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Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics

Nature Structural & Molecular Biology volume 11, pages 73–80 (2004)Cite this article

Abstract

During erythroid differentiation, β-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for β-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of β-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of β-globin transcription in committed cells before terminal differentiation.

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Figure 1
Figure 2: Western blot analysis of MafK-associated proteins purified before and after MEL cell differentiation.
Figure 3: Gel-filtration analysis of MEL cell nuclear extracts before and after differentiation.
Figure 4: Analysis of the occupancy of the β-globin locus by MafK-interacting proteins during MEL cell differentiation.
Figure 5: The exchange of MafK-heterodimerization partner leads to the activation of transcription of the adult β-major globin (βmaj) gene during terminal erythroid differentiation in MEL cells.

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Acknowledgements

We thank F.J. Dilworth and T. Sawado for helpful discussions, F.J. Dilworth for critically reading the manuscript, J. Eng and A. Nesvizhskii for help in the analysis of the mass spectrometry data, E. Soutoglou for helpful advice on the ChIP experiments, J. Sun for the generation of Bach1 antiserum, P. Chambon for the gift of the HP1γ antibody and V. Blank for the gift of the MafG antibody. M.B. is supported by a long-term fellowship from the Human Frontier Science Program Organization. This project has been funded in part with federal funds as part of the NHLBI Proteomics Initiative from the National Heart, Lung and Blood Institute, US National Institutes of Health (NIH), under contract no. N01-HV-28179 (R.A.), and with NIH grants DK44746 and HL57620 (M.G.).

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  1. Claire Francastel

    Present address: Institut National de la Santé et de la Recherche Médicale U474, Institut Cochin de Génétique Moléculaire, Paris, 75014, France

Authors and Affiliations

  1. Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, 98109, Washington, USA

    Marjorie Brand, Nicolas T Kummer, Joan Hamilton, Claire Francastel & Mark Groudine

  2. Institute for Systems Biology, Seattle, 98103, Washington, USA

    Jeffrey A Ranish & Ruedi Aebersold

  3. Department of Biochemistry, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Hiroshima, 734-8551, Japan

    Kazuhiko Igarashi

  4. Departments of Pathology and Developmental Biology, Howard Hughes Medical Institute, Stanford University Medical School, Palo Alto, 94305, California, USA

    Tian H Chi & Gerald R Crabtree

  5. Department of Radiation Oncology, University of Washington School of Medicine, Seattle, 98104, Washington, USA

    Mark Groudine

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Brand, M., Ranish, J., Kummer, N. et al. Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics. Nat Struct Mol Biol 11, 73–80 (2004). https://doi.org/10.1038/nsmb713

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