Cytoplasmic IκBα Increases NF-κB-independent Transcription through Binding to Histone Deacetylase (HDAC) 1 and HDAC3

Abstract

IB␣ is an inhibitory molecule that sequesters NF-B dimers in the cytoplasm of unstimulated cells. Upon stimulation, NF-B moves to the nucleus and induces the expression of a variety of genes including IB␣. This newly synthesized IB␣ also translocates to the nucleus, removes activated NF-B from its target genes, and brings it back to the cytoplasm to terminate the phase of NF-B activation. We show here that IB␣ enhances the transactivation potential of several homeodomain-containing proteins such as HOXB7 and Pit-1 through a NF-B-independent association with histone deacetylase (HDAC) 1 and HDAC3 but not with HDAC2, -4, -5, and -6. IB␣ bound both HDAC proteins through its ankyrin repeats, and this interaction was disrupted by p65. Immunofluorescence experiments demonstrated further that IB␣ acts by partially redirecting HDAC3 to the cytoplasm. At the same time, an IB␣ mutant, which lacked a functional nuclear localization sequence, interacted very efficiently with HDAC1 and -3 and intensively enhanced the transactivation potential of Pit-1. Our results support the hypothesis that the NF-B inhibitor IB␣ regulates the transcriptional activity of homeodomain-containing proteins positively through cytoplasmic sequestration of HDAC1 and HDAC3, a mechanism that would assign a new and unexpected role to IB␣.The paradigm of NF-B activation is based on its cytoplasmic sequestration by IB␣ through the masking of its nuclear localization sequence (NLS) 1 in unstimulated cells (1). Recent studies have challenged this hypothesis suggesting that IB␣ constantly shuttles in and out of the nucleus via nuclear import (2) and export sequences (3, 4). Because IB␣ nuclear export is more efficient than its nuclear import process, the NF-B⅐IB␣ complex remains mainly in the cytoplasm of unstimulated cells. Upon stimulation by proinflammatory cytokines, viral infection, or bacterial pathogens, IB␣ is phosphorylated by the IB kinase complex and degraded through the proteasome pathway (5). Then NF-B enters into the nucleus and activates the expression of multiple genes including IB␣. This newly synthesized IB␣ protein moves to the nucleus, removes NF-B from its target genes, and brings it back to the cytoplasm to terminate the phase of NF-B activation. IB␣ is part of a family of ankyrin-containing proteins that also includes IB␤ and IB⑀ (6) as well as p100, p105, and BCL-3 (7). In contrast to the IB proteins, BCL-3 is mainly nuclear and harbors some transactivation potential through heterodimer formation with the NF-B proteins p50 or p52 (8) and recruitment of coactivators such as JAB1 and BARD1 (9).We previously demonstrated that IB␣ enhances the transactivation potential of HOXB7, a homeodomain-containing protein (10). In this study, we elucidated further the underlying mechanism and showed that cytoplasmic IB␣ enhances the transactivation potential of other homeodomain-containing proteins such as Pit-1 and PAX8 through binding to some but not all HDAC proteins, a family of proteins sharing the ability to deacetylate their substrates a...

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