1 research outputs found
Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and <i>Acta2</i> Repression by Purine-Rich Element-Binding Protein B
Myofibroblast
differentiation is characterized by an increased
level of expression of cytoskeletal smooth muscle Ī±-actin. In
human and murine fibroblasts, the gene encoding smooth muscle Ī±-actin
(<i>Acta2</i>) is tightly regulated by a network of transcription
factors that either activate or repress the 5ā² promoterāenhancer
in response to environmental cues signaling tissue repair and remodeling.
Purine-rich element-binding protein B (PurĪ²) suppresses the
expression of <i>Acta2</i> by cooperatively interacting
with the sense strand of a 5ā² polypurine sequence containing
an inverted MCAT <i>cis</i> element required for gene activation.
In this study, we evaluated the chemical basis of nucleoprotein complex
formation between the PurĪ² repressor and the purine-rich strand
of the MCAT element in the mouse <i>Acta2</i> promoter.
Quantitative single-stranded DNA (ssDNA) binding assays conducted
in the presence of increasing concentrations of monovalent salt or
anionic detergent suggested that the assembly of a high-affinity nucleoprotein
complex is driven by a combination of electrostatic and hydrophobic
interactions. Consistent with the results of pH titration analysis,
site-directed mutagenesis revealed several basic amino acid residues
in the intermolecular (R267) and intramolecular (K82 and R159) subdomains
that are essential for PurĪ² transcriptional repressor function
in <i>Acta2</i> promoterāreporter assays. In keeping
with their diminished <i>Acta2</i> repressor activity in
fibroblasts, purified PurĪ² variants containing an R267A mutation
exhibited reduced binding affinity for purine-rich ssDNA. Moreover,
certain double and triple-point mutants were also defective in binding
to the <i>Acta2</i> corepressor protein, Y-box-binding protein
1. Collectively, these findings establish the repertoire of noncovalent
interactions that account for the unique structural and functional
properties of PurĪ²