Assembly of major histocompatability complex (MHC) class II transcription factors: Association and promoter recognition of RFX proteins
Assembly of major histocompatability complex (MHC) class II transcription factors: Association and promoter recognition of RFX proteins
Engineering Village 2
2006 Elsevier Inc.
Accession number: 04438414487
Title: Assembly of major histocompatability complex (MHC) class II transcription factors: Association and promoter recognition of RFX proteins
Authors: Burd, Amy L.; Ingraham, Richard H.; Goldrick, Susan E.; Kroe, Rachel R.; Crute, James J.; Grygon, Christine A.
Author affiliation: Dept. of Immunology and Inflammation, Boehringer Ingelheim Pharmaceut., I., Ridgefield, CT 06877-0368, United States
Serial title: Biochemistry
Abbreviated serial title: Biochemistry
Volume: v 43
Issue: n 40
Issue date: Oct 12 2004
Publication year: 2004
Pages: p 12750-12760
Language: English
ISSN: 0006-2960
CODEN: BICHAW
Document type: Journal article (JA)
Publisher: American Chemical Society, Columbus, OH 43210-3337, United States
Abstract: Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (Kd = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.
Number of references: 48
Ei main heading: Proteins
Ei controlled terms: Genes - Fluorescence - Anisotropy - DNA - Stoichiometry
Uncontrolled terms: Class II transcriptional activator (CIITA) - Major histocompatibility complex (MHC)
Ei classification codes: 804.1 Organic Compounds - 461.2 Biological Materials - 741.1 Light/Optics - 931.2 Physical Properties of Gases, Liquids & Solids - 801.4 Physical Chemistry
Treatment: Experimental (EXP)
DOI: 10.1021/bi030262o
Database: Compendex
Compilation and indexing terms, ? 2006 Elsevier Inc. All rights reserved
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