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Results for “Forkhead Box Protein O1”

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4 recordsLinked to original sources
PubMed2025

Progesterone receptor isoform modulation via enhancer activation regulates progesterone signaling in endometrial stromal cells.

OBJECTIVE: To investigate enhancer-mediated regulation of progesterone receptor (PGR) isoforms, PGR-A and PGR-B, in human endometrial stromal cells, and to determine how isoform modulation shapes the progesterone-responsive transcriptome and cistrome relevant to endometrial function. DESIGN: A clustered regularly interspaced short palindromic repeats-based functional genomic screen was used to identify distal enhancers in telomerase-immortalized human endometrial stromal cells. Subsequent clustered regularly interspaced short palindromic repeats targeting of identified enhancers and the PGR promoter was used to modulate PGR isoform balance and assess functional consequences. SUBJECTS: None. EXPOSURE: Engineered endometrial stromal cells were treated with medroxyprogesterone acetate or vehicle. MAIN OUTCOME MEASURES: PGR isoform expression was assessed by western blot, the progesterone-responsive transcriptome was characterized by bulk ribonucleic acid sequencing, and the PGR cistrome was characterized by Cut&Run. RESULTS: Two distal PGR enhancers were identified in endometrial stromal cells located approximately 60 and 220 kb upstream of the PGR transcription start site. Clustered regularly interspaced short palindromic repeats-based activation of these enhancers upregulated both PGR-A and PGR-B, whereas promoter activation primarily upregulated PGR-B. Bulk ribonucleic acid sequencing revealed that shifting the PGR isoform balance altered the progesterone-regulated transcriptome: PGR-A/B-equivalent cells exhibited proinflammatory gene signatures, whereas PGR-B-dominant cells demonstrated suppression of inflammatory signaling and altered cell cycle programs. The PGR Cut&Run profiling revealed distinct genomic binding patterns associated with each isoform profile. Integration of the PGR cistrome with chromatin interaction maps suggested that these isoforms directly regulate distinct gene subsets involved in inflammation and fibrosis. Mechanistically, estrogen receptor alpha (ESR1) indirectly activated PGR-A expression, potentially through recruitment of Forkhead box protein O1 (FOXO1) at the distal enhancer, suggesting a noncanonical, enhancer-mediated mechanism of PGR regulation. CONCLUSIONS: Distal enhancers regulate the PGR isoform balance and shape the progesterone-responsive transcriptome in human endometrial stromal cells. This enhancer-mediated mechanism expands current models of PGR regulation beyond promoter-level control and may offer potential therapeutic targets to restore normal progesterone responsiveness in conditions marked by PGR isoform imbalance.

Humans↗
PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗
PubMed2025

Exploring the therapeutic potential of extract in targeting localized adiposity.

OBJECTIVE: To determine direct targeting of localized adiposity through Morus alba Linne bark injection based on pharmacology network analysis. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD) to induce obesity. After 6 weeks on HFD, the water extract of Morus alba L.bark (MAB, 2 mg/mL) was locally injected into one inguinal fat pad, while saline was injected into the other side, 3 times/week for 6 weeks (n = 6/group). The water extract of MAB was freeze-dried and then diluted in saline before use. RESULTS: HFD-fed mice treated with local MAB topical injection showed reduced adipocyte weight and size in inguinal fat pads by dual-energy X-ray absorptiometry. No toxicity changes seen in liver, spleen, kidney tissue, or alanine aminotransferase / aspartate aminotransferase levels in serum by MAB injection. Protein levels of phosphorylated insulin receptor substrate-1 and glucose transporter type 4, and mRNA expression of adiponectin, were increased in inguinal adipose tissue injected with MAB locally. Locally MAB injection led to a decrease in glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, linked to gluconeogenesis, while forkhead box protein O1, which regulates these factors, was increased. Moreover, there was an increase in adenosine 5'-monophosphate-activated protein kinase, related to lipogenesis, as well as elevated levels of hormone-sensitive lipase and fatty acid synthase, both associated with lipolysis. These results support the 'insulin signaling pathway' and 'regulation of lipolysis in adipocytes' identified in the Kyoto Encyclopedia of Genes and Genomes pathway through network analysis. CONCLUSION: This study suggests that MAB topical injection exhibits localized fat reduction by inhibiting insulin resistance, gluconeogenesis and lipogenesis mediator, while activating lipolysis enzymes within targeted adipose site.

Animals↗
PubMed2025

Functional Motif Discovery in FOXO1 Through CRISPR/Cas9 Exon Tiling Scan.

The study of FOXO1, a pivotal transcription factor, has garnered significant attention due to its critical role in diverse cellular processes, including lineage differentiation, apoptosis, cell cycle regulation, and metabolism. To comprehensively understand the functional intricacies of FOXO1, an innovative approach is essential. This chapter highlights employing CRISPR exon scanning as a strategic tool to dissect the functional domains of FOXO1 and unravel its diverse regulatory functions. CRISPR exon scan allows for the identification of functionally important domains based on the levels of sgRNA depletion or enrichment within the FOXO1 gene, providing a unique opportunity to investigate the domain function under relevant biological contexts. This approach enables the systematic exploration of FOXO1's structural domains, shedding light on how distinct regions contribute to its overall function. The comprehensive exon scan analysis using CRISPR technology allows gaining a nuanced understanding of FOXO1's functional diversity and regulatory mechanisms.

Forkhead Box Protein O1↗