Abstract
Y-box protein-1 involvement in cyclin A and B1 gene regulation has recently been demonstrated. A more generalized role of this protein for cell replication is hypothesized as numerous regulatory sequences of cell cycle-related genes contain putative binding sites. In the present study the DNA polymerase alpha (DPA) gene is identified as another YB-1-responsive gene with a Y-box and 3' inverted repeat sequence, designated DPA RE-1, in the serum-responsive promoter region. Overexpressed YB-1 concentration-dependently trans-activated DPA gene expression in reporter assays and Southwestern blotting as well as DNA binding analyses revealed binding of distinct endogenous proteins to the RE-1 with molecular sizes of 26, 32 and 52 kDa. Among these, YB-1 binding was confirmed using recombinant as well as endogenous proteins, with preferential single-stranded DNA binding. Early serum growth response in mesangial cells was accompanied by a nuclear YB-1 shift and nucleocomplex formation at the RE-1. Fine mapping of the DPA RE-1 sequence unraveled a dependence on co-factors for trans-regulation with gene activation in the context of a heterologous SV40 promoter but suppression in the context of the abbreviated homologous promoter sequence. A YB-1 knock down resulted in decreased DPA transcription rates and abrogated the serum-dependent induction of DPA transcription. These results link YB-1 with serum responsiveness of DPA gene expression and provide insight into the required sequence and protein binding context.
Highlights
Y-box protein-1 involvement in cyclin A and B1 gene regulation has recently been demonstrated
Our findings add the DNA polymerase ␣ gene to this list, which is in accord with previous reports indicating that Y-box protein 1 (YB-1) is a crucial regulator of DNA replication [23] and cell proliferation [24]
Wahl et al reported [7] that in actively cycling cells, DNA polymerase ␣ mRNA, protein, and in vitro activity levels are constitutively expressed through the cell cycle
Summary
Close inspection of the DPA gene sequences Ϫ45/Ϫ17 revealed the presence of an inverted CCAAT box on the opposite strand with an inverse repeat sequence extending from Ϫ37 to Ϫ10 bps relative to the transcription start site (see Fig. 3). These motifs exhibit striking similarities to a previously identified enhancer cell; AS, antisense; BrdUrd, bromodeoxyuridine; RE-1, response element-1; MBN, mung bean nuclease; IR, inverted repeat. The current study examined the potential YB-1 interaction with the inverted CCAAT-box and demonstrates that YB-1 functions as a positive trans-activator of DPA gene expression, underscoring the pivotal role of YB-1 in the regulation of cellular proliferation
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