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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 - Protect the chromosome terminus from unwanted nuclease and DNA repair activities - Provide a mechanism to compensate for the inability of DNA polymerase to replicate the 5′ end of a linear chromosome Considerations - Consist of several kilobases of TTAGGG DNA-repeats. - Shorten with every round of replication - Causes telomeres to lose their protective activity - 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 - Unique set of proteins composed of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. - Protect telomeres from enzymatic attack. - Telomeres contain a 3′ G-rich single-strand overhang that binds POT1 - 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 - DNA hybridization for capture is not sensitive to high concentrations of ionic detergent - Allows for stringent capture and purification conditions that reduce nonspecific binding of proteins. - Each cell contains ∼100 telomeres, which greatly decreases the amount of input material needed. - Capture probes contain locked nucleic acids, which increase the melting temperature by enhanced base stacking. Weaknesses - DNA-probe design - Many cases such as damage caused by a genotoxic agent, site-specificity is not available. - 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 - Inability for locus-specific design - Unlike locus-tagging system in iChIP, isolation of specific target alleles such as maternal or paternal alleles is not yet feasible. - 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.