YY1-binding sites provide central switch functions in the PARP-1 gene expression network.

Martina Doetsch,Melita Vidaković,Goran Poznanović,Angela Gluch,Juergen Bode,Andre Van Wijnen

doi:10.1371/journal.pone.0044125

Martina Doetsch, Melita Vidaković + Show 4 more

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https://doi.org/10.1371/journal.pone.0044125

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Abstract

Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (−574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (−587/−558), BM4 (−348/−319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis.

Highlights

Poly(ADP-ribose) polymerase-1 (PARP-1) is the principal member of the PARP family of enzymes that utilize b-NAD+ as a substrate to synthesize and transfer ADP-ribose polymers to acceptor proteins, including itself
This study explores the transcriptional regulation of the muPARP-1 promoter by Yin Yang 1 (YY1) and extends our earlier work which dealt with an autoregulatory loop by which PARP-1 can limit its own expression [31]
YY1 binding to the muPARP-1 core promoter was subsequently examined in electrophoretic mobility shift assay (EMSA) experiments

Summary

Introduction

Poly(ADP-ribose) polymerase-1 (PARP-1) is the principal member of the PARP family of enzymes that utilize b-NAD+ as a substrate to synthesize and transfer ADP-ribose polymers to acceptor proteins, including itself (automodification). Subsequent studies have expanded the list of its functions and have led to the conclusion that PARP-1 is a constitutivelyexpressed, multifunctional enzyme for which DNA damageinduced hyper activation is just one out of several options [1,2]. In addition to its function as a DNA-damage sensor, the enzyme contributes to DNA methylation and imprinting [3], insulator activity [4], chromosome organization [5], the regulation of telomere length [6] and aging [7,8]. PARP-1 is involved in transcription regulation [9] and acts as an important modulator of transcriptional processes, enabling cells to cope with noxious stimuli [10]

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