cis -Regulatory Circuits Regulating NEK6 Kinase Overexpression in Transformed B Cells Are Super-Enhancer Independent

Abstract

SUMMARY Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested. Here, we dissect cis-regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis-regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets.

High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human

“…c HiDRA identifies enhancer activity within an intron in the immunoglobulin heavy chain locus. Red bar, DNA segment active in luciferase assay performed by Huang et al 23 . Orange bar and highlight, region identified by HiDRA as having transcriptional regulatory activity.…”

“…e Comparison of HiDRA-called active regions with luciferase assay results for 13 enhancers at the NEK6 locus. Luciferase experiments are colored in red or gray depending on whether DNA fragments drive luciferase activity in GM12878 cells as determined by Huang et al 23 …”

“…2c). To assess the quantitative accuracy of HiDRA relative to luciferase assays, we compared active HiDRA regions and luciferase results across 21 putative enhancers predicted and tested independently by Huang et al 23 . We found a 0.88 Pearson correlation between measured luciferase activity and HiDRA activity, confirming the accuracy and quantitative nature of our high-throughput approach (Fig.…”

High-resolution genome-wide functional dissection of transcriptional regulatory regions in human

“…To assess the quantitative accuracy of HiDRA relative to luciferase assays, we compared active HiDRA regions and luciferase results across 21 putative enhancers predicted and tested independently by Huang et al 19 . We found a 0.88 Pearson correlation between measured luciferase activity and HiDRA activity, confirming the accuracy and quantitative nature of our high-throughput approach (Fig.…”

HIFI: estimating DNA-DNA interaction frequency from Hi-C data at restriction-fragment resolution

“…e IF matrices produced by HIFI-MRF (top) and fixed binning (bottom) for the 200-kb NEK6 locus (chr9:126879748-127079891). Regulatory regions identified in Huang et al [62] are marked SE (super enhancer), CE1 (conventional enhancer), and NEK6-TSS1 and NEK6-TSS2 (alternative promoters). Notice how all these regions lie between visible subTADs chromosomes to produce lower-resolution matrices [10].…”