genética , Regiões Promotoras Genéticas , Cromatina/genéticaRESUMO
The evolutionary relationship of cells within tissues having a similar function but located in different anatomical sites is of considerable biological interest. The development of single-cell RNA sequencing (scRNA-seq) protocols has greatly enhanced opportunities to address this topic. Here we focus on cells in the epithelium which lines two regions of the human respiratory tract and the male genital ducts to delineate the shared, differentiated functions of the different cell populations. Transcriptomic data were used to assess the gene expression profiles of human bronchial, nasal, and epididymal epithelium (HBE, HNE, and HEE). Bulk RNA-seq showed many shared genes expressed in cells from the nasal and bronchial epithelium and highlighted their divergence from the epididymal epithelium. ScRNA-seq in HBE and HNE cells demonstrated overlapping gene expression patterns within basal and secretory cell populations. Moreover, the distribution of cell types was altered in HNE cells derived from donors with cystic fibrosis (CF) when compared to cells from healthy donors. Next, the HBE and HNE datasets were merged and confirmed intersection of cell type gene expression profiles from the two sites. However, secretory and ciliated cells were the most abundant types in the HBE samples, while more basal cells were seen in the HNE populations. We then merged single-cell data from the epididymis to determine if overlapping functions of these cells corresponded to those in the airway. Of note, only the pulmonary ionocytes/epididymis clear cells showed a strongly conserved identity, which was confirmed by imputation in bulk RNA-seq datasets from the same cells.
Assuntos
Epididimo , Análise de Célula Única , Brônquios/metabolismo , Epitélio , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Análise de Célula Única/métodos , TranscriptomaRESUMO
INTRODUCTION: The primary aim of this study was to evaluate patient-reported outcome measures in patients undergoing mastectomy with and without breast reconstruction (immediate or delayed) with and without nipple preservation. METHODS: All female patients undergoing mastectomy between 2011 and 2015 at Mayo Clinic Rochester were identified and were mailed the BREAST-Q survey. Breast satisfaction, psychosocial well-being, and sexual well-being were evaluated and compared by surgery type using Wilcoxon rank-sum tests for univariate analysis and linear regression for multivariable analysis adjusting for potential confounders. RESULTS: Of 1547 patients, 771 completed the BREAST-Q survey (response rate 50%). Of these 771 respondents, 237 (31%) did not have reconstruction, 198 (26%) had nipple-sparing mastectomy with reconstruction (NSM), and 336 (44%) had skin-sparing mastectomy with reconstruction (SSM) ± nipple-areolar complex (NAC) reconstruction (via surgery ± tattoo). Patients with breast reconstruction had consistently higher BREAST-Q scores versus those without. Comparing NSM with all SSMs, there was no difference in satisfaction with breasts (mean 71.8 vs. 70.2, p = 0.21) or psychosocial well-being (mean 81.9 vs. 81.3, p = 0.47); however, sexual well-being was significantly higher in the NSM group on univariate (mean 64.5 vs. 58.0, p = 0.002) and multivariable (ß = -4.69, p = 0.03) analysis. Sexual well-being scores were similar for NSM and the SSM subgroups with any type of NAC reconstruction. CONCLUSIONS: This study demonstrates that NSM positively impacts patient sexual well-being after breast reconstruction compared with SSM, particularly SSM without nipple reconstruction or tattoo. SSM with any type of NAC reconstruction achieved similar satisfaction and sexual well-being to those undergoing NSM.
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Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.
Assuntos
Cromatina , Regulador de Condutância Transmembrana em Fibrose Cística , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like , Ativação Transcricional , Cromatina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismoRESUMO
Robust protocols to examine 3D chromatin structure have greatly advanced knowledge of gene regulatory mechanisms. Here we focus on the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which provides a paradigm for validating models of gene regulation built upon genome-wide analysis. We examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR gene coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results suggest a mechanism whereby the locus responds when CREs are lost, which may involve activating transcription factors such as FOXA2. Also, by removing the 5' topologically-associating domain (TAD) boundary, we illustrate its impact on CFTR gene expression and architecture. These data suggest a multi-layered regulatory hierarchy that is highly sensitive to perturbations.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Elementos Facilitadores Genéticos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cromatina , Regulação da Expressão Gênica , Fatores Ativadores da Transcrição/genética , Fatores Ativadores da Transcrição/metabolismoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB domain and CNC homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here, we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (â¼8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.
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Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Estresse Oxidativo/genética , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxigênio/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del / F508del and CFTRG542X / G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR -/- sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del / F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X / G542X lambs showed modest restoration of CFTR function following inhibition of nonsense-mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life-limiting disorder.
