15 research outputs found
Defining regions of PARP-1 that interact with HOXB7.
<p>A. The molecular structure of PARP-1 and the deletion constructs used in the present study are shown in the panel. B, C. Full-length PARP-1 and different truncations of PARP-1 proteins as indicated were <i>in vitro</i> transcribed and translated (TNT) in presence of the <sup>35</sup>S-methionine and subjected to GST-HOXB7 or GST pull-down assay (left panels Fig. 2B and 2C). The right panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040644#pone-0040644-g002" target="_blank">Figure 2B and 2C</a> show the input (20%) of the PARP-1 full-length and truncation proteins from TNT products. D. Plasmids coding Flag-tagged HOXB7 and Myc-tagged wild type PARP1 or first zinc-finger deleted PARP1 were cotransfected into MCF-7 cells. Cell lysates were immunoprecipitated with anti-Flag antibody. PARP-1 and HOXB7 protein were detected with anti-Myc or anti-Flag antibodies.</p
HOXB7 is the substrate of PARP-1 and is poly(ADP ribosyl)ated by PARP-1.
<p>A. Different amounts of Flag tagged HOXB7 plasmids were transfected into SKBR3 cells, cells were harvested and permeabilized by 0.01% digitonin in PARP-1 activity reaction buffer. PARP-1 auto-modification was visualized by autoradiograph after the cell lysates were separated by SDS-PAGE (top panel). Immunoblot with anti-Flag, PARP-1 and β-actin antibodies were performed after autoradiography (bottom panels). B. GST and GST-HOXB7 fusion proteins were incubated with or without purified PARP-1 protein or <sup>32</sup>P NAD+. After 30 minutes incubation, free 32P NAD+ and unbound PARP-1 proteins were washed off with reaction buffer. Proteins on glutathione sepharose beads were then separated by SDS-PAGE and transferred to PVDF membrane and stained with Ponceau (left panel). The poly(ADP ribosyl)ated proteins were visualized by autoradiography (right panel). C. Vector control and Flag-tagged HOXB7 plasmids were transfected into SKBR3 cells. Cells were harvested and incubated with PARP-1 activity assay buffer including <sup>32</sup>P NAD+ or PARP-1 inhibitor (20 µM DPQ). Cell lysates were immunoprecipitated with anti-Flag antibody and separated by SDS-PAGE followed by autoradiography (top panel) and immunoblotting with anti-Flag antibodies (bottom panel).</p
PARP-1 interacts and modifies other HOX proteins.
<p>A. SKBR3 cells were transfected with empty vector, or Flag-tagged HOXA5, B6, B7, C6 and C8, respectively. Cell lysates were immunoprecipitated with anti-Flag antibody, and western blotted with anti-PARP-1 (top panel) and Flag antibodies (bottom panel), respectively. HOXC6 transfected SKBR3 cell lysate was used as loading control. B. Vector control and Flag-tagged HOX plasmids were transfected into SKBR3 cells as indicated. Cells were harvested and incubated with PARP activity reaction buffer containing 0.01% digitonin and <sup>32</sup>P NAD+. Cells were then lysed after incubation and immunoprecipitated with anti-Flag antibody. The precipitated complexes were then separated by SDS-PAGE, transferred to the nitrocellulose membrane that was used for autoradiography (top panel) and western blot (bottom panel) using anti-PARP-1 and anti-Flag antibodies. C. Multiple sequence alignment of the C-terminal peptides of HOXA5, HOXA7, HOXB6, HOXB7, HOXC6 and HOXC8 show extent of homology. D. The ONP assay was performed with the SKBR3 cells transfected with Flag HOXA7 with or without PARP-1, and one set of the HOXA7 and PARP-1 co-transfected cells was treated with DPQ 6 hours post transfection. Cell lysates were used for ONP (top panel) and western blots (bottom panel). E. ONP assays were performed with SKBR3 cells transfected with Flag HOXA1, Flag HOXA5, Flag HOXC6 or Flag HOXB6 plasmids with or without PARP-1 plasmids, and one set of each of the HOX and PARP-1 co-transfected cells was treated with DPQ 6 hours post transfection. Cell lysates were used for ONP assays (top panel) and western blots (bottom panel). F. ONP assay was performed with the SKBR3 cells transfected with Flag-tagged HOXB6 or the Lys to Glu mutant, HOXB6K221E, with or without PARP-1. Cell lysates were used for ONP assays (top panel) and western blots (bottom panel).</p
Poly(ADP ribosyl)ation on HOXB7 by PARP-1 reduces HOXB7 transcriptional activity.
<p>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 <sup>32</sup>P 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 <sup>32</sup>P 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.</p
HOXB7 interacts with PARP-1.
<p>A. Co-immunoprecipitation of PARP-1 with HOXB7-YFP in SKBR3 cells. SKBR3 cells were stably transfected with HOXB7-YFP (lanes 1, 4, and 7) or YFP alone as a vector control (lanes 2, 5, and 8) prior to immunoprecipitation with GFP antibodies and subsequent Western blotting of precipitated proteins. Parental SKBR3 cells, which lack detectable HOXB7, were used as controls as well (lanes 3, 6, and 9). Lanes 1 to 3, protein levels in 100µg of total cell extract (5% of input); lanes 4 to 6, proteins that precipitated with HOXB7-YFP or controls that did not express HOXB7 (SKBR3-YFP and parental cells). Normal rabbit serum (NRS) was used to control for specificity (lanes 7–9). B. Endogenous interaction between HOXB7 and PARP-1. Extracts of MCF-7 cells were co-immunoprecipitated with antibodies to PARP-1, HOXB7 or p53 as a nonspecific IgG (NS IgG). Subsequent immunoblotting was done with the antibodies indicated. C. Flag-tagged HOXB7 or constructs in which select regions were deleted or mutated, were transiently transfected into CHO cells together with a PARP expression construct (PARPpCR3.1, where indicated, empty plasmid was used as control) to determine if a specific region of HOXB7 mediated its interaction with PARP. Co-immunoprecipitation with FLAG antibodies (top panel) was performed followed by immunoblot with PARP antibodies. The lower panel shows protein expression of all transfected plasmids. Structure of FLAG-HOXB7 showing locations of point mutations and deletions is shown below. F: Flag tagged HOXB7, P: pentapeptide, H: homeodomain, h: helix 3 of the homeodomain. D. Full length HOXB7 or deletion constructs B7-D1 or B7-D2 were transcribed and translated <i>in vitro</i> in the presence of <sup>35</sup>S-methionine prior to mixing with <i>in vitro</i> transcribed and translated PARP-1. Immunoprecipitation was performed with PARP-1 monoclonal antibodies (lanes 1, 3 and 5). Complexes were resolved by SDS-PAGE and subjected to autoradiography for 24 hours. Lanes 2, 4 and 6 are input (20%) from the TNT reactions. Deletions D1 and D2 of full length HOXB7 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040644#pone-0040644-g002" target="_blank">Figure 2</a>. Asterisks point to the specific bands for HOXB7 full-length or deletion proteins. P: pentapeptide, H: homeodomain.</p