March 6, 2014 D145 Presentation Manpreet Singh and Veronica Ortiz

1. March 6, 2014 D145 Presentation Manpreet Singh and Veronica Ortiz

2. Background • Problem: • Eukaryotic DNA is bound and interpreted by various protein complexes in the context of chromatin • Understanding the proteins that regulate specific loci is critical to understanding regulation • However, chromosomes remain poorly understood due to the inability to purify chromatin segments

3. Past strategies • Achieved enrichment of the targeted regions • However, none gave a sufficient amount and purity to allow identification of bound factors • Yeast one-hybrid and nucleic acid affinity capture • Identifies sequence –specific DNA-binding proteins • Problem: does not provide a complete description of what is found at the loci in vivo • ChIP • Finds if a protein of interest is bound to a given genomic region • Problem: it relies on the use of antibodies, thus resulting to limitations

4. Goal • To develop a strategy to purify an endogenous segment of chromatin in sufficient quantity and purity to identify the associated proteins • Methods had to be: • Direct • Relatively quantitative • Achievable without the requirement for genetic engineering • Proteomics of Isolated Chromatin (PICh)

5. PICh • Uses DNA to retrieve the protein information, which is the opposite of ChIP (uses protein antigens to retrieve DNA) • Used nucleic acid hybridization as the basis for purification • Isolated specific formaldehyde-crosslinkedchromatin-probe regions and identified the proteins bound to those loci using mass spec.

6. Cells are fixed • Chromatin is solubilized • Specific probe was hybridized to the chromatin • Hybridized chromatin was captured on magnetic beads • Hybrids were eluted • Associated proteins were identified with MS

7. Mass Spectrometry Nature Reviews Cancer 10, 639-646 (September 2010)

8. Fine Tuning PICh • Hybridization is insensitive to the presence of ionic detergents • Limits contamination • Stability of probe-chromatin interactions was increased by the use of LNA • Have altered backbones, which favors base staking and increases the melting point • Makes the probe to chromatin interactions harder to break • Minimize steric hindranceby adding long spacers between the immobilization tag and the LNA probe • Improves yield • Limited co-elution of nonspecific factors was achieved by using desthiobiotin • Biotin analog with weaker affinity • Allows a competitive but gentle elution

9. Chromosomes reach a critical length where it becomes “old” and the cell dies • Popular because of the “immortal cell” theory • Telomerase adds to the telomeres so cells don’t age, thus important in cancer and aging fields

10. Pathways for telomeres aTelomerase lengthening of telomeres

11. PICh Tested on Telomeres • Attractive target to test PICh because 1) They are abundant • ~100 telomeres per cell 2) Are of significant biological interest they have been well characterized • Perfect to validate PICh

12. PICh on Telomeres • Used PICh on three human cell lines 1) 2 HeLa clones that are telomerase positive with different telomere length 2) WI38-VA13 ALT cell line with ALT pathway • Probes were designed to hybridize with telomeres • Control involved a probe with the same sequence but in a scrambled order Figure 2. Purification of Telomeric Chromatin from Transformed Human Cell Lines A) Silver staining of material obtained from PICh purified telomere chromatin.

13. Results of PICh and Telomeres PICh was able to retrieve 85% of the known components of the targeted locus

14. Figure 2. Purification of Telomeric Chromatin from Transformed Human Cell Lines (C) Coimmunostaining with the Flag-tagged HMBOX1 and RAP1 in the WI38-VA13 and HeLa 1.2.11 cell lines (B) Validation of selected PICh associations to ALT telomeres by immunostaining

15. Orphan Receptors • When characterizing the proteins of ALT telomeres, they found 2 orphan receptors (COUP-TF3 and TR4) • Orphan receptors: receptors whose endogenous ligands are yet to be characterized • Orphan receptor and ALT telomere’s association and specificity was analyzed

16. PICh • Past methods were powerful but did not provide information concerning the complete composition of a locus • PICh has the potential to characterize chromosomes by providing a method to examine the entire set of interacting proteins and how the composition changes during regulation • With PICh previous known factors were identified and new proteins, as well

17. Cons Pros • Need for sufficient amount of protein for MS • Optimization of probe design to ensure maximum protein identification • Can be used on repeat or low-copy elements • No genetic engineering required • Can characterize non-chromatin targets

18. Future Goals • To identify proteins bound to regulatory sequences based solely on DNA, in order to allow unbiased discovery of regulatory interactions at key genomic loci