Structural Basis of Multisite Single-Stranded DNA Recognition and ACTA2 Repression by Purine-Rich Element Binding Protein B (Purβ)

Abstract

A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purβ) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purβ as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purβ in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purβ knockdown MEFs confirmed that these changes were attributable, in part, to de-repression of ACTA2 transcription. To map the domains in Purβ responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally-derived models of the tertiary and quaternary structure of Purβ. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purβ homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purβ repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.

Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

“…Primers were selected using the QuikChange Primer Design Program (Supporting Information Table S1) and mutated plasmids were amplified using procedures specified by the manufacturer of the QuikChange II XL kit. Mixtures contained 1 μg of empty pCI plasmid or pCI-NHis-Purβ plasmid plus 0.9 μg of Acta2 promoterluciferase reporter plasmid (pVSMP8-Luc) 23 and 0.1 μg of pSV40-βGal control reporter plasmid. Mutated plasmids were isolated from bacterial host cells via double cesium chloride gradient centrifugation.…”

“…16,18,19 Conversely, repression of Acta2 in fibroblasts appears to be governed by the purinerich element-binding proteins A and B (Purα and Purβ) and Y-box binding protein 1 (YB1), which bind in a strand-specific manner to DNA sequences that overlap the G/C-rich and MCAT elements. 22,23 The solved x-ray crystal structures of the truncated repeat I-II region of Drosophila melanogaster (Dm) Purα and fulllength Borrelia burgdorferi (Bb) Purα showed that certain β-strand and α-helix forming sequences located in each repeat associate to form a so-called PUR domain. 16,20 Insights into the specific molecular mechanism by which Purα and Purβ repress Acta2 have been provided by studies that have uncovered the unique structural features of each protein.…”

“…The primary sequences of Purα and Purβ each contain three regions of internal homology known as Pur repeats I, II, and III. 23 Yet despite sharing similar structural features, the isolated repeat III domain from Purβ binds with high affinity and specificity to ssDNA, while the isolated repeat III domain from Purα is apparently deficient in binding to ssDNA. Importantly, this domain adopts a whirly-like fold and is capable of binding to single-stranded DNA (ssDNA).…”

“…22,23 The solved x-ray crystal structures of the truncated repeat I-II region of Drosophila melanogaster (Dm) Purα and fulllength Borrelia burgdorferi (Bb) Purα showed that certain β-strand and α-helix forming sequences located in each repeat associate to form a so-called PUR domain. 23,25 This distinction along with other structural differences may explain why Purβ functions as a dominant repressor of Acta2 in comparison to Purα when evaluated using both gain-of-function and loss-offunction approaches. 22,24 The more recently reported crystal structure of repeat III from Dm Purα confirmed that another PUR domain is formed by the intermolecular association of two repeat III sequences in a manner which mediates Purα dimerization.…”

“…23 Yet despite sharing similar structural features, the isolated repeat III domain from Purβ binds with high affinity and specificity to ssDNA, while the isolated repeat III domain from Purα is apparently deficient in binding to ssDNA. 22,23,30 Briefly, amino acid sequences corresponding to Mm Purβ residues 1 to 236 and 180 to 324 were separately submitted to the web-based modeling server I-TASSER 31,32 using 3K44 and 5FGO, respectively, as templates. 26,27 The National Heart Lung and Blood Institute (NHLBI) Grand Opportunity Exome Sequencing Project (GO-ESP) has analyzed the exomes of over 200 000 individuals enrolled in a number of well-known epidemiological studies to identify variants within the population.…”