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A complex network of transcription factors regulates genes involved in establishing and maintaining key biological properties of the human airway epithelium. However, detailed knowledge of the contributing factors is incomplete. Here we characterize the role of Krüppel-like factor 5 (KLF5), in controlling essential pathways of epithelial cell identity and function in the human lung. RNA-seq following siRNA-mediated depletion of KLF5 in the Calu-3 lung epithelial cell line identified significant enrichment of genes encoding chemokines and cytokines involved in the proinflammatory response and also components of the junctional complexes mediating cell adhesion. To determine direct gene targets of KLF5, we defined the cistrome of KLF5 using ChIP-seq in both Calu-3 and 16HBE14o- lung epithelial cell lines. Occupancy site concordance analysis revealed that KLF5 colocalized with the active histone modification H3K27ac and also with binding sites for the transcription factor CCAAT enhancer-binding protein beta (C/EBPß). Depletion of KLF5 increased both the expression and secretion of cytokines including IL-1ß, a response that was enhanced following exposure to Pseudomonas aeruginosa lipopolysaccharide. Calu-3 cells exhibited faster rates of repair after KLF5 depletion compared with negative controls in wound scratch assays. Similarly, CRISPR-mediated KLF5-null 16HBE14o- cells also showed enhanced wound closure. These data reveal a pivotal role for KLF5 in coordinating epithelial functions relevant to human lung disease.
Assuntos
Células Epiteliais , Imunidade Inata , Fatores de Transcrição Kruppel-Like , Linhagem Celular , HumanosRESUMO
The causative gene in cystic fibrosis (CF) was identified in 1989, 3 years before the publication of the first issue of Human Molecular Genetics. The cystic fibrosis transmembrane conductance regulator (CFTR) gene was among the first underlying a common inherited disorder to be cloned, and hence, its subsequent utilization toward a cure for CF provides a roadmap for other monogenic diseases. Over the past 30 years, the advances that built upon knowledge of the gene and the CFTR protein to develop effective therapeutics have been remarkable, and yet, the setbacks have also been challenging. Technological progress in other fields has often circumvented the barriers. This review focuses on key aspects of CF diagnostics and current approaches to develop new therapies for all CFTR mutations. It also highlights the major research advances that underpinned progress toward treatments and considers the remaining obstacles.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Fibrose Cística/terapia , Estudos de Associação Genética , Predisposição Genética para Doença , Animais , Sistemas CRISPR-Cas , Gerenciamento Clínico , Edição de Genes , Regulação da Expressão Gênica , Estudos de Associação Genética/métodos , Terapia Genética , Humanos , Técnicas de Diagnóstico Molecular , Mutação , Análise de Célula ÚnicaRESUMO
Mechanisms regulating gene expression in the airway epithelium underlie its response to the environment. A network of transcription factors (TFs) and architectural proteins, modulate chromatin accessibility and recruit activating or repressive signals. Bromodomain-containing proteins function as TFs or by engaging methyltransferase or acetyltransferase activity to induce chromatin modifications. Here we investigate the role of Bromodomain Containing 8 (BRD8) in coordinating lung epithelial function. Sites of BRD8 occupancy genome-wide were mapped in human lung epithelial cell lines (Calu-3 and 16HBE14o-). CCCTC-Binding Factor (CTCF) was identified as a predicted co-factor of BRD8, based upon motif over-representation under BRD8 ChIP-seq peaks. Following siRNA-mediated depletion of BRD8, differentially expressed genes with nearby peaks of BRD8 occupancy were subject to gene ontology process enrichment analysis. BRD8 targets are enriched for genes involved in the innate immune response and the cell cycle. Depletion of BRD8 increased the secretion of the antimicrobial peptide beta-defensin 1 and multiple chemokines, and reduced cell proliferation.
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Células Epiteliais/metabolismo , Redes Reguladoras de Genes , Pulmão/citologia , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Proliferação de Células/genética , Quimiocinas/metabolismo , Genoma Humano , Humanos , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells differentiated into pseudostratified airway epithelial cells to study these mechanisms. RESULTS: Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, two novel open chromatin regions, at +19.6 kb and +22.6 kb 3' to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6 kb site alone does not affect CFTR expression in DE or AFE cells. CONCLUSIONS: These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation.
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Diferenciação Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fatores de Transcrição Otx/metabolismo , Elementos Reguladores de Transcrição , Mucosa Respiratória/citologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Endoderma/citologia , Humanos , Células-Tronco Pluripotentes InduzidasRESUMO
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 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.