Mechanism of Strand-Specific Smooth Muscle α-Actin Enhancer Interaction by Purine-Rich Element Binding Protein B (Purβ)

Abstract

Expression of the smooth muscle α-actin gene in growth-activated vascular smooth muscle cells and stromal fibroblasts is negatively regulated by members of the Pur family of single-stranded DNA/RNA-binding proteins. In particular, Purα and Purβ are postulated to repress transcription by forming helix-destabilizing complexes with the sense strand of an asymmetric polypurine-polypyrimidine tract containing a canonical MCAT enhancer motif in the 5′ region of the gene. Herein, we establish the mechanism of Purβ binding to the purine-rich strand of the enhancer using quantitative methods and purified components. Initial evaluation of DNA-binding specificity and equilibrium stoichiometry via colorimetric-, autoradiographic-, and fluorescence-based assays suggested that Purβ interacts with two distinct G/A-rich sites within the nominal single-stranded enhancer element to form a high affinity 2:1 protein:DNA complex. Statistical mechanical analyses of band shift titrations of the nominal element in conjunction with DNase I footprint titrations of the extended smooth muscle α-actin 5′-flanking region demonstrated that assembly of the nucleoprotein complex likely occurs in a sequential, cooperative, and monomer-dependent fashion. Resolution of the microscopic energetics of the system indicated that monomer association with two non-identical sites flanking the core MCAT motif accounts for the majority of the intrinsic binding affinity of Purβ with intersite cooperativity contributing a ~12-fold increase to the stability of the nucleoprotein complex. These findings offer new insights into the mechanism, energetics, and sequence determinants of Purβ repressor binding to a biologically relevant, contractile phenotype-regulating cis-element while also revealing the thermodynamic confines of putative Purβ-mediated effects on DNA structure.

Isolation and characterization of the core single-stranded DNA-binding domain of purine-rich element binding protein B (Purβ)

“…In support of this contention, the calculated IC50 values for the full-length protein and the core fragment were 1.3 and 0.5 nM, respectively. These values are consistent with previous estimates of the macroscopic binding affinity of Purβ for this sequence element based on quantitative band-shift and footprinting assays [17]. …”

“…4B, the isolated core domain preferentially interacts with purine-rich sense (forward) strands of the PE32 and SPUR32 elements. With respect to the PE32 sequence, individual or combined mutation of the high affinity 3′ and 5′ binding sites and low affinity internal site [17], reduced the interaction of full-length NHis-Purβ and the core tryptic fragment in an analogous fashion (Fig. 4C).…”

“…A unique role for Purβ in actin and myosin isoform class switching has also been suggested in the context of cardiac transplant remodeling, heart failure, and skeletal muscle performance [14–16]. Biochemical explanations for the seemingly dominant repressor activity of Purβ have included its preferred binding to certain trans -activators [12] and its ability to interact cooperatively with multiple ssDNA-binding sites [17]. …”

hnRNP C1/C2 and Pur-beta proteins mediate induction of senescence by oligonucleotides homologous to the telomere overhang

“…This study identified proteins, such as hnRNP A, hnRNP C1/C2, hnRNP D, VDAC 1 and 2, MSI-1, Pur-alpha, and Pur-beta, which are known to be involved in DDR22,25,34–40 (Table S1). In the present study, hnRNP C1/C2, Pur-alpha and Pur-beta, identified by Ingenuity Pathways Analysis, were further investigated due to their key roles in nucleic acid binding, regulation of transcription/translation, and association with telomeres and DDR 18,21,24,26,32…”

“…Further, hnRNP C1/C2 may play an important role in regulating p53 transcription in response to cytostatic drugs 22. Pur-alpha and Pur-beta are single-stranded DNA binding proteins, which form a heterodimeric complex23 and regulate DNA replication and transcription 24. Both Pur-alpha and Pur-beta have been linked with acute myelogenous leukemia and brain tumors 25,26.…”