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Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

Bottom Line: A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering.We find that all depend directly or indirectly on caspase-3 activation.CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Dynamics Unit, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

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HMGB1 immobilization is not mediated by binding to DNA or disulfide bonds.(A) The substitution of F37, F102 and I121 residues with alanines do not impact HMGB1 apoptotic immobilization. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wt and mutHMGB1. The results are expressed as mean +/− standard deviation (n = 16). (B) The three cysteine residues in HMGB1 were mutated to serine. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wtHMGB1-GFP, singly mutated HMGB1 (HMGB1C22S-GFP, HMGB1C44S-GFP and HMGB1C105S-GFP), and triply mutated HMGB1 (HMGB1C22/44/105S-GFP). The results are expressed as the mean +/− standard deviation (n = 17).
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pone-0006234-g002: HMGB1 immobilization is not mediated by binding to DNA or disulfide bonds.(A) The substitution of F37, F102 and I121 residues with alanines do not impact HMGB1 apoptotic immobilization. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wt and mutHMGB1. The results are expressed as mean +/− standard deviation (n = 16). (B) The three cysteine residues in HMGB1 were mutated to serine. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wtHMGB1-GFP, singly mutated HMGB1 (HMGB1C22S-GFP, HMGB1C44S-GFP and HMGB1C105S-GFP), and triply mutated HMGB1 (HMGB1C22/44/105S-GFP). The results are expressed as the mean +/− standard deviation (n = 17).

Mentions: The two HMG boxes have hydrophobic residues in conserved positions (F37, F102, I121) that anchor them to the minor groove of DNA [14], and three cysteine residues, of which two (Cys22 and Cys44) can form a disulfide bond, while the third (Cys105) is free (Figure 2) [15]. Triple-mutated HMGB1-GFP (mutHMGB1-GFP), where the 3 critical hydrophobic residues have been replaced by alanine, was already shown to have reduced binding to chromatin [16], [17]. We also constructed cysteine-to-serine single and triple mutants. We tested each mutant by FRAP in proliferating HeLa cells and in cells forced to undergo apoptosis. For all HMGB1 mutants the mobile fraction in apoptotic cells corresponds roughly to 25% of the total pool of fluorescent protein, not unlike the wt (Figure 2). Therefore, the severe reduction of either the ability to bind DNA or to form disulfide bonds does not impair HMGB1 immobilization in the nucleus of apoptotic cells. These data also imply that oxidation of HMGB1, that occurs on C105 [18], does not modify HMGB1 binding to chromatin.


Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

HMGB1 immobilization is not mediated by binding to DNA or disulfide bonds.(A) The substitution of F37, F102 and I121 residues with alanines do not impact HMGB1 apoptotic immobilization. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wt and mutHMGB1. The results are expressed as mean +/− standard deviation (n = 16). (B) The three cysteine residues in HMGB1 were mutated to serine. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wtHMGB1-GFP, singly mutated HMGB1 (HMGB1C22S-GFP, HMGB1C44S-GFP and HMGB1C105S-GFP), and triply mutated HMGB1 (HMGB1C22/44/105S-GFP). The results are expressed as the mean +/− standard deviation (n = 17).
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Related In: Results  -  Collection

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pone-0006234-g002: HMGB1 immobilization is not mediated by binding to DNA or disulfide bonds.(A) The substitution of F37, F102 and I121 residues with alanines do not impact HMGB1 apoptotic immobilization. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wt and mutHMGB1. The results are expressed as mean +/− standard deviation (n = 16). (B) The three cysteine residues in HMGB1 were mutated to serine. FRAP experiments were performed in proliferating and apoptotic HeLa cells transfected respectively with wtHMGB1-GFP, singly mutated HMGB1 (HMGB1C22S-GFP, HMGB1C44S-GFP and HMGB1C105S-GFP), and triply mutated HMGB1 (HMGB1C22/44/105S-GFP). The results are expressed as the mean +/− standard deviation (n = 17).
Mentions: The two HMG boxes have hydrophobic residues in conserved positions (F37, F102, I121) that anchor them to the minor groove of DNA [14], and three cysteine residues, of which two (Cys22 and Cys44) can form a disulfide bond, while the third (Cys105) is free (Figure 2) [15]. Triple-mutated HMGB1-GFP (mutHMGB1-GFP), where the 3 critical hydrophobic residues have been replaced by alanine, was already shown to have reduced binding to chromatin [16], [17]. We also constructed cysteine-to-serine single and triple mutants. We tested each mutant by FRAP in proliferating HeLa cells and in cells forced to undergo apoptosis. For all HMGB1 mutants the mobile fraction in apoptotic cells corresponds roughly to 25% of the total pool of fluorescent protein, not unlike the wt (Figure 2). Therefore, the severe reduction of either the ability to bind DNA or to form disulfide bonds does not impair HMGB1 immobilization in the nucleus of apoptotic cells. These data also imply that oxidation of HMGB1, that occurs on C105 [18], does not modify HMGB1 binding to chromatin.

Bottom Line: A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering.We find that all depend directly or indirectly on caspase-3 activation.CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Dynamics Unit, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

Show MeSH