Chromatin Accessibility, Transcriptional Networks, And Fatty Acid (Fa) Synthesis During Adipogenesis In Subcutaneous (Sc) Adipose Stem Cells (Ascs) Of Polycystic Ovary Syndrome (Pcos) Women.

To examine dynamic changes of chromatin accessibility, transcriptional regulation, and total vs. de novo fatty acid (FA) synthesis in subcutaneous (SC) adipose stem cells (ASCs) of normal-weight PCOS women vs. age- and BMI-matched controls. Prospective cohort study. SC abdominal ASCs were obtained from 3 non-Hispanic Caucasian normal-weight PCOS women who had previously shown higher PPARg and CEBPa mRNA expression in newly-formed adipocytes compared to age- and BMI-matched controls. ASCs were cultured in adipogenic medium for 0-12 days. Chromatin and RNA were isolated at days 0, 3, 7 and 12 for Assay Transposase Accessible Chromatin (ATAC)- and RNA-sequencing (RNA-seq). Accessible regions and enriched transcription factor (TF) binding motifs were identified from ATAC-seq using MACS2 and Homer (Hypergeometric Optimization of Motif EnRichment), respectively. Differentially expressed genes identified from RNA-seq were filtered for significance (padj<0.05) and fold-change (>2-fold); gene ontology (GO) functions and canonical pathways were determined using Ingenuity Pathway Analysis, and gene set enrichment analysis (GSEA) was used to identify enriched cellular processes. A subset of ASCs were exposed to adipogenic medium containing 13C-glucose 48 hours before cell harvest at days 7 and 12 to analyze de novo FA synthesis. Different FAs were detected by high pressure liquid chromatograph-mass spectrometry and quantified with Fatty Acid Source Analysis. Paired and unpaired t-test were used for statistical analyses. PCOS cells showed a substantial shift in chromatin accessibility between days 0 and 12, when less accessible chromatin at day 0 became more accessible than control cells by day 12. In correspondence of open chromatin, binding motifs for crucial adipogenic TFs FRA1, ATF3, BATF, FRA2, JUNB, AP-1 and FOSL2 were significantly enriched at days 0, 3 and 12. RNA-seq indicated adipogenic genes (PPARγ, CEBPα, ADIPOQ, AGPAT2, FABP4, LPL, PLIN1; 2-6 fold changes) were upregulated in PCOS cells, while lipid oxidation and FA β-oxidation (z>2, p<0.05) were upregulated GO functions at days 3, 7, and 12. In parallel, up-regulated pathways in PCOS included oxidative phosphorylation and cholesterol biosynthesis at days 3, 7, and 12. GSEA confirmed significantly increased transcripts related to oxidative phosphorylation, peroxisome activity and adipogenesis at days 3, 7, and 12 in PCOS cells (padj<0.05). De novo FA synthesis was 30% of total FA content. During adipocyte formation at day 7, total and de novo synthesis of myristic, palmitic, palmitoleic, and oleic acid increased in ASCs of both female types (day 7 to 12, p<0.02), but were significantly greater in PCOS than control cells at day 12 (p<0.05). ASCs of PCOS women exhibit dynamic changes in chromatin accessibility during adipogenesis, with little accessibility at day 0 to enhanced accessibility by day 12. These developmental programming events may enhance transcriptional activation of crucial adipogenic genes and pathways, promoting greater FA production and fat storage in mature adipocytes.