A secretome CRISPR screen identifies SFRP1 as a regulator of epidermal self-renewal [ATAC-seq]
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PROVIDER: PRJNA1122788 | ENA |
REPOSITORIES: ENA
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Project description:Secreted epidermal proteins play pivotal roles in cell-cell communication, extracellular matrix remodeling, and antimicrobial defense. However, the complete spectrum of secreted proteins in the epidermis has not been experimentally defined. In this study, we performed mass spectrometry on conditioned media from primary human keratinocytes, identifying 406 proteins that constitute the keratinocyte secretome. To assess functional impacts of uncharacterized secreted proteins on epidermal stem cell behavior, we devised a colony formation assay-based CRISPR screen. The screen identified six candidate proteins that promoted proliferation of epidermal progenitors and two proteins that inhibited it. Secreted frizzled related protein-1 (SFRP1), an inhibitor of Wnt signaling, was the most potent inhibitor of progenitor proliferation in the screen. We discovered that in addition to regulating Wnt activity, SFRP1 restrained epidermal stem cell proliferation by inhibiting ectopic expression of leukemia inhibitory factor (LIF). Collectively, our study defines the keratinocyte secretome, demonstrates the application of a CRISPR screen to assess the function of non-cell autonomous factors, and highlights SFRP1’s role in regulating epidermal balance.
2025-04-30 | GSE269627 | GEO
Project description:Secreted epidermal proteins play pivotal roles in cell-cell communication, extracellular matrix remodeling, and antimicrobial defense. However, the complete spectrum of secreted proteins in the epidermis has not been experimentally defined. In this study, we performed mass spectrometry on conditioned media from primary human keratinocytes, identifying 406 proteins that constitute the keratinocyte secretome. To assess functional impacts of uncharacterized secreted proteins on epidermal stem cell behavior, we devised a colony formation assay-based CRISPR screen. The screen identified six candidate proteins that promoted proliferation of epidermal progenitors and two proteins that inhibited it. Secreted frizzled related protein-1 (SFRP1), an inhibitor of Wnt signaling, was the most potent inhibitor of progenitor proliferation in the screen. We discovered that in addition to regulating Wnt activity, SFRP1 restrained epidermal stem cell proliferation by inhibiting ectopic expression of leukemia inhibitory factor (LIF). Collectively, our study defines the keratinocyte secretome, demonstrates the application of a CRISPR screen to assess the function of non-cell autonomous factors, and highlights SFRP1’s role in regulating epidermal balance.
2025-04-30 | GSE269626 | GEO
Project description:A secretome CRISPR screen identifies SFRP1 as a regulator of epidermal self-renewal [RNA-seq]
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Project description:Troy/Tnfrsf19 marks epidermal cells that govern interfollicular epidermal renewal and cornification
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Project description:The skin epidermis is a highly compartmentalised tissue consisting of a cornifying epithelium called the interfollicular epidermis (IFE) and associated hair follicles (HFs). Several stem cell populations have been described that mark specific sub compartments in the skin but none of them is IFE-specific. Here we identify Troy as a marker of IFE and HF infundibulum basal layer cells in embryonic and adult human and mouse epidermis. Genetic lineage-tracing experiments demonstrate that Troy-expressing basal cells contribute to long-term renewal of all layers of the cornifying epithelium. Single-cell transcriptomics and organoid assays of Troy-expressing cells as well as their progeny confirmed stem cell identity as well as the ability to generate differentiating daughter cells. In conclusion, we define Troy as a marker of epidermal basal cells that govern interfollicular epidermal renewal and cornification.
2021-08-05 | GSE165379 | GEO
Project description:Cell lines geneticially engineered to undergo conditional asymmetric self-renewal were used to identify genes whose expression is asymmetric self-renewal associated (ASRA). Non-random sister chromatid segregation occurs concordantly with asymmetric self-renewal in these cell lines. Asymmetric self-renewal occurs when murine embryo fibroblasts that are otherwise p53-null are induced to express physiological levels of wildtype p53 protein (Asym). To distinguish p53-responsive genes that also require induction of asymmetric self renewal (i.e., ASRA genes) and/or non-random sister chromatid segregation for change, an additional control cell line, which continues to symmetrically self-renew (with random sister chromatid segregation) even when p53 is induced, was also compared (Symp53). This congenic cell line constitutively expresses the type II inosine monophosphate dehydrogenase (IMPDH II; the rate-limiting enzmye for guanine ribonucleotide biosynthesis) and, thereby, prevents p53-induced asymmetric self-renewal and non-random sister chromatid segregation. Three biological replicates of asymmetrically self-renewing cultures (Asym1-3) were compared with cultures that were symmetrically self-renewing - either because they did not express p53 (3 biological replicates, Sym1-3) or they expressed constitutive IMPDH II (i.e., not regulated by p53) as well as p53 (2 biological replicates, Symp53_1 and 2.)
