Functional Consequences of a Polymorphism Affecting NF-κB p50-p50 Binding to the TNF Promoter Region

Abstract

Stimulation of the NF-B pathway often causes p65-p50 and p50-p50 dimers to be simultaneously present in the cell nucleus. A natural polymorphism at nucleotide ؊863 in the human TNF promoter (encoding tumor necrosis factor [TNF]) region provides an opportunity to dissect the functional interaction of p65-p50 and p50-p50 at a single NF-B binding site. We found that this site normally binds both p65-p50 and p50-p50, but a single base change specifically inhibits p50-p50 binding. Reporter gene analysis in COS-7 cells expressing both p65-p50 and p50-p50 shows that the ability to bind p50-p50 reduces the enhancer effect of this NF-B site. Using an adenoviral reporter assay, we found that the variant which binds p50-p50 results in a reduction of lipopolysaccharide-inducible gene expression in primary human monocytes. This finding adds to a growing body of experimental evidence that p50-p50 can inhibit the transactivating effects of p65-p50 and illustrates the potential for genetic modulation of inflammatory gene regulation in humans by subtle nucleotide changes that alter the relative binding affinities of different forms of the NF-B complex.The NF-B/Rel family of transcription factors is involved in many physiological processes, including regulation of a wide range of inflammatory mediators (2). Inflammation is a critical component of host defense, but it is also responsible for many of the clinical symptoms of infection and injury and can be fatal if elicited in excess. This raises the fundamental question of how the level of response to NF-B is optimized across a large number of different inflammatory genes. Often this may involve functional interactions between NF-B and other transcription factors (24). This paper considers another mechanism, which has received relatively little attention, namely, through variation in the composition of NF-B dimers that bind to a specific regulatory site. The canonical form of NF-B is a heterodimer comprising a p65 subunit, containing both a DNA binding domain and a domain that is essential for transcriptional activation, plus a p50 subunit which has a DNA binding domain but no activation domain. The biological role of p50 was initially thought to relate solely to its DNA binding properties within the active p65-p50 complex, but more recent evidence suggests that p50-p50 homodimers are capable of acting as transcriptional repressors. For example, artificial overexpression of p50 acts to suppress the transactivating effects of p65 at some NF-B sites (15,17), and this mechanism has been implicated in the downregulation of major histocompatibility complex expression (16) and in viral postinduction repression of the beta interferon gene (23). Since the optimal binding sequences for p65 and p50 are similar but not identical (12,15,23), it is possible that NF-B-induced responses might be fine-tuned by minor sequence variations that alter the relative binding of p65-p50 and p50-p50 to different regulatory elements. To examine this question, we have investigated the functional properties o...

MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia

“…other groups indicates that p50/p50 homodimers are generally repressors of transcription (Mori et al, 1999;Udalova et al, 2000). However, there are several reports that support the ability of p50/p50 homodimers to activate transcription (Dechend et al, 1999;Kurland et al, 2001;Priel et al, 2005).…”

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

“…The NF-kB/Rel family of transcription factors is involved in a wide range of inflammatory signaling accomplished either by functional interaction with other transcription factors or by variation in the composition of the NF-kB dimers that translocate to the nucleus and bind DNA. 36 Subtle differences in NF-kBbinding sites may favor certain dimer combinations with varied functional transcriptional consequences. 36 We plan to study the effects of the putative gain of a NF-kB TFBS in the future.…”

“…36 Subtle differences in NF-kBbinding sites may favor certain dimer combinations with varied functional transcriptional consequences. 36 We plan to study the effects of the putative gain of a NF-kB TFBS in the future.…”

An analysis of tumor necrosis factor α gene polymorphisms and haplotypes with natural clearance of hepatitis C virus infection

“…The effects of À863A are dependent on the proportion of the p65 and p50 NF-kB subunits present, which varies with the cell type and its activation factors. 28,29 In vivo, it is unclear how the interplay of these various transcription factors and variables would ultimately affect the complex process of TNF expression.…”