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Elucidating Selective Gene Regulation by NF-B and Nuclear IκB in Macrophages

  • Author(s): Yeh, George
  • Advisor(s): Smale, Stephen T
  • et al.
Abstract

The NF-κB family of transcription factors (RelA [RELA], c-Rel [REL], RelB [RELB], p50 [NFKB1], and p52 [NFKB2]) regulates many critical biological processes including development, immunity, and inflammatory responses. It has a dominant role in regulating gene transcription in almost all mammalian cell types in response to a wide range of external, internal and environmental cues. Understandably, its misregulation results in the development of severe diseases, such as chronic inflammatory disorders, autoimmune diseases, and cancer. While the NF-κB signaling cascade has been extensively studied and each individual NF-κB knockout strain (Rela-/- for RELA protein, Rel-/- for CREL protein, Relb-/- for RELB protein, Nfkb2-/- for NFKB2 protein, and Nfkb1 -/- for NFKB1 protein) exhibited different phenotypes in mouse, it is still poorly understood whether there exists a distinctive subset of target genes for each NF-κB family member. Our lab previously reported that Il12b gene transcription is strongly dependent on a unique 46-residue segment of c-Rel which promotes strong binding of the protein to two non-consensus κB sequences in the Il12b promote. Here we further investigate NF-κB selectivity at a genomic scale using chromatin RNA-seq and ChIP-seq technologies. Strikingly, c-Rel appears to be exclusively dedicated to controlling the expression of Il12b during lipid A-stimulated mouse bone marrow-derived macrophages (BMDM), as no other genes demonstrated the same dependence and preference for c-Rel binding.

To carefully modulate NF-κB signaling, numerous regulatory layers are used by the cell, one of which is mediated by the nuclear IκB proteins (BCL3 or Bcl-3, NFKBID or IκBNS, and NFKBIZ or IκBz). Unlike prototypic IκBs (IκBa, IκBb, and IκBe), which sequester NF-κB in the cytoplasm during homeostasis and release it upon stimulation, nuclear IκBs are located in the cell nucleus and are thought to serve as co-activators or co-repressors. Furthermore, nuclear IκB selectively interacts with p50 (NFKB1) and p52 (NFKB2), two of the NF-κB proteins that do not contain transactivation domains (TADs). Both Bcl-3 and IκBz, but not IκBNS, contain TADs and thus are able to alter the repressive nature of p50 or p52 via protein-protein interaction. To study the regulatory effects of nuclear IκBs in transcription, we performed chromatin RNA-seq and ChIP-seq on lipid A-stimulated BMDM. Lipid A induces the expression of both primary and secondary response genes in BMDMs, including Nfkb1, Nfkb2, and all three nuclear IκBs. RNA-seq was performed with Nfkb1-/-, Nfkb2-/-, Bcl3-/-, Nfkbid-/-, and Nfkbiz-/- cells. Our results indicate that the absence of p52, Bcl-3, or IκBNS has only minor effects on Lipid A induced TLR4-mediated gene transcription. However, a subset of inducible genes depends strongly on the expression of p50 and IκBz. Taking into account the corroborative data from RNA- and ChIP-seq experiments, we delineate and classify genes that are either directly or indirectly regulated by p50 and IκBz.

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