2010-12-22 | E-GEOD-25334 | biostudies-arrayexpress
Project description:Long noncoding RNAs (lncRNAs) are a major component of the noncoding genome, but their functions in human development and disease are still incompletely understood. CRISPR interference screens allow for systematic identification of functional lncRNAs that regulate fundamental biological processes such as tissue development and homeostasis. We identified 2,263 lncRNAs transcribed in human epidermis, and performed a comprehensive CRISPR interference screen against all these elements in primary human epidermal progenitors. Our screen identified 209 lncRNAs controlling epidermal progenitor renewal, with the majority (94%) being positive regulators of proliferation. To validate the results, we performed deeper characterization of a novel lncRNA identified from this screen, PRANCR (LINC01481), using RNA interference-mediated transcript knockdown and phenotype analysis in organotypic human tissue. PRANCR depletion inhibited progenitor proliferation through a G2/M cell cycle arrest, reduced clonogenic potential, and impaired functional epidermal stratification. Transcriptome and computational analysis indicated that PRANCR governs progenitor renewal by controlling the expression of 1,136 genes in trans, in part via the p53-E2F4-CHR pathway. This pathway controls the expression of G2/M cell cycle genes and is a novel mechanism of regulating cell proliferation in epidermal progenitors.
2019-11-07 | GSE125400 | GEO
Project description:Cell lines geneticially engineered to undergo conditional asymmetric self-renewal were used to identify genes whose expression is asymmetric self-renewal associated (ASRA). Non-random sister chromatid segregation occurs concordantly with asymmetric self-renewal in these cell lines. Asymmetric self-renewal occurs when murine embryo fibroblasts that are otherwise p53-null are induced to express physiological levels of wildtype p53 protein (Asym). To distinguish p53-responsive genes that also require induction of asymmetric self renewal (i.e., ASRA genes) and/or non-random sister chromatid segregation for change, an additional control cell line, which continues to symmetrically self-renew (with random sister chromatid segregation) even when p53 is induced, was also compared (Symp53). This congenic cell line constitutively expresses the type II inosine monophosphate dehydrogenase (IMPDH II; the rate-limiting enzmye for guanine ribonucleotide biosynthesis) and, thereby, prevents p53-induced asymmetric self-renewal and non-random sister chromatid segregation.
2010-12-22 | GSE25334 | GEO
Project description:Fibroblast to myofibroblast conversion is a major driver of tissue remodeling in organ fibrosis. Several distinct lineages of fibroblasts support homeostatic tissue niche functions, yet, specific activation states and phenotypic trajectories of fibroblasts during injury repair have remained unclear. Here, we combined spatial transcriptomics, longitudinal single-cell RNA-seq and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. We discovered a transitional fibroblast state characterized by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1+ cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1+ transitional fibroblasts and Cthrc1+ myofibroblasts. Tgfβ1 downregulated Sfrp1 in non-invasive transitional cells and induced their switch to an invasive Cthrc1+ myofibroblast identity. Finally, using loss of function experiments we show that autocrine Sfrp1 directly inhibits fibroblast invasion by regulating the RhoA pathway. In summary, our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies Sfrp1 as an autocrine inhibitor of fibroblast invasion during early stages of fibrogenesis.
2023-11-14 | GSE207561 | GEO
Project description:Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterize their contribution to fibrosis. EVs accumulated 14 days post-bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of broncho-alveolar lavage fluid (BALF)-EVs collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in Secreted Frizzled Related Protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as Krt8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 is expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast derived vesicular SFRP1 as fibrotic mediator and potential therapeutic target for IPF.
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