Structural Basis and Specificity of Acetylated Transcription Factor GATA1 Recognition by BET Family Bromodomain Protein Brd3

ABSTRACT Recent data demonstrate that small synthetic compounds specifically targeting bromodomain proteins can modulate the expression of cancer-related or inflammatory genes. Although these studies have focused on the ability of bromodomains to recognize acetylated histones, it is increasingly becoming clear that histone-like modifications exist on other important proteins, such as transcription factors. However, our understanding of the molecular mechanisms through which these modifications modulate protein function is far from complete. The transcription factor GATA1 can be acetylated at lysine residues adjacent to the zinc finger domains, and this acetylation is essential for the normal chromatin occupancy of GATA1. We have recently identified the bromodomain-containing protein Brd3 as a cofactor that interacts with acetylated GATA1 and shown that this interaction is essential for the targeting of GATA1 to chromatin. Here we describe the structural basis for this interaction. Our data reveal for the first time the molecular details of an interaction between a transcription factor bearing multiple acetylation modifications and its cognate recognition module. We also show that this interaction can be inhibited by an acetyllysine mimic, highlighting the importance of further increasing the specificity of compounds that target bromodomain and extraterminal (BET) bromodomains in order to fully realize their therapeutic potential.

[1]  A. Breeze.  Isotope-filtered NMR methods for the study of biomolecular structure and interactions , 2000 .

[2]  Wei Gu,et al.  Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain , 1997, Cell.

[3]  S. Beck,et al.  Chromosomal localization, gene structure and transcription pattern of the ORFX gene, a homologue of the MHC-linked RING3 gene. , 1997, Gene.

[4]  G. Blobel,et al.  Acetylation of GATA-1 is required for chromatin occupancy. , 2006, Blood.

[5]  R. Hardison,et al.  Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes , 2011, Proceedings of the National Academy of Sciences of the United States of America.

[6]  David E Neal,et al.  Tip60 and Histone Deacetylase 1 Regulate Androgen Receptor Activity through Changes to the Acetylation Status of the Receptor* , 2002, The Journal of Biological Chemistry.

[7]  J. Lippincott-Schwartz,et al.  A Bromodomain Protein, MCAP, Associates with Mitotic Chromosomes and Affects G2-to-M Transition , 2000, Molecular and Cellular Biology.

[8]  Yixue Li,et al.  Regulation of Cellular Metabolism by Protein Lysine Acetylation , 2010, Science.

[9]  Michael Nilges,et al.  ARIA: automated NOE assignment and NMR structure calculation , 2003, Bioinform..

[10]  S. Mujtaba,et al.  Structure and acetyl-lysine recognition of the bromodomain , 2007, Oncogene.

[11]  Xin Cai,et al.  An acetylation switch in p53 mediates holo-TFIID recruitment. , 2007, Molecular cell.

[12]  J. Bieker,et al.  Acetylation of EKLF Is Essential for Epigenetic Modification and Transcriptional Activation of the β-Globin Locus , 2008, Molecular and Cellular Biology.

[13]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[14]  T. Yamagata,et al.  Acetylation of GATA‐3 affects T‐cell survival and homing to secondary lymphoid organs , 2000, The EMBO journal.

[15]  V. Ogryzko,et al.  p300 and p300/cAMP-response Element-binding Protein-associated Factor Acetylate the Androgen Receptor at Sites Governing Hormone-dependent Transactivation* , 2000, The Journal of Biological Chemistry.

[16]  V. Ogryzko,et al.  Regulation of activity of the transcription factor GATA-1 by acetylation , 1998, Nature.

[17]  J. Mackay,et al.  Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions. , 2000, Structure.

[18]  R. Clubb,et al.  Improved NMR spectra of a protein–DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment , 2001, Journal of biomolecular NMR.

[19]  M. Stallcup,et al.  Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. , 2009, Molecular endocrinology.

[20]  E. Seto,et al.  Acetylation and deacetylation of non-histone proteins. , 2005, Gene.

[21]  Jason A. Lowry,et al.  GATA-1: one protein, many partners. , 2006, The international journal of biochemistry & cell biology.

[22]  A. Harel-Bellan,et al.  Acetylation of MyoD by p300 Requires More Than Its Histone Acetyltransferase Domain* , 2001, The Journal of Biological Chemistry.

[23]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[24]  J. Hayes,et al.  Structures and interactions of the core histone tail domains. , 2003, Biopolymers.

[25]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[26]  Ming-Ming Zhou,et al.  Brd4 Coactivates Transcriptional Activation of NF-κB via Specific Binding to Acetylated RelA , 2008, Molecular and Cellular Biology.

[27]  J. Visvader,et al.  Structural basis for the recognition of ldb1 by the N‐terminal LIM domains of LMO2 and LMO4 , 2003, The EMBO journal.

[28]  Danny Reinberg,et al.  Is there a code embedded in proteins that is based on post-translational modifications? , 2008, Nature Reviews Molecular Cell Biology.

