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Principal Investigator: Douglas Glenn Brownfield
Organization: MAYO CLINIC ROCHESTER
Fiscal Year: 2024
Award: $638,986
Funding agency: National Heart Lung and Blood Institute
PROJECT SUMMARY
The alveolus is lined by two epithelial cell types: thin gas-exchanging alveolar type 1 (AT1) cells and cuboidal
surfactant-producing AT2 cells. Both are selected from a common distal progenitor by FGF, Notch, and stretch
signaling. Injury in the adult lung activates AT2 into a facultative progenitor state to regenerate lost AT1 and AT2
cells. While the inductive cues that select fate have been identified, it is less clear what role – if any – repressive
cues play in fate maturation. Two gaps of understanding in the lung field are 1) when and how is stemness lost
after fate selection and 2) once lost, how is stemness re-accessed by AT2s after injury to regenerate the alveolar
epithelium. Our recent work indicates perinatal AT2s retain stemness for weeks after fate selection, despite being
transcriptionally and functionally differentiated. This proposal focuses on a putative repressor of AT2 stemness,
determining its target and modes of action, whether and how it is overridden in adult AT2s to re-access stemness,
and investigating the extent to which it is dysfunctional in pulmonary fibrosis - wherein aberrant AT2 states are
observed. Our first aim focuses on a putative repressor of stemness we identified, the CCAAT enhancer binding
protein C/EBPα. Using transgenic mouse models, we will test the requirement of Cebpa for AT2 stemness and
determine whether the targeted stemness program acts in a cell autonomous or non-autonomous manner by
mosaic deletion. Next, scRNAseq, ATAC- and ChIP-seq will identify genes targeted by, as well as molecules
interacting with, C/EBPα. Finally, we will use organotypic culture and transgenic mouse experiments to determine
whether timing of Cebpa expression is itself regulated earlier in development by the polycomb repressive
complex PRC2. The second aim of the proposal is to determine whether and how C/EBPα regulation is
overridden following injury to re-access AT2 stemness and promote repair. We will determine whether C/EBPα
downregulation and AT2 stemness is dependent on AT1 cell death in vivo by performing genetically targeted cell
type specific ablation. Finally, we will compare the spatiotemporal program of C/EBPα regulation we identify
during injury and repair to the fibrotic lung and investigate whether it is dysfunctional in its associated aberrant
AT2 cells. Our approach is innovative, as no transgenic mouse experiments using mosaic deletion or lineage
tracing have been performed for Cebpa in the lung, nor have precise cell ablation studies been conducted in the
adult lung to study C/EBPα regulation. Further, the observation of retained stemness in perinatal AT2 cells is
novel and thus no investigation has implemented a multi-omics approach to understand its underlying
mechanism. Finally, little is known on the role repressive cues play in AT2 cells differentiation and maturation. If
successful, we will have uncovered a novel mechanism of AT2 stemness regulation for the lung field which will
be informative in understanding pulmonary diseases with aberrant epithelial differentiation. Further, our findings
will guide development of novel diagnostics or therapeutics, either for direct use in patients or in culture to control
stem cell differentiation through reprogramming patient’s adult AT2 cells.
Terms: <0-4 weeks old><21+ years old><ATAC sequencing><ATAC-seq><ATACseq><Ablation><Adult><Adult Human><Alveolar><Alveolus><Assay for Transposase-Accessible Chromatin using sequencing><Attenuated><Basal Transcription Factor><Basal transcription factor genes><Bio-Informatics><Bioinformatics><Birth><Bleo><Bleomycin><Body Tissues><Breathing><Bronchial Alveolus><C-EBP Nuclear Protein><C-EBP Proteins><C/EBP><CAAT-Enhancer-Binding Proteins><CCAAT Sequence-Specific DNA-Binding Proteins><CCAAT-Enhancer-Binding Proteins><Cell Body><Cell Communication and Signaling><Cell Death><Cell Differentiation><Cell Differentiation process><Cell Maturation><Cell Signaling><Cell secretion><Cells><Cellular Secretion><ChIP Sequencing><ChIP-seq><ChIPseq><Complex><Cues><DNA Synthesis Factor><Data><Defect><Development><Distal><Down-Regulation><Embryo Development><Embryogenesis><Embryonic Development><Endothelial Cell Growth Factor><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Epithelial Cells><Epithelium><FGF><Fibroblast Growth Factor><Fibroblast Growth Factor Gene Family><Fibroblast Growth Regulatory Factor><Fibrosis><Fibrotic lesions in lung><Gases><Gene Down-Regulation><Gene Expression><Gene Targeting><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic><Genetic Transcription><Image><Injury><Intracellular Communication and Signaling><Investigation><Leanness><Lung><Lung Alveolar Epithelia><Lung Diseases><Lung Respiratory System><Lung Surfactant><Lung Tissue Fibrosis><Lung scar><Lung tissue regeneration><Lung tissue scar><Maintenance><Mediating><Modeling><Modification><Molecular><Natural regeneration><Newborn Infant><Newborns><Parturition><Patients><Perinatal><Peripartum><Play><Polycomb><Protein C><Pulmonary Alveoli><Pulmonary Diseases><Pulmonary Disorder><Pulmonary Fibrosis><Pulmonary Scar><Pulmonary Surfactants><Pulmonary Tissue fibrosis><Pulmonary alveolar structure><RNA Expression><Regeneration><Regulation><Reporting><Repression><Respiratory Aspiration><Respiratory Inspiration><Role><Scarring at the lung><Scarring in the lung><Signal Transduction><Signal Transduction Systems><Signaling><Stem Cell like><Stretching><Surface><Testing><Therapeutic><Thinness><Tissues><Transcription><Transcription Factor Proto-Oncogene><Transcription Repression><Transcription factor genes><Transcriptional Repression><Transgenic Mice><Update><Work><adulthood><alveolar epithelium><assay for transposase accessible chromatin followed by sequencing><assay for transposase accessible chromatin seq><assay for transposase accessible chromatin sequencing><assay for transposase-accessible chromatin with sequencing><attenuate><attenuates><biological signal transduction><cell type><cellular differentiation><chromatin immunoprecipitation-sequencing><developmental><disease of the lung><disorder of the lung><epigenetically><experiment><experimental research><experimental study><experiments><fibrosis in the lung><fibrotic lung><functional status><gene repression><imaging><in vivo><injuries><injury and repair><innovate><innovation><innovative><insight><inspiration><lung alveolus><lung development><lung disorder><lung fibrosis><lung regeneration><lung repair><lung tissue repair><mosaic><mouse model><multiomics><multiple omics><murine model><necrocytosis><new diagnostics><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><newborn child><newborn children><next generation diagnostics><next generation therapeutics><notch><notch protein><notch receptors><novel><novel diagnostics><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><overexpress><overexpression><panomics><premature><prematurity><progenitor><progenitor cell differentiation><progenitor differentiation><programs><pulmonary><pulmonary regeneration><pulmonary repair><regenerate><repair><repaired><scRNA-seq><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><social role><spatiotemporal><stem and progenitor differentiation><stem cell characteristics><stem cell differentiation><stemness><surfactant><transcription factor>