Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1–3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic–osteogenic coupling. Wealso demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly,we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic–osteogenic coupling

Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells

Analysis of p53 Transactivation Domain Mutants Reveals Acad11 as a Metabolic Target Important for p53 Pro-Survival Function

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation

Suppression of PGC-1α Is Critical for Reprogramming Oxidative Metabolism in Renal Cell Carcinoma

Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC), frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC

Additional file 1 of ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma

Additional file 1: Supplemental Figure 1. A) Comparison of cholesterol ester levels in RCC4 cells with and without VHL as measured by LCMS. Prior to lipid extraction cells were cultured overnight in media supplemented with CSFBS +/- 100 μM oleic acid. B) FACS analysis of BODIPY 493/503 fluorescent intensity in RCC4 cells transfected with siACSL3 or non-targeting control and cultured in media supplemented with FBS or FBS and 100uM Oleic Acid. C) qPCR results showing mRNA expression levels of ACSL family members in ccRCC tumors compared to normal renal cortex. Data was generated from TCGA KIRC dataset accessed using cBioPortal. Note that ACSL6 was excluded from presentation due to low expression levels in the renal cortex and associated tumor tissue

Additional file 2 of ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma

Additional file 2: Supplemental Figure 2. A) qPCR results showing ACSL3 mRNA levels in parental RCC4 cells and RCC4 cells expressing siRNA resistant ACSL3 and transfected with ACSL3 siRNA or non-targeting control. ACSL3 expression levels were normalized to TBP levels as a loading control. n=3, error bars depict standard deviation from the mean. B) Oil Red O images of parental RCC4 cells and RCC4 cells expressing siRNA resistant ACSL3 exposed to 50 uM oleic acid for 18 hrs. n=2, images presented at the same magnification. C) qPCR results showing ACSL3 mRNA levels in RCC4 cells transfected with the individual siRNA oligonucleotides from the ACSL3 SMARTpool siRNA that is used throughout the manuscript. n=2, error bars depict the standard deviation from the mean. D) qPCR results showing ACSL3 mRNA expression levels in ccRCC cells transfected with shRNA against ACSL3 or scrambled negative control shRNA. n=3, error bars depict the standard deviation from the mean. E) Images of Oil Red O stained RCC4 cells transfected with the individual ACSL3 siRNA oligonucleotides from the ACSL3 SMARTpool siRNA. n =2, images presented at the same magnification