Transcriptional determinants of lipid mobilization in human adipocytes

Defects in adipocyte lipolysis drive multiple aspects of cardiometabolic disease but the transcriptional framework controlling this process has not been established. To address this, we performed a targeted perturbation screen in primary human adipocytes. Our analyses identified 37 transcriptional regulators of lipid mobilization, which we classified as: i) transcription factors, ii) histone chaperones, and iii) mRNA processing proteins. Based on its strong relationship with multiple readouts of lipolysis in patient samples, we performed mechanistic studies on one hit, ZNF189, which encodes the Zinc Finger Protein 189. Using mass-spectrometry and chromatin profiling techniques, we show that ZNF189 interacts with the tripartite motif family member TRIM28 and represses the transcription of an adipocyte-specific isoform of Phosphodiesterase 1B (PDE1B2). The regulation of lipid mobilization by ZNF189 requires PDE1B2 and overexpression of PDE1B2 is sufficient to attenuate hormone-stimulated lipolysis. Thus, our work identifies the ZNF189-PDE1B2 axis as a determinant of human adipocyte lipolysis and highlights a link between chromatin architecture and lipid mobilization.

Methods

RNAi Screen - Glycerol Release: RNA interference screening experiment, in adipocytes differentiated from CD55+/DPP4+ progenitor cells, was performed with the siGENOME® SMARTpool® siRNA Human Transcription Factor Library (Dharmacon, Lafayette, CO, USA). Lipolysis was assessed by fluorometric detection of glycerol in phenol-red-free media (ThermoFisher Scientific, 21041-033) collected from cells after 72 hours after siRNA transfection. Cytotoxicity was assessed by measuring LDH activity using the Cytotoxicity Detection KitPLUS 548 (Roche, 4744926001) according to the manufacturer’s instructions. Glycerol release and LDH data were analyzed using R v4.2.2. Outliers were identified using the IQR method, additionally triplicates with CV > 20% were excluded from further analysis. The reproducibility of the glycerol release assays was assessed by calculation of the Pearson correlation coefficient between the experimental replicates. Data were normalized and scaled using the robust z-score method. Replicates with LDH release with rz-score > |5| were excluded from the statistical analysis. Targets that significantly affect glycerol release (“hits”) were identified using a One-way ANOVA with Dunnet’s post-hoc test.

 Meta-analysis of the relationship between ZNF189 mRNA clinical parameters
To assess the relationship between ZNF189 mRNA levels and metabolic parameters, we performed a meta-analysis of its expression in subcutaneous WAT samples across nine published clinical cohorts. For the meta-analysis of the relationship between ZNF189 mRNA and clinical parameters, both random and common effects from Spearman correlations are presented.

Lipidomics
Samples were analyzed in both positive and negative ion modes with a resolution of Rm/z=200=500000 for MS and Rm/z=200=30000 for MS/MS experiments. MS/MS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1Da increments. Both MS and MS/MS data were combined to monitor CE, DAG, and TAG ions as ammonium adducts; PC, PC O-, as acetate adducts; and CL, PA, PE, PE O-, PG, PI, and PS as deprotonated anions. MS only was used to monitor LPA, LPE, LPE O-,LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts. Data were analyzed with in-house developed lipid identification software, based on LipidXplorer. Data post-processing and normalization were performed using an in-house developed data management system. Only lipid identifications with a signal-to-noise ratio >and a signal intensity five-fold higher than in corresponding blank samples were considered for further data analysis. Lipid species were included in statistical analysis if they had detectable values across all 6 replicates for both siRNA conditions. Principal components analysis was performed with the FactoMineR package in R. Confidence ellipses were calculated for a 0.95 confidence level. Species-level differences in lipids were assessed by pairwise comparisons using eBayes with the limma package in R, with FDR correction.