A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage

Daniella Brasacchio, Tahereh Noori, Colin M House, Amelia J Brennan, Kaylene J Simpson, Olivia Susanto, Phillip Ian Bird, Ricky W Johnstone, Joseph A Trapani

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.
Original languageEnglish
Pages (from-to)748 - 760
Number of pages13
JournalCell Death and Differentiation
Volume21
Issue number5
DOIs
Publication statusPublished - 2014

Cite this

Brasacchio, Daniella ; Noori, Tahereh ; House, Colin M ; Brennan, Amelia J ; Simpson, Kaylene J ; Susanto, Olivia ; Bird, Phillip Ian ; Johnstone, Ricky W ; Trapani, Joseph A. / A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage. In: Cell Death and Differentiation. 2014 ; Vol. 21, No. 5. pp. 748 - 760.
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title = "A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage",
abstract = "The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.",
author = "Daniella Brasacchio and Tahereh Noori and House, {Colin M} and Brennan, {Amelia J} and Simpson, {Kaylene J} and Olivia Susanto and Bird, {Phillip Ian} and Johnstone, {Ricky W} and Trapani, {Joseph A}",
year = "2014",
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A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage. / Brasacchio, Daniella; Noori, Tahereh; House, Colin M; Brennan, Amelia J; Simpson, Kaylene J; Susanto, Olivia; Bird, Phillip Ian; Johnstone, Ricky W; Trapani, Joseph A.

In: Cell Death and Differentiation, Vol. 21, No. 5, 2014, p. 748 - 760.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage

AU - Brasacchio, Daniella

AU - Noori, Tahereh

AU - House, Colin M

AU - Brennan, Amelia J

AU - Simpson, Kaylene J

AU - Susanto, Olivia

AU - Bird, Phillip Ian

AU - Johnstone, Ricky W

AU - Trapani, Joseph A

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AB - The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.

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