The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly.
HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.
Mitsouras
Katherine
K
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737, USA.
Wong
Ben
B
Arayata
Charina
C
Johnson
Reid C
RC
Carey
Michael
M
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism*; D017665:Hydroxyl Radical; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
C068382:BRLF1 protein, Human herpesvirus 4; C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D017665:Hydroxyl Radical; D004247:DNA