Abstract
SummaryCCAAT enhancer-binding protein beta (C/EBPβ) is a pioneer transcription factor that specifies cell differentiation. C/EBPβ is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPβ differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPβ alters protein interactions and cell differentiation, suggesting that a C/EBPβ PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPβ using an array technique based on spot-synthesized C/EBPβ-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ∼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPβ acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function.
Highlights
CCAAT enhancer-binding proteins (C/EBPa, b, d, ) are basic leucine zipper transcription factors that regulate chromatin structure and gene expression by dimerization and binding to cis-regulatory, palindromic 50ATTGC$GCAAT30, or quasi-palindromic DNA sites in gene enhancers and promoters
The CCAAT enhancer-binding protein beta (C/EBPb) Peptide Matrix The occurrence of novel post-translational modifications (PTM) on endogenous C/EBPb was investigated by mass spectrometry of C/EBPb immunoprecipitates derived from the human anaplastic lymphoma cell line SU-DHL1 that critically depends for growth and survival on C/EBPb (Anastasov et al, 2010; Jundt et al, 2005)
To systematically explore the C/EBPb interactome and its PTM-specific regulation, we developed the PRISMA technology, which is based on a solid matrix consisting of immobilized peptides spanning the entire primary structure of rat C/EBPb (297 amino acids), 352 iScience 13, 351–370, March 29, 2019
Summary
CCAAT enhancer-binding proteins (C/EBPa, b, d, ) are basic leucine zipper (bZip) transcription factors that regulate chromatin structure and gene expression by dimerization and binding to cis-regulatory, palindromic 50ATTGC$GCAAT30, or quasi-palindromic DNA sites in gene enhancers and promoters. Prototypic C/EBPb is widely expressed, highly regulated at the post-transcriptional level, and integrated in many signaling events communicating extracellular cues to epigenetic changes, examples of which include adipogenesis, hematopoiesis, innate immunity, female fertility, skin function, apoptosis, and cellular senescence (Nerlov, 2008; Rodier and Campisi, 2011; Tsukada et al, 2011). C/EBPb communicates with numerous other transcription factors, co-factors, histone modifiers, and chromatin remodeling complexes to alter the susceptibility of chromatin to the gene regulatory machinery. The chromatin and gene regulatory functionality of C/EBPb is linked to distinct regions and post-translational modifications (PTMs) that suspend auto-inhibition, direct the activity of C/EBPb, and regulate recruitment of chromatin remodelers and writers of histone modifications (Kowenz-Leutz and Leutz, 1999; Kowenz-Leutz et al, 1994, 2010; Lee et al, 2010b; Mo et al, 2004; Pless et al, 2008; Siersbaek et al, 2011)
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