p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage.
Here, we identify a p53-induced IncRNA suicidal PARP-1 cleavage enhancer(SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis.
SPARCLE does not alter gene expression
Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.
Keywords
- p53: master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage.
- SPARCLE: a ~770-nt, nuclear lncRNA induced 1 day after DNA damage.
- PARP-1: NAD+ ADP-ribosyltransferase 1 or poly[ADP-ribose] synthase 1 is an enzyme that in humans is encoded by the PARP1 gene.
SPARCLE binds to PARP-1, causing apoptosis by PARP-1 cleavage. Producing NT-PARP-1, inhibiting DNA repair.
Introduction
p53
Master transcriptional regulator of the cellular geno- toxic stress response.
Inactivating mutation of TP53, the gene encoding p53, occur in almost every type of cancer and are linked to poor prognosis.
TP53 is the most frequently mutated gene in cancer, indicaitng its potency as a tumor suppressor.
p53 activates the transcription of over a hundred genes, including the CDK4/6 inhibitor CDKN1A/p21, to block cell-cycle progression and the bcl-2 family genes to promote apoptosis.
No singlel protein-coding p53 transcriptional target gene explains the strong effect of p53 on tumorigenesis and malignancy, prompting researchers to investigate p53-induced ncRNAs.
The miR-34 family is the most studied p53-regulated ncRNA.
- miR-34a on chromosome 1. Its overexpression enhances p53-mediated cell-cycle arrest or apoptosis.
- miR-34b and miR-34c, located in the same transcriptional unit on chromosome 11.
However, miR-34a deletion has minot effects on apoptosis and cell-cycle progression after genotoxic stress, which may be explained if other miR-34 family members, substitute for miR-34a.
However deletion ofo all 3 miR-34 miRNAs in mice did not impair the p53 response to genotoxic damage, unlike deletion of p53.
miR-34b/c deletion (unlike miR-34a deletion) reduced DNA-damage- induced apoptosis as much as p53 deficiency.
Here, we show that a lncRNA adjacent to the miR-34b/c cluster that we named suicidal PARP-1 cleavage enhancer (SPARCLE), which is not expressed in miR-34b/c- deleted cells and is induced late after DNA damage by p53 binding to a p53 response element (p53RE) that also induces miR-34b/c, regulates apoptosis after DNA damage.
Results
miR-34b/c knockout cells are resistant to DNA-damage- induced apoptosis
Deletion of miR-34b and miR-34c reduced DOX-induced apoptosis almost as much as p53 KO. Resulting in fewer sub G1 cells, and an increase in arrested cells.
Surprisingly, mature miR-34b and miR-34c mimics did not rescue apoptosis of miR-34b/c KO cells, suggesting that lack of these two miRNAs was not responsible for protection from apoptosis
SPARCLE is a ~770-nt nuclear lncRNA
SPARCLE’s expression was not detected above background by qRT-PCR under basal conditions in WT and p53 KO HCT116 but was induced after DOX treatment only in p53 WT cells.
Thus, SPARCLE is a low abun- dance, DNA-damage-induced, nuclear lncRNA.
SPARCLE’s promoter contains one functional p53 response element
Thus, p53 activation is all that is needed to induce SPARCLE.
p53RE2 is the only func- tional p53RE.
SPARCLE is a p53-induced lncRNA induced at low levels after genotoxic stress.
Cells lacking p53RE2 resist DNA-damage-induced apoptosis that is rescued by SPARCLE overexpression
SPARCLE and miR-34b/c are co-regulated or may form part of the same transcriptional unit.
SPARCLE is likely a trans-acting lncRNA rather than a local chromatin modifier.
Thus, SPARCLE is needed for apoptosis and the first 275 bases of SPARCLE is active.
SPARCLE deficiency reduces apoptosis in response to DNA damage
Thus, SPARCLE, but not miR-34b/c, acti- vates DNA-damage- and p53-induced apoptosis in multiple cell types.
SPARCLE promotes apoptosis in response to single- and double-stranded DNA damage
SPARCLE plays a major role in p53-mediated apoptosis in response to both SSB and DSB but may not be important in responding to oxidative DNA damage.
SPARCLE does not regulate gene expression
SPARCLE binds to PARP-1
RAP-WB confirmed an interac- tion of SPARCLE with both full-length (FL) and the caspase-3- cleaved N-terminal (NT) fragment of PARP-1 in WT cells.
SPARCLE deficiency enhances DNA-damage repair
SPARCLE reduced both HR- and NHEJ-mediated DNA repair.
SPARCLE enhances caspase-3-mediated cleavage of PARP-1
By which SPARCLE increases apoptosis is by enhancing PARP-1 cleavage by caspase-3, rather than by directly altering its catalytic activity.
NT-PARP-1 interferes with DNA repair and restores DNA-damage-induced apoptosis in SPARCLE KO cells
Because NT-PARP-1 expression rescues SPARCLE deficiency, the main mechanism by which SPARCLE inhibits DNA repair and increases apoptosis is likely by enhancing cas- pase-3 cleavage of PARP-1.
SPARCLE KO tumors are relatively resistant to chemotherapy
SPARCLE inhibits DNA repair and promotes apoptosis after DNA damage in vivo and its absence modestly, but significantly, promotes HCT116 tumor growth.