Identification of a Distal Locus Enhancer Element That Controls Cell Type–Specific TNF and LTA Gene Expression in Human T Cells

Abstract

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type–specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type–specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.

Human germline biallelic complete NFAT1 deficiency causes the triad of progressive joint contractures, osteochondromas, and susceptibility to B cell malignancy

“…Consistent with the documented NFAT1 deficiency, classical NFAT1 targets such as TNF and LTA 56,57 were significantly decreased in patient naïve B cells.…”

Inflammatory Immune-Associated eRNA: Mechanisms, Functions and Therapeutic Prospects

“…Additionally, smoking-altered methylation and intragenic AHRR enhancer-produced eRNA were found to be necessary prerequisites for monocyte type-specific AHRR transcription ( 124 ). Moreover in THP-1 monocytes, hHS-8 was shown to target dCas9-KRAB at the IRF1 binding site to impair IFN-gamma expression on LPS-induced TNF genes and eRNA, thereby affecting monocyte inflammatory immune action ( 125 ). Current studies suggest that eRNAs regulate the direction of monocyte function; however, their functional mechanism remains unknown.…”

“…In Th1 cells, eRNAs of IFNG upstream SEs were transcribed, while the downstream enhancer exhibited lower levels of P-TEFb occupancy and eRNA production, which affects Th cell differentiation. Luke et al ( 125 ) found that activated CD4+ T cell increased hHS-8, TNF and LTA promoter H3K27 acetylation and nuclease sensitivity while synergistically inducing TNF, LTA and hHS-8 eRNA transcription to regulate TNF mRNA and LTA mRNA, affecting T cell pathology.…”

Enhancers that regulateTNFgene transcription in human macrophages in response to TLR3 stimulation

“…Previous studies reported that a distal hypersensitive site ~8 kb upstream of the human TNF TSS (human hypersensitive site −8kb, hHS-8) is required for LPS-induced TNF and LTA gene expression in macrophages and T cells (21,22). Although our results showed that deletion of the TNF E-16.0 did not affect LTA or LTB gene transcription in human macrophages, we should take caution in interpreting this finding because human macrophages do not transcribe LTA and LTB genes at levels comparable to T cells (28,29).…”

“…Additionally, A distal hypersensitive site ~8 kb upstream of the human TNF TSS (human hypersensitive site -8kb, hHS-8) is required for and mediates IFN-γ-stimulated augmentation of LPS-induced TNF gene expression via binding of IRF1 to a cognate hHS-8 site in human monocytes/macrophages (21). hHS-8 is also coordinately regulated with TNF and LTA gene expression in activated human T cells via a discrete and highly conserved NFAT binding site (22). However, distal enhancers that regulate TNF gene transcription in human macrophages is incompletely understood.…”