Week 13: Journal Club

Question 1

HPGS

Answer 1

1. This disorder causes premature aging features
2. Mutation in lamin A gene = called progerin
   2a. Lamin organizes the NE, therefore when its mutated to progerin the organization of the NE is destroyed in these cells

Question 2

What do they show in this article

Answer 2

1. They know that heterochromatin anchored to nuclear lamina=LADs=repressed DNA …. In HPGS, nuclear defects=loss of heterochromatin regions =alterations in LADs
   1a. Telomere elongation by telomerase expression prevents progerin defects THEREFORE connection between telomere and lamins in the nucleoplasm plays a role in telomere protection
2. They find out: HGPS related lamina defects (progerin) trigger an altered 3D telomere organization with increased contact sites between telomeres and nuclear lamina; causes accelerated telomere shortening (early replication occurs which agrees w the loss of heterochromatin model (late replication))

Question 3

What are telomeres

Answer 3

1. Specialized nucleoprotein complexes capping the ends of linear chromosomes
2. Have repetitive nucleotide sequences: TTAGGG that end w 3’ overhang and are shielded by shelterin
   2a. Shelterin: repress DNA damage response at chromosome ends … deletion of shelterin= activation of ATM or ATR
3. Primary function of telomeres: delineate chromosome ends and avoid genomic instability

Question 4

Figure 1A/B/C/D

Answer 4

1. Hypothesis: expression of progerin in HGPS cells may also affect human telomere organization and be detrimental to their organization
2. Figure 1a: control (modelled HGPS fibroblasts by making normal HDF cell lines expressing EGFP-PG and used EGFP-LA and EGFP-NLS as controls): EGFP-LA is smaller because it was spliced: experiment worked!
3. Figure 1b: EGFP-PG-> lead to misshapen nuclei; EGFP-LA went to NE but didnt change morphology
4. Figure 1c: to assess impact of progerin on telomere distribution, imaged telomeres by staining TRF1 shelterin BUT there was too much NE blebbing so they developed madID (probes telomere-NE contact sites)
5. Figure 1d: MADID techniques: probes telomere-NE contact sites which relies on a bacterial methyltransferase which catalyzes the methylation of adenine residues (m6A): saw contact sites way better

Question 5

Figure 1E/F/G/H/I

Answer 5

1. Figure 1e: M6a signal proportional to DNA: control
2. Figure 1F: control: no difference to fold enrichment with or without m6a methylation (qPCR is suitable approach to assess purified telomeres in HDF(left) they successfully purified telomeres in HDF samples (right)
3. Figure 1g: lamin was able to methylate NLS-EGFP, EGFP-LA, EGFP-PG
4. Figure 1h: progerin expression induced 1.5x fold increase in m6a signal at telomeres: telomeres contact lamina more frequently in progerin cells (makes sense bc they shorten)
5. Figure 1i: CHIP analysis where they looked at two histone modifications; less histone modification in progerin cells; potentially diff 3D structure
6. Overall: results indicate that progerin expression affects telomere 3D organization and their chromatin state (heterochromatin loss)

Question 6

Figure 2

Answer 6

1. Critically short telomeres induce senescence in progerin expressing cells =growth arrest
2. Figure 2a: SB of terminal restriction fragments (TRFs), confirmed ectopic expresssion of progerin shortens telomeres
3. Figure 2b: Telomeres shortened (2a) at a rate of 100/150bp/PD
4. Figure 2c: SB of TRFs from normal HDF, HGPS M14/F8; initial telomere length was shorter in HGPS
5. Figure 2d/e: high hTert=increase in LaminB1=telomere length increases (hTert (telomerase) rescues short telomeres)
6. Figure 2f: telomere length was stabilizes in both; progerin doesn’t impair telomere elongation regulation by telomerase

Question 7

Figure 3

Answer 7

1. To find mechanism of action of progerin on telomere length (progerin=DNA replication defect=defect in telomere replication)
2. Figure 3a: monitored telomere integrity in EGFP-PG/LA/NLS and observed that shelterin subunits were unaffected by progerin expression (progerin doesn’t affect telomerase)
3. Figure 3b: confirm that TRF1/2 (core shelterin proteins) bind to TTAGGG repeats on telomeres (doesn’t affect shelterin on telomeres)
4. Figure 3c: fragile telomeres were found at both progerin expressing HGPS cell lines
5. Figure 3d: phenotype from 3c was more pronounced in HGPS M14 indicating patient-patient variability / link to age? Also increase of MTS (multi telomere signals) in HDF expressing EGFP-PG ; telomere replication affected in progerin cells
6. Figure 3e: used UFBs (anaphase bridges/ BAD/PICH): diffuse EGFP staining reflected NE breakdown (because they had anaphase bridged): problem in replication?
7. Figure 3f: (3e) found UFBs per anaphase was higher in progerin (problem in replication)
8. Conclusion: DNA replication defect triggered by progerin expression affects telomere replication

Question 8

Figure 4

Answer 8

1. From figure 3, they know that replication defects triggers HGPS so now they want to know what DNA damage repossess are activated
2. Figure 4a/b: confirm that endogenous levels of H2AX (ATM) was higher in HGPS cell lines compared to normal HDFs
3. Figure 4c: H2AX (ATM) was found enriched in HDF expressing EGFP-PG
4. Figure 4d: higher intensity of H2AX (c) in S and G2/M phase; transmission of the damage signal outside of S phase
5. Figure 4E: 53BP1 levels dropped in EGFP-PG: confirm defective recruitment of 53BP1 to sites of damage in progerin (it gets degraded by proteases)
6. Figure 4f: H2AX was detetected in nuclear fraction confirming checkpoint activation following induced DNA damage ; confirms specificity of 53BP1-LaminA interactions =progerin expression didnt prevent this interaction
7. Suggest progerin cells are sensitive to drugs challenging DNA replication =slow damage checkpoint activation=pathway is impaired due to decreased 53BP1