[29]  P. Wolf,et al.  A critical reappraisal of Waddell's technique for ultraviolet spectrophotometric protein estimation. , 1983, Analytical biochemistry.

[30]  K. Senger,et al.  Acetylation of HMG I(Y) by CBP turns off IFN beta expression by disrupting the enhanceosome. , 1998, Molecular cell.

[31]  W. Greene,et al.  Duration of nuclear NF-kappaB action regulated by reversible acetylation. , 2001, Science.

[32]  Lei Zeng,et al.  Structure and ligand of a histone acetyltransferase bromodomain , 1999, Nature.

[33]  C. Rice,et al.  Suppression of inflammation by a synthetic histone mimic , 2010, Nature.

[34]  Eric Verdin,et al.  Structural basis of lysine-acetylated HIV-1 Tat recognition by PCAF bromodomain. , 2002, Molecular cell.

[35]  C. Allis,et al.  Translating the Histone Code , 2001, Science.

[36]  A. Harel-Bellan,et al.  Acetylation is important for MyoD function in adult mice , 2006, EMBO reports.

[37]  Wenjun Zhang,et al.  Site-Specific Acetylation by p300 or CREB Binding Protein Regulates Erythroid Krüppel-Like Factor Transcriptional Activity via Its Interaction with the SWI-SNF Complex , 2001, Molecular and Cellular Biology.

[38]  Roberto Sanchez,et al.  Structural mechanism of the bromodomain of the coactivator CBP in p53 transcriptional activation. , 2004, Molecular cell.

[39]  T. Kouzarides,et al.  Regulation of E2F1 activity by acetylation , 2000, The EMBO journal.

[40]  Stephanie Spange,et al.  Acetylation of non-histone proteins modulates cellular signalling at multiple levels. , 2009, The international journal of biochemistry & cell biology.

[41]  H. Saito,et al.  Functional regulation of GATA‐2 by acetylation , 2003, Journal of leukocyte biology.

[42]  A. Harel-Bellan,et al.  CREB-binding Protein/p300 Activates MyoD by Acetylation* , 2000, Journal of Biological Chemistry.

[43]  Masayuki Yamamoto,et al.  GATA1 Function, a Paradigm for Transcription Factors in Hematopoiesis , 2005, Molecular and Cellular Biology.

[44]  Binh Nguyen,et al.  Biosensor-surface plasmon resonance: quantitative analysis of small molecule-nucleic acid interactions. , 2007, Methods.

[45]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[46]  Andrew J. Bannister,et al.  Regulation of gene expression by transcription factor acetylation , 2000, Cellular and Molecular Life Sciences CMLS.

[47]  Toru Kita,et al.  Differentiation of embryonic stem cells into cardiac myocytes , 2002 .

[48]  W. Greene,et al.  Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF‐κB , 2002, The EMBO journal.

[49]  Marko Marttila,et al.  An Arginine Switch in the Species B Adenovirus Knob Determines High-Affinity Engagement of Cellular Receptor CD46 , 2008, Journal of Virology.

[50]  L. Nieba,et al.  Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics. , 1996, Analytical biochemistry.

[51]  William B. Smith,et al.  Selective inhibition of BET bromodomains , 2010, Nature.

[52]  C. Allis,et al.  Roles of histone acetyltransferases and deacetylases in gene regulation , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[53]  G. Blobel,et al.  CREB-Binding Protein Acetylates Hematopoietic Transcription Factor GATA-1 at Functionally Important Sites , 1999, Molecular and Cellular Biology.

[54]  K. Wüthrich,et al.  Torsion angle dynamics for NMR structure calculation with the new program DYANA. , 1997, Journal of molecular biology.

[55]  S. Orkin,et al.  CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[56]  T. Umehara,et al.  Structural implications for K5/K12‐di‐acetylated histone H4 recognition by the second bromodomain of BRD2 , 2010, FEBS letters.

[57]  M. Nilges,et al.  Refinement of protein structures in explicit solvent , 2003, Proteins.

[58]  K. S. Egorova,et al.  Lysine methylation of nonhistone proteins is a way to regulate their stability and function , 2010, Biochemistry (Moscow).

[59]  D. Torchia,et al.  Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated IIIGlc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques , 1993, Protein Science.

[60]  Jeroen Krijgsveld,et al.  Cooperative binding of two acetylation marks on a histone tail by a single bromodomain , 2009, Nature.

[61]  G M Clore,et al.  An efficient and cost-effective isotope labeling protocol for proteins expressed in shape Escherichia coli , 1998, Journal of biomolecular NMR.

[62]  L. Kay,et al.  Latent and active p53 are identical in conformation , 2001, Nature Structural Biology.

[63]  Eric Verdin,et al.  Duration of Nuclear NF-κB Action Regulated by Reversible Acetylation , 2001, Science.

[64]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[65]  Jack Greenblatt,et al.  Methods for Measurement of Intermolecular NOEs by Multinuclear NMR Spectroscopy: Application to a Bacteriophage λ N-Peptide/boxB RNA Complex , 1997 .