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Purification of Proteins
Associated with Specific
Genomic Loci
Jérôme Déjardin and Robert E. Kingston
Presented by Eric Labachyan and Lester Chiu
Telomeres and their significance
Two main functions
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Protect the chromosome terminus from unwanted nuclease and DNA repair activities
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Provide a mechanism to compensate for the inability of DNA polymerase to replicate the 5′ end of a linear
chromosome
Considerations
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Consist of several kilobases of TTAGGG DNA-repeats.
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Shorten with every round of replication
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Causes telomeres to lose their protective activity
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Dividing cells can only avoid telomere deprotection when able to induce sufficient activity of the telomerase
enzyme to add telomere repeats.
The shelterin protein
complex
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Unique set of proteins composed of TRF1, TRF2, RAP1, TIN2,
TPP1, and POT1.
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Protect telomeres from enzymatic attack.
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Telomeres contain a 3′ G-rich single-strand overhang that binds
POT1
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Forms a loop (t-loop) that hides the extreme end of the
chromosome from activity.
PICh protocol
Proteomics of isolated chromatin segments
Locked nucleic acid (LNA)
Increases stability of probe-chromatin
interaction through base stacking;
increases melting temperature
Spacer
Minimizes steric hindrance
Desthiobiotin
Immobilization tag
Proteomics of isolated chromatin segments
(PICh)
Uses DNA and nucleic acid hybridization
Correlates composition at locus and phenotype
Quantitative, does not require genetic engineering
Reversible formaldehyde crosslinking
Good cell permeability, fast cross-linking kinetics, short cross-linker length
Downstream advantages:
Protein Analysis - SDS-PAGE and Western Blot
Nucleic Acid Analysis - Library Prep and Sequencing (ChIP-Seq)
Allows for stringent capture and purification
Not sensitive to ionic detergents
Reduces non-specific binding of proteins
Hypothesis
Proof of principle
Validate whether PICh could:
Identify the majority of previously characterized associations with these telomeres
Identify novel proteins whose association with telomeres could be verified using independent
means
PICh Reveals Telomere Composition
Silver Staining of material obtained from PICh
S: “scrambled” probe
T: telomere-specific probe
Validation of Telomere Associations
Ranked list of factors based on abundance
(peptide number/protein size)
Validated association of proteins not previously
reported to interact w/ telomeres.
Two of the five lowest ranked proteins
(Fanc-J and RIP140) and NXP2
HMBOX1
Colocalized w/ RAP1 in 70% of
WI38VA13 but only 10% of
HeLa.
False positive
7/8 showed clear association. Low false
Orphan receptors at ALT Telomeres
Orphan nuclear receptors and Telomere PML-NB
colocalization
Proposed that critical step in ALT occurs in PMLNBs
Used shRNA to knock down COUP-TF2.
Colocalization of telomeres decreased from 8090% to 60%
Subtle shortening of telomeres but no obvious
proliferation defect.
Complicated by TR4 upregulation
Need to knock down all orphan receptors to
confirm
Summary/strengths,weaknesses/limitations
Strengths
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DNA hybridization for capture is not sensitive to high concentrations of ionic detergent
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Allows for stringent capture and purification conditions that reduce nonspecific binding of proteins.
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Each cell contains ∼100 telomeres, which greatly decreases the amount of input material needed.
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Capture probes contain locked nucleic acids, which increase the melting temperature by enhanced base stacking.
Weaknesses
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DNA-probe design
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Many cases such as damage caused by a genotoxic agent, site-specificity is not available.
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May not be easily adapted to identifying proteins at DNA sequences that are found at one or few copies in the genome.
Summary/strengths,weaknesses/limitations
Weaknesses
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Inability for locus-specific design
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Unlike locus-tagging system in iChIP, isolation of specific target alleles such as maternal or paternal alleles is not yet
feasible.
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Good example is genomic imprinting
References
Antao J.M., Mason J.M., Dejardin J., Kingston R.E. (2012) Protein landscape at Drosophila melanogaster telomere-associated sequence
repeats. Mol. Cell. Biol., 32, 2170–2182.
Jacobs, Jacqueline J. L. “Loss of Telomere Protection: Consequences and Opportunities.” Frontiers in Oncology 3 (2013): 88. PMC.
Guillen-Ahlers, Hector et al. “Advanced Methods for the Analysis of Chromatin-Associated Proteins.” Physiological Genomics 46.13
(2014): 441–447. PMC.
Fujita, Toshitsugu, and Hodaka Fujii. “Locus-Specific Biochemical Epigenetics/Chromatin Biochemistry by Insertional Chromatin
Immunoprecipitation.” ISRN biochemistry 2013 (2013): 913273.