E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

PLoS One. 2018 Oct 25;13(10):e0206253. doi: 10.1371/journal.pone.0206253. eCollection 2018.

Abstract

The cellular inhibitor of apoptosis 1 (cIAP1) is an E3-ubiquitin ligase that regulates cell signaling pathways involved in fundamental cellular processes including cell death, cell proliferation, cell differentiation and inflammation. It recruits ubiquitination substrates thanks to the presence of three baculoviral IAP repeat (BIR) domains at its N-terminal extremity. We previously demonstrated that cIAP1 promoted the ubiquitination of the E2 factor 1 (E2F1) transcription factor. Moreover, we showed that cIAP1 was required for E2F1 stabilization during the S phase of cell cycle and in response to DNA damage. Here, we report that E2F1 binds within the cIAP1 BIR3 domain. The BIR3 contains a surface hydrophobic groove that specifically anchors a conserved IAP binding motif (IBM) found in a number of intracellular proteins including Smac. The Smac N-7 peptide that includes the IBM, as well as a Smac mimetic, competed with E2F1 for interaction with cIAP1 demonstrating the importance of the BIR surface hydrophobic groove. We demonstrated that the first alpha-helix of BIR3 was required for E2F1 binding, as well as for the binding of Smac and Smac mimetics. Overexpression of cIAP1 modified the ubiquitination profile of E2F1, increasing the ratio of E2F1 conjugated with K11- and K63-linked ubiquitin chains, and decreasing the proportion of E2F1 modified by K48-linked ubiquitin chains. ChIP-seq analysis demonstrated that cIAP1 was required for the recruitment of E2F1 onto chromatin. Lastly, we identified an E2F-binding site on the cIAP1-encoding birc2 gene promoter, suggesting a retro-control regulation loop.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Communication / genetics
  • Cell Line
  • Chromatin / metabolism*
  • E2F1 Transcription Factor / chemistry
  • E2F1 Transcription Factor / metabolism*
  • HeLa Cells
  • Humans
  • Inhibitor of Apoptosis Proteins / chemistry
  • Inhibitor of Apoptosis Proteins / genetics
  • Inhibitor of Apoptosis Proteins / metabolism*
  • Protein Binding
  • Protein Domains
  • Signal Transduction
  • Ubiquitination

Substances

  • Chromatin
  • E2F1 Transcription Factor
  • Inhibitor of Apoptosis Proteins

Grants and funding

This work was supported by grants from the ’Comités de Côte d’Or et de l’Yonne‘ of the ’Ligue Contre le Cancer‘ (LD), the ‘European Union’ and the ’Conseil Régional de Bourgogne‘, a French Government grant managed by the ‘French National Research Agency’ under the program “Investissements d’Avenir” (reference ANR-11-LABX-0021), and fellowships from the ’Ministère de l’Enseignement Supérieur et de la Recherche‘ of France (to JA, JB, JC, BD), ’Ligue Nationale Contre le Cancer‘ (VG) and the ’Fondation pour la Recherche Médicale‘ (FRM)(to JB).