Week 8: Journal Club

Question 1

Highlights of this paper

Answer 1

1. Elephants have an extra LIF genes; LIF6 is expressed in response to dna damage
2. LIF6 induces Bak/Bax apoptosis
3. LIF6 is a refunctionalized pseudogene

Question 2

Petos paradox

Answer 2

1. When there is no correlation between body size and cancer risk across species
2. People think this is because large bodied and/or long lived species evolved enhanced cancer protection mechanisms

Question 3

What does this paper show

Answer 3

1. That elephants and their extinct relatives may have resolved petos paradox by refunctionalizing a leukemia inhibitory factor (LIF6) with pro Apoptotic function
2. LIF6 is upregulated by TP53 in response to DNA damage=apoptosis
   2a. Suggests that refunctionalizing of a proapoptotic LIF pseudogene may have been permissive for the evolution of large body sizes in proboscideans

Question 4

Figure 2a/b/c

Answer 4

1. Figure a: studied duplications of the LIF gene and showed none of the duplicates have 5’UTR, coding exon 1 and paired low complexity CT rich repeat compared to the common LIF genes that do; these duplicates may result form independent duplication events in the past
2. Figure b: showed the LIF gene tree which showed the duplications and loss events in paenungulates; identified well supported classes containing loci from only a single species
3. Figure c: tree was reconstructed which showed 17 duplications (red) and 14 loss events (purple): additional LIF genes show from duplication and recombination

Question 5

Figure 4a

Answer 5

1. 4a: They identified 3 transcripts of the canonical LIF1 and 1 transcript for the duplicate LIF6 (low expression)
2. This establishes that LIF6 is transcribed as a response to DNA damage, supporting its potential role in a DNA damage response pathway controlled by TP53.

Question 6

Figure 4B

Answer 6

1. Found TP53 binding sites (as it induces LIF) in some animal’s LIF1 genes and elephants LIF6
2. Therefore they treated elephants with DOX (dna damage) or mdm2 antagonist (pro apoptosis) and measured the transcription of LIF1, LIF6 and TP53 target gene Bax (pro apoptosis)
3. DOX or n3a= increase in LIF6 and Bax (no effect on LIF1)
4. Suggests: LIF6 encodes a transcribed gene in elephants
5. To test whether TP53 regulates LIF6 expression in response to DNA damage.
6. Experiment Summary: The authors treated elephant fibroblasts with DNA-damaging agents (DOX or nutlin-3a) and used qRT-PCR to measure the expression of LIF6, TP53 target genes (e.g., BAX), and canonical LIF. They also used siRNA to knock down TP53 expression to test its role.
7. Results: LIF6 expression increased significantly after treatment with DOX and nutlin-3a. Knockdown of TP53 nearly abolished this induction, indicating that TP53 is necessary for LIF6 upregulation. Canonical LIF was not upregulated by the same treatments.
8. Big Picture: This demonstrates that LIF6 is a bona fide TP53 target gene, specifically induced in response to genotoxic stress.

Question 7

Figure 4c

Answer 7

1. Objective: To determine whether the upstream regulatory region of LIF6 contains a functional TP53 binding site.
2. Experiment Summary: The authors cloned the upstream regulatory region of LIF6 into a luciferase reporter and performed luciferase assays in elephant fibroblasts treated with DNA-damaging agents. They also deleted the TP53 binding site to test its necessity.
3. Results: The LIF6 regulatory region strongly activated luciferase expression in response to DOX and nutlin-3a, but this effect was abolished when the TP53 binding site was deleted.
4. Big Picture: This confirms that the TP53 binding site upstream of LIF6 is functional and critical for its transcriptional activation in response to DNA damage.

Question 8

Figure 4d

Answer 8

1. Objective: To verify that TP53 physically binds to the LIF6 regulatory region in vivo.
2. Experiment Summary: The authors performed ChIP-qPCR using a TP53 antibody to measure its binding to the LIF6 regulatory region after treatment with DNA-damaging agents (DOX or nutlin-3a).
3. Results: TP53 binding to the LIF6 regulatory region increased significantly after treatment with DOX or nutlin-3a. This binding was greatly reduced by siRNA-mediated TP53 knockdown.
4. Big Picture: This provides direct evidence that TP53 physically interacts with the LIF6 regulatory region in response to DNA damage, further validating it as a TP53 target gene.

Question 9

Figure 4E

Answer 9

1. Objective: To determine whether LIF6 contributes to DNA damage-induced apoptosis.
2. Experiment Summary: The authors transiently transfected fibroblasts with either control siRNA or TP53 siRNA and tested the effects of LIF6 expression on apoptosis using an ApoTox-Glo assay.
3. Results: Overexpression of LIF6 augmented apoptosis in response to DNA-damaging agents. Knockdown of TP53 reduced apoptosis, but LIF6 overexpression still induced apoptosis independently of TP53.
4. Big Picture: This demonstrates that LIF6 is both sufficient to induce apoptosis and acts as a downstream effector of TP53 in the DNA damage response.

Question 10

Figure 5A/B

Answer 10

1. Test contribution of LIF6 to the sensitivity of elephant cells to stress they designed 3 siRNAs that target LIF6
2. Figure a: Treated elephant cells w DOX or n3a: both DOX and n3a reduced cell viability and increased apoptosis (more stress when LIF6 was knockdown)
   2a. LIF6 inhibition increased cell viability and reduced apoptosis
3. Figure b: determine whether LIF6 expression was sufficient to induce apoptosis; LIF6 overexpression induced apoptosis in absense of DNA damage … suggests that LIF6 contributes to enhanced apoptosis response

Question 11

Figure 6

Answer 11

1. Figure a: To infer the mechanisms which LIF6 contributes to the induction of apoptosis, they determined the localization of LF6 in elephant fibroblasts: mitochondria
2. Figure b: to test whether LIF6 induced apoptosis was specific to elephant cells and independent of LIFR mediated signaling, they transferred a hamster w LIF6 which induced apoptosis
3. Figure c: to test whether LIF6 apoptosis is dependant on Bax/Bak; they over expressed LIF6 in Bax/Bak knowckdown; no apoptosis (it is dependant on Bax/bak)
4. Figure d: LIF6 overexpression, treatment w DOX or N3A induces loss of MMP (apoptosis=loss of MMP (collapses))
5. THEREFORE: LIF6 is sufficient to induce mitochondrial dysfunction and apoptosis mediated through Bax/Bak and independent of MPTP opening

Question 12

Figure 7

Answer 12

1. A: phylogeny of when LIF6 was refunctionalized: found the divergence rate 59 million years ago
2. B: LIF6 refunctionalized during evolutionary origin of large body
3. Therefore, LIF6 was reanimated sometime before the demands of maintaining a larger body