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Science 323 (5917): 1057-1060

Copyright © 2009 by the American Association for the Advancement of Science

HIN-200 Proteins Regulate Caspase Activation in Response to Foreign Cytoplasmic DNA

Tara L. Roberts1,2*, Adi Idris1*, Jasmyn A. Dunn1, Greg M. Kelly1, Carol M. Burnton1, Samantha Hodgson1, Lani L. Hardy1, Valerie Garceau1{dagger}, Matthew J. Sweet1,3, Ian L. Ross1, David A. Hume1{dagger}, and Katryn J. Stacey1,3{ddagger}

1 The University of Queensland, Institute for Molecular Bioscience, QLD 4072, Australia.
2 Queensland Institute of Medical Research, Brisbane, QLD 4029, Australia.
3 The University of Queensland, School of Chemistry and Biomolecular Science, QLD 4072, Australia.

Figure 1 Fig. 1.. Transfected DNA–induced BMM death is dependent on DNA length and strandedness and is independent of TLR9. Mitochondrial activity was measured by cleavage of the MTT reagent as an index of cell viability. Except as noted, the assay was performed at 24 hours after transfection, and bars show the mean and error bars the range of duplicate electroporations. Results are representative of two to five experiments. (A) Wild-type and TLR9–/– BMMs were electroporated either without DNA or with 10 µg of calf thymus genomic DNA (CT DNA). Results were normalized to the "no DNA" samples. (B) Cell death was caused by transfected dsDNA but not ssDNA. C57BL/6 BMMs were electroporated without DNA or with 10 µg of poly(dA) or poly(dA):(dT). (C) Loss of mitochondrial function occurred as early as 3 hours after transfection of poly(dA):(dT). TLR9–/– BMMs were electroporated either without DNA, with the indicated amounts of poly(dA):(dT), with 3 µg of DNase I–digested poly(dA):(dT) ("DNase 3µg"), or with DNase I in digestion buffer as a control ("DNase alone"). The assay was perfomed at 3 hours after electroporation. Bars show the mean and error bars the range of values from two independent experiments. (D) The length of electroporated DNA fragments determines the degree of cell death. BALB/c BMMs were electroporated without DNA or with 10 µg of polymerase chain reaction (PCR) products of various length, as indicated, or with CT DNA. (E) Caspase 3 is rapidly activated after electroporation with dsDNA. BALB/c BMMs were either untreated ("no zap") or electroporated alone ("–DNA") or with 20 µg of CT DNA ("+DNA"). Cells were harvested after 5, 15, or 20 min. Total caspase 3 was detected by Western blotting. Pro–caspase 3 is 35 kD in size and activated cleaved forms are 17 and 19 kD in size. [View Larger Version of this Image (54K GIF file)]

Figure 2 Fig. 2.. p202 is a dsDNA-specific binding protein found in the ultracentrifuge pellet fraction of cytoplasmic extract. (A) EMSA shows that cytoplasmic extract contains a dsDNA-specific binding protein (arrow). Protein extract was bound to 44-bp dsDNA or 44-base ssDNA probe alone ("no comp.") or with a 70-fold molar excess of unlabeled ds or ss probe, showing that ssDNA did not compete for binding. "Probe" indicates probe without extract. (B) The dsDNA binding protein requires no specific sequence motif. Cytoplasmic protein was incubated with probe and 7- or 13-fold molar excess of competitors, either unlabeled probe (ds44bp) or a sequence-scrambled version (dsSCR). (C) Binding depends on DNA length. Cytoplasmic protein was incubated with 0.77 ng of 44-bp probe and 1.2 ng of unlabeled 22-, 44-, or 100-bp DNA (1.6-fold ng excess). (D) Purification of dsDNA-binding protein. Cytoplasmic extract was incubated with beads bound to either dsDNA, ssDNA, or no DNA ("beads"). Analysis of bound proteins revealed a 52-kD dsDNA binding protein (arrow). (E) EMSA supershift showing that the dsDNA-specific binding protein is p202. Cytoplasmic protein and probe were incubated with or without antisera against p202, p204, or vimentin. (F) p202 stably interacts with cytosolic DNA. Biotinylated plasmid DNA ("biotin-dsDNA"), unlabeled plasmid ("dsDNA"), or biotinylated 44-base oligonucleotide ("biotin-ssDNA") was electroporated into RAW264 cells stably expressing p202-V5. After 1 hour, cells were lysed and proteins associated with the biotinylated DNA were isolated by binding to streptavidin-Sepharose ("pellet"). The location of p202 in the biotinylated DNA-bound pellet fraction or unbound supernatant (S/N) was assessed by Western blotting for V5. (G) Purified recombinant SUMO-p202 binds directly to plasmid DNA. N-terminally SUMO/His-tagged p202 was incubated with streptavidin beads with or without bound biotinylated dsDNA. Bound protein was analyzed by Western blotting with anti-His. Analysis of input protein (right) shows full-length SUMO-p202 (arrow) and two C-terminal truncations (asterisks). A contaminating cross-reactive protein of 15kD bound nonspecifically to beads. (H) Purified SUMO tag does not bind to DNA. The experiment was performed as in (G). [View Larger Version of this Image (80K GIF file)]

Figure 3 Fig. 3.. Colocalization of microinjected Cy3-labeled CT DNA with p202. One hour after microinjection with labeled DNA, NZB BMMs were methanol fixed and antibody stained to show p202 localization. p202 (green, left panels) completely colocalized with CT DNA (red, center top panel) and slightly with ds44bp (red, center panel) and not at all with ds22bp (red, center bottom panel). Colocalization is indicated in yellow (right panels). [View Larger Version of this Image (112K GIF file)]

Figure 4 Fig. 4.. HIN-200 factors regulate DNA-dependent caspase activation. (A) Knockdown of p202 enhances DNA-induced caspase 3 and caspase 1 activation. BALB/c BMMs were electroporated with the indicated siRNAs and left for 24 hours before electroporation with either no DNA ("cont.") or 1 µg of poly(dA):(dT) ("AT"). After 20 min, cells were lysed and protein extracts analyzed by Western blotting for cleaved caspase 3, S6 ribosomal protein as loading control, and caspase 1. A separate gel with the same samples is shown for caspase 1. The large arrow indicates full-length pro-caspase 1 (45kD), a small arrow the active cleaved product (10 kD), and the asterisk a nonspecific band. Results are representative of five experiments with three different siRNAs against p202 (fig. S10). (B) p202 and p204 mRNA expression relative to hypoxanthine-guanine phosphoribosyltransferase (HPRT) measured by real time PCR, 24 hours after electroporation with siRNAs used in (A). Bars show the mean of duplicate assays and error bars the SD as defined in the online methods. (C) Knockdown of AIM2 prevents DNA-induced caspase 3 and caspase 1 activation. BALB/c BMMs were electroporated with the indicated siRNAs and left for 24 hours before electroporation with either no DNA ("cont."), 10 µg of CT DNA ("CT"), or 1 µg of poly(dA):(dT) ("AT"). Samples were analyzed as in (A). Results are representative of four experiments. (D) AIM2 mRNA expression relative to HPRT measured by real time PCR as in (B), 24 hours after electroporation of siRNAs used in (C). (E) Expression of p202 and AIM2 mRNAs in BMMs from C57BL/6, BALB/c, and NZB mouse strains. Shown are the mean and range of results for two independent RNA preparations. (F) Activation of caspases in BMMs from three mouse strains electroporated with DNAs as in (C). Arrows and asterisk are as in (A). [View Larger Version of this Image (52K GIF file)]

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