Regulation at multiple levels of NF-κB-mediated transactivation by protein acetylation

Abstract

Evidence has accumulated that deacetylation and acetylation events are implicated in the regulation of NF-κB transcriptional activity. Several groups have reported potentiation of NF-κB-mediated gene induction [by specific inducers (such as TNFα)], following deacetylase inhibition by trichostatin A or sodium butyrate. This potentiation reflects a complex acetylation-dependent regulation of NF-κB-dependent transactivation. This acetylation-dependent regulation occurs at multiple levels. First, acetylation of histones regulates the NF-κB-dependent gene accessibility. Second, unidentified acetylation events modulate temporally the IKK activity and subsequently the duration of NF-κB presence and DNA-binding in the nucleus. Third, direct acetylation of the NF-κB subunits p65 and p50 regulates different NF-κB functions, including transcriptional activation, DNA-binding affinity and IκBα assembly. Finally, acetyltransferases and deacetylases interact directly with several proteins involved in the NF-κB signaling pathway, including NF-κB itself, IκBα, IKKα and IKKγ. These interactions probably allow acetylation of NF-κB itself, of other transcription factors and of histones associated with NF-κB-regulated genes. The present review discusses these recent data obtained on the role of protein acetylation in the regulation of the NF-κB cascade.

Introduction

NF-κB is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in inflammatory and immune responses and cellular proliferation (reviewed in [1], [2], [3], [4]). In mammals, there are five known members of NF-κB/Rel family: p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-Rel and RelB. The most abundant form of NF-κB is a heterodimer of p50 and p65. In unstimulated cells, NF-κB is sequestered in the cytoplasm in an inactive form through interaction with the IκB inhibitory proteins (including IκBα, IκBβ and IκBε, of which the best studied is IκBα). In the canonical activation pathway (Fig. 1, top panel), upon stimulation of cells by specific inducers [such as the proinflammatory cytokine tumor necrosis factor α (TNFα)], IκBα is phosphorylated on two specific serine residues by a large cytoplasmic IκB kinase (IKK) complex, that consists of the kinase catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO/IKKγ (reviewed in [1], [5]). This phosphorylation marks IκBα for polyubiquitination by the E3-SCF^β-TrCP ubiquitin ligase complex, a specific ubiquitin ligase belonging to the SCF (Skp-1/Cul/Fbox) family, and for degradation by the 26S proteasome (reviewed in [3]). Degradation of IκBα allows a rapid and transient translocation of NF-κB to the nucleus, where it activates transcription from a wide variety of promoters, including that of its own inhibitor IκBα. The newly synthesized IκBα enters the nucleus and removes NF-κB from its DNA-binding sites and transports it back to the cytoplasm, thereby terminating NF-κB-dependent transcription (reviewed in [1], [4]).

Protein acetylation influences a broad set of cellular processes including diverse aspects of transcriptional regulation through the recruitment of enzymes: the deacetylases (HDACs) and the acetyltransferases (HATs). The packaging of eukaryotic DNA into chromatin plays an active role in transcriptional regulation by interfering with the accessibility to the transcription factors. Acetylation of specific lysine residues within the amino-terminal tails of nucleosomal histones is generally linked to chromatin disruption and transcriptional activation of genes. Consistent with their role in altering chromatin structure, many transcriptional coactivators (including hGCN5, CBP/p300, P/CAF, SRC-1) possess intrinsic acetyltransferase activity that is critical for their function [6], [7], [8]. Similarly, corepressor complexes include proteins that have deacetylase activity (reviewed in [9], [10], [11], [12], [13]). Moreover, reversible acetylation has also been identified as a critical post-translational modification of non-histone proteins, including general and specific transcription factors, non-histone structural chromosomal proteins, HATs themselves, the HIV-1 Tat protein, non-nuclear proteins (α-tubulin) and nuclear import factors (such as human importin-α). Depending on the functional domain that is modified, acetylation can regulate different functions of these non-histone proteins such as DNA recognition, protein stability, protein–protein interaction and subcellular localisation (reviewed in [7], [14], [15], [16], [17]).

It is now well established that NF-κB-dependent transcription requires multiple coactivators possessing HAT activity: CBP and its paralogue p300, p300/CBP-associated factor (P/CAF) and SRC-1/NcoA-1 [18], [19], [20], [21], [22]. The interactions between NF-κB and these HATs suggest the existence of a link between acetylation events and NF-κB-mediated transactivation. A role for acetylation in the regulation of NF-κB-mediated transactivation has definitively emerged with the finding by our laboratory and other groups, that deacetylase inhibitors (HDACi) (such as trichostatin A (TSA) or sodium butyrate) enhance NF-κB-dependent gene expression in the presence of TNFα [23], [24], [25], [26], [27], [28], [29], [30]. However, the data of these different groups do not converge to a simple link between protein acetylation and NF-κB-dependent regulation, but rather demonstrate that acetylation regulates NF-κB action at multiple levels (Fig. 1). First, it has been shown that in addition to its interactions with acetyltransferases, NF-κB also interacts directly with several deacetylases [25], [26], [27], [31], [32], [33], [34]. A subtle competition between HAT and HDAC activities regulates the acetylation rate of histones and non-histone proteins. The use of HDACi causes a global hyperacetylation of all acetylable proteins in the cell. Second, the most studied NF-κB heterodimer is composed of two subunits p50 and p65, which are both acetylated at multiple lysine residues; the HATs p300/CBP play a major role in this latter process in vivo [27], [34], [35], [36], [37], [38], [39]. The acetylation of different lysines in p65 and p50 regulates different functions of NF-κB, including transcriptional activation, DNA-binding affinity and IκBα assembly. Acetylated forms of p65 are subjected to deacetylation by histone deacetylase 3 (HDAC3). Third, we have demonstrated that HDACi enhance the duration of TNFα-induced NF-κB translocation in the nucleus, thereby participating in the strong transcriptional synergism observed between HDACi and TNFα [29], [30]. Fourth, two distinct classes of NF-κB-activable genes exist: those constitutively and immediately accessible to NF-κB and those that have to be conformationally modified to become accessible to NF-κB. The second class of NF-κB-activable genes are hyperacetylated after stimulation, before NF-κB recruitment [40]. HDACi could thereby increase the accessibility to these latter genes and thus favour their NF-κB-dependent transcription.

In this review, we will describe and discuss these recent data demonstrating the complex involvement of protein acetylation in the regulation of NF-κB-dependent transactivation.