Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells

The three-dimensional context of endogenous chromosomal regions may contribute to the regulation of gene clusters by influencing interactions between transcriptional regulatory elements. In this study, we investigated the effects of tumor necrosis factor (TNF) signaling on spatiotemporal enhancer-promoter interactions in the human tumor necrosis factor (TNF)/lymphotoxin (LT) gene locus, mediated by CCCTC-binding factor (CTCF)-dependent chromatin insulators. The cytokine genes LT alpha, TNF, and LT beta are differentially regulated by NF-kappa B signaling in inflammatory and oncogenic responses. We identified at least four CTCF-enriched sites with enhancer-blocking activities and a TNF-responsive TE2 enhancer in the TNF/LT locus. One of the CTCF-enriched sites is located between the early-inducible LT alpha/TNF promoters and the late-inducible LT beta promoter. Depletion of CTCF reduced TNF expression and accelerated LT beta induction. After TNF stimulation, via intrachromosomal dynamics, these insulators mediated interactions between the enhancer and the LT alpha/TNF promoters, followed by interaction with the LT beta promoter. These results suggest that insulators mediate the spatiotemporal control of enhancer-promoter associations in the TNF/LT gene cluster.