Structural Requirements for Assembly of the CSL·Intracellular Notch1·Mastermind-like 1 Transcriptional Activation Complex*

Ligand binding by Notch receptors triggers a series of proteolytic cleavages that liberate the intracellular portion of Notch (ICN) from the cell membrane, permitting it to translocate to the nucleus. Nuclear ICN binds to a highly conserved DNA-binding transcription factor called CSL (also known as RBP-Jκ, CBF1, Suppressor of Hairless, and Lag-1) and recruits Mastermind-like transcriptional co-activators to form a transcriptional activation complex. Using bioinformatics tools, we identified a Rel homology region (RHR) within CSL that was used as a guide to determine the minimal protein requirements for ternary complex formation. The RHR of CSL contains both the N- and C-terminal β-sheet domains (RHR-n and RHR-c) of typical Rel transcription factors, as judged by circular dichroism spectra. Binding of monomeric CSL to DNA requires the entire RHR of CSL and an additional 125-residue N-terminal sequence, whereas binding to ICN requires only the RHR-n domain. Although the RAM (RBP-Jκ (recombination-signal-sequence-binding protein for Jκ genes)-associated molecule) domain of ICN is flexible and relatively unstructured as an isolated polypeptide in solution, it associates stably with CSL on DNA. Recruitment of Mastermind-like 1 (MAML1) to CSL·ICN complexes on DNA requires inclusion of the ankyrin repeat domain of ICN, and N- and C-terminal sequences of CSL extending beyond the DNA-binding region. The requirement for cooperative assembly of the MAML1·ICN·CSL·DNA complex suggests that a primary function of ICN is to render CSL competent for MAML loading. On the basis of our results, we present a working structural model for the organization of the MAML1·ICN·CSL·DNA complex.

[1]  James D. Griffin,et al.  Growth Suppression of Pre-T Acute Lymphoblastic Leukemia Cells by Inhibition of Notch Signaling , 2003, Molecular and Cellular Biology.

[2]  U. Lendahl,et al.  p300 and PCAF Act Cooperatively To Mediate Transcriptional Activation from Chromatin Templates by Notch Intracellular Domains In Vitro , 2002, Molecular and Cellular Biology.

[3]  J. Aster,et al.  Notch signaling as a therapeutic target. , 2002, Current opinion in chemical biology.

[4]  A. Capobianco,et al.  Characterization of a High-Molecular-Weight Notch Complex in the Nucleus of Notchic-Transformed RKE Cells and in a Human T-Cell Leukemia Cell Line , 2002, Molecular and Cellular Biology.

[5]  K. Jones,et al.  Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. , 2002, Genes & development.

[6]  J. Stroud,et al.  Structure of a TonEBP–DNA complex reveals DNA encircled by a transcription factor , 2002, Nature Structural Biology.

[7]  D. Barrick,et al.  Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 1. Solution conformational and hydrodynamic properties. , 2001, Biochemistry.

[8]  D. Barrick,et al.  Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding. , 2001, Biochemistry.

[9]  P. Cramer,et al.  Crystal structure of the ankyrin repeat domain of Bcl‐3: a unique member of the IκB protein family , 2001, The EMBO journal.

[10]  N. Sreerama,et al.  Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. , 2000, Analytical biochemistry.

[11]  James D. Griffin,et al.  MAML1, a human homologue of Drosophila Mastermind, is a transcriptional co-activator for NOTCH receptors , 2000, Nature Genetics.

[12]  Jon C. Aster,et al.  Essential Roles for Ankyrin Repeat and Transactivation Domains in Induction of T-Cell Leukemia by Notch1 , 2000, Molecular and Cellular Biology.

[13]  M. Sternberg,et al.  Enhanced genome annotation using structural profiles in the program 3D-PSSM. , 2000, Journal of molecular biology.

[14]  J. Kimble,et al.  Mastermind is a putative activator for Notch , 2000, Current Biology.

[15]  J. Kimble,et al.  LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway , 2000, Nature.

[16]  S. Artavanis-Tsakonas,et al.  Notch signaling: cell fate control and signal integration in development. , 1999, Science.

[17]  J. Hsieh,et al.  CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[18]  G. Ghosh,et al.  The Crystal Structure of the IκBα/NF-κB Complex Reveals Mechanisms of NF-κB Inactivation , 1998, Cell.

[19]  R. Evans,et al.  A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. , 1998, Genes & development.

[20]  S. Harrison,et al.  Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA , 1998, Nature.

[21]  G. Ghosh,et al.  Crystal structure of p50/p65 heterodimer of transcription factor NF-κB bound to DNA , 1998, Nature.

[22]  T. Honjo,et al.  LIM Protein KyoT2 Negatively Regulates Transcription by Association with the RBP-J DNA-Binding Protein , 1998, Molecular and Cellular Biology.

[23]  M J May,et al.  NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. , 1998, Annual review of immunology.

[24]  S. Minoguchi,et al.  Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives. , 1997, Development.

[25]  S. Minoguchi,et al.  RBP-L, a transcription factor related to RBP-Jkappa , 1997, Molecular and cellular biology.

[26]  E. Kieff,et al.  Oncogenic Forms of NOTCH1 Lacking Either the Primary Binding Site for RBP-Jκ or Nuclear Localization Sequences Retain the Ability to Associate with RBP-Jκ and Activate Transcription* , 1997, The Journal of Biological Chemistry.

[27]  M. Bosenberg,et al.  lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H). , 1996, Development.

[28]  J. Hsieh,et al.  Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2 , 1996, Molecular and cellular biology.

[29]  P. Sigler,et al.  Structure of NF-κB p50 homodimer bound to a κB site , 1998, Nature.

[30]  Christel Brou,et al.  Signalling downstream of activated mammalian Notch , 1995, Nature.

[31]  Gregory L. Verdine,et al.  Structure of the NF-κB p50 homodimer bound to DNA , 1995, Nature.

[32]  M. Fortini,et al.  The suppressor of hairless protein participates in notch receptor signaling , 1994, Cell.

[33]  T. Honjo,et al.  Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-Jx , 1994 .

[34]  B. Rost,et al.  Combining evolutionary information and neural networks to predict protein secondary structure , 1994, Proteins.

[35]  T. Honjo,et al.  Recognition sequence of a highly conserved DNA binding protein RBP-Jx , 1994 .

[36]  J. Sklar,et al.  Functional analysis of the TAN-1 gene, a human homolog of Drosophila notch. , 1994, Cold Spring Harbor symposia on quantitative biology.

[37]  B. Rost,et al.  Prediction of protein secondary structure at better than 70% accuracy. , 1993, Journal of molecular biology.

[38]  J. Posakony,et al.  Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates , 1992, Cell.

[39]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

[40]  D. Smoller,et al.  Molecular analysis of the neurogenic locus mastermind of Drosophila melanogaster. , 1988, Genetics.

[41]  W F van Gunsteren,et al.  Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: Application to the DNA binding domain of lac repressor from Escherichia coli , 1988, Proteins.

[42]  S. Artavanis-Tsakonas,et al.  Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[43]  H. Edelhoch,et al.  Spectroscopic determination of tryptophan and tyrosine in proteins. , 1967, Biochemistry.