Nanoscale imaging of DNA-RNA identifies transcriptional plasticity at heterochromatin.

The three-dimensional structure of DNA is a biophysical determinant of transcription. The density of chromatin condensation is one determinant of transcriptional output. Chromatin condensation is generally viewed as enforcing transcriptional suppression, and therefore, transcriptional output should be inversely proportional to DNA compaction. We coupled stable isotope tracers with multi-isotope imaging mass spectrometry to quantify and image nanovolumetric relationships between DNA density and newly made RNA within individual nuclei. Proliferative cell lines and cycling cells in the murine small intestine unexpectedly demonstrated no consistent relationship between DNA density and newly made RNA, even though localized examples of this phenomenon were detected at nuclear-cytoplasmic transitions. In contrast, non-dividing hepatocytes demonstrated global reduction in newly made RNA and an inverse relationship between DNA density and transcription, driven by DNA condensates at the nuclear periphery devoid of newly made RNA. Collectively, these data support an evolving model of transcriptional plasticity that extends at least to a subset of chromatin at the extreme of condensation as expected of heterochromatin.

Transcriptional synergy in human aortic endothelial cells is vulnerable to combination p300/CBP and BET bromodomain inhibition.

Combinatorial signaling by proinflammatory cytokines synergizes to exacerbate toxicity to cells and tissue injury during acute infections. To explore synergism at the gene-regulatory level, we investigated the dynamics of transcription and chromatin signaling in response to dual cytokines by integrating nascent RNA imaging mass spectrometry, RNA sequencing, amplification-independent mRNA quantification, assay for transposase-accessible chromatin using sequencing (ATAC-seq), and transcription factor profiling. Costimulation with interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) synergistically induced a small subset of genes, including the chemokines CXCL9, -10, and -11. Gene induction coincided with increased chromatin accessibility at non-coding regions enriched for p65 and STAT1 binding sites. To discover coactivator dependencies, we conducted a targeted chemogenomic screen of transcriptional inhibitors followed by modeling of inhibitor dose-response curves. These results identified high efficacy of either p300/CREB-binding protein (CBP) or bromodomain and extra-terminal (BET) bromodomain inhibitors to disrupt induction of synergy genes. Combination p300/CBP and BET bromodomain inhibition at half-maximal inhibitory concentrations (subIC50) synergistically abrogated IFNγ/TNFα-induced chemokine gene and protein levels.