An RNA polymerase III tissue and tumor atlas uncovers context-specific activities linked to 3D epigenome regulatory mechanisms

RNA polymerase III (Pol III) produces a plethora of small noncoding RNA species involved in diverse cellular processes, from transcription regulation and splicing to RNA stability, translation, and proteostasis. Though Pol III activity is broadly coupled with cellular demands for protein synthesis and growth, a more precise understanding of gene-level dynamics and context-specific expression patterns remains missing, in part due to multiple challenges related to sequencing and mapping Pol III-derived small ncRNAs. Here, we establish a predictive multi-tissue map of Pol III activity across 19 tissues and 22 primary cancers by comprehensively profiling the chromatin accessibility of canonical Pol III-transcribed gene classes. Our framework relies on the unique relationship between gene accessibility and Pol III transcription, inferring activity through uniform binary classification of ATAC-seq enrichment at Pol III-transcribed genes. By characterizing multi-context gene uniformity, we provide a definition of the core Pol III transcriptome, broadly active across specialized tissues, and catalog genes with varied levels of context specificity. Our genomic Pol III atlas uncovers variable levels of activity across tissues, including sharp contraction of the Pol III transcriptome in heart and brain tissues and frequent expansion across diverse cancers. We show that both tissue- and tumor-specific genes are significantly enriched within lamina-associated domains (LADs), and that aberrant expression of nuclear lamin proteins is sufficient to induce Pol III- emergent patterns at tumor-specific genes. Together, these findings link Pol III dynamics to subnuclear compartment- alization and provide a resource for better understanding Pol III expansion and small RNA biogenesis in cancer. Overall design: H1 stem cells and H1-derived cardiomyocytes were harvested (~10 million cells per ChIP experiment) and resuspended in growth media at 1 × 106 cells/mL and cross-linked with rotation at room temperature in 1% formaldehyde for 10 min. Cross-linking was quenched with the addition of 200 mM glycine and an additional 5 min of rotation at room temperature. Cross-linked cells were then spun down and resuspended in 1× RIPA lysis buffer, followed by chromatin shearing via sonication (3 cycles using a Branson sonicator: 30 s on, 60 s off; 20 additional cycles on a Bioruptor sonicator: 30 s on, 30 s off). Individual ChIP experiments were performed on pre-cleared chromatin using antibody-coupled ChIP grade Protein G magnetic beads (Cell Signaling Technology). POLR3A antibody was obtained from ThermoFisher Scientific (PA5-58170). 5 ug of antibody per ChIP was coupled to 18 uL of beads and rotated overnight with sheared chromatin at 4°C. Beads were then washed 5× in ChIP wash buffer (Santa Cruz), 1× in TE, and chromatin eluted in TE + 1% SDS. Cross-linking was then reversed by incubation at 65°C overnight, followed by digestion of RNA (30 min RNase incubation at 37°C) and digestion of protein (30 min proteinase K incubation at 45°C). ChIP DNA was then purified on a minElute column (Qiagen), followed by DNA library preparation (NEBNext Ultra II DNA Library Prep Kit for Illumina) and size selection of 350-550 bp fragments via gel extraction (Qiagen).