Figure 4. Poly(ADP ribosyl)ation on HOXB7 by PARP-1 reduces HOXB7 transcriptional activity.

A. Luciferase reporter assay were performed 24 hours after transfection of HOXB7, different amounts of PARP-1 plasmid as indicated, along with TTATpGL3 reporting reporter plasmid containing HOXB7 binding motifs, into HeLa cells. B. Different amounts of HOXB7 plasmids as indicated, with or without 400 ng PARP-1 plasmid and/or its inhibitor (10 nM DPQ), were transfected into HeLa cells, and luciferase activity assay was performed 24 hours after transfection. Paired student t-test was applied to compare the differences between both groups with or without the overexpression of PARP-1. C. Left panel: GST or GST-HOXB7 proteins were incubated with ³²P labeled oligonucleotide consisting of the HOXB7-consensus binding sequence (HBS) in presence or absence of PARP-1 proteins. Right panel: GST-HOXB7 proteins were incubated with ³²P labeled oligonucleotide consisting of the HOXB7-consensus binding sequence (lane 2) or in combination with 100-fold excess of unlabeled oligonucleotide (lane 3), or purified PARP-1 protein (lane 4), or purified PARP-1 and increasing amounts of NAD+ (lane 5, 6). D. Oligonucleotide precipitation assays (ONP) were performed by using SKBR3 cells transfected with vector, Flag HOXB7 and/or PARP-1 plasmids. One group of HOXB7 and PARP-1 transfectants were treated with DPQ 6 hours after transfection. Cell lysates were incubated with biotinylated HBS and/or DPQ. DNA protein complexes immobilized with streptavidin agarose beads were subjected to western blot with Flag antibody. Biotinylated SP1 binding site probe was used as negative control. Cell lysates were also blotted with anti-Flag, PARP-1 or β-actin antibody as loading control. E. Top panel: HOXB7 expressing and TTATpGL3 reporting plasmids were co-transfected with siPARP or siGFP control oligomer into HeLa cells, and luciferase activity was measured 24-hours post transfection. P value, derived from the Student t-test is shown. The efficacy of siPARP oligomer was tested by transient transfection of siRNA into HeLa cells and evaluated by western blot (bottom panel). F. Flag-HOXB7 plasmids were co-transfected with PARP-1 full-length or PARP-1 mutant plasmids, as indicated, into SKBR3 cells. Cell lysates were used for ONP assay in presence or absence of poly ADP ribose glycohydrolase (PARG) (top panel). The cell lysates were also used for western blot with anti-Flag, PARP-1 or β-actin antibody as loading control.