Purification of Proteins Associated with Specific Genomic Loci

Jérôme Déjardin and Robert E. Kingston

Background info

• Chromatin is essential to study entire proteome and regulation of cellular mechanisms in vivo• General lack of methods that preserve specific genomic regions

including associated factors

Background info

• Methods acknowledged by the authors that seek to identify proteins thus far include:• Yeast one-hybrid• Affinity purification

• Setbacks of these methods are that it “baits” proteins utilizing sequences nearby, thus taking it out of genomic context• GOAL: To be able to isolate genomic regions with associated factors

WITHOUT “baiting” out of context

Overview

Establishing a protocol called proteomics of isolated chromatin segments (PICh)

•A useful tool for finding what proteins are associated with a specific region on chromatin•Validated their technology by analyzing proteins that

associated with telomeres

Purpose

Looked at proteins found at telomerase-maintained telomeres and

Alternative Lengthening of Telomeres (ALT)

Validated PICh by asking whether PICh could:1. Associate previously known proteins with these telomeres2. Identify novel proteins that associate with telomeres

Telomerase-maintained telomeres

Alternative lengthening of telomeres

Finding the protein of interest

Detect the protein of interest located on telomeres

• purified proteins from a genomic locus using nucleic acid hybridization • Protein crosslinking was used to preserve protein-DNA

and protein-protein interactions• mass spectrometry to identify the associated factors

1. Associate previously known proteins with ALT

Identified and validated several proteins that specifically bind to ALT telomeres

• PICh on three human cell lines.• Two HeLa clones that are

telomerase positive and show different telomere length • One ALT telomeres of the WI38-

VA13 cells • Probe designed to hybridize with

telomere sequences• a probe with the same base

composition but in a scrambled order

•PICh identified 210 proteins associated with HeLa 1.2.11 telomeres and 190 proteins associated with ALT telomeres of the WI38-VA13 cells

• Consistent with MS results

• Found 33 proteins previously shown to associate with telomeres

• 4 proteins were not found

Telomere Associations

• Assembled a list of factors based on abundance. • Among the top 25 scoring proteins from each of the two lists

18 Telomerase (HeLa 1.2.11)

17 ALT (WI38-VA13)

• Seven out of eight novel proteins identified by PICh proved to be telomere-associated

are telomere-interacting proteins

2. Identify novel proteins that associate with telomeres

• Out of the seven novel proteins, two of the lowest ranked proteins from the ALT list and NXP-2 were analyzed

• All three proteins were found to

associate with ALT telomeres by immunostaining

• Out of the seven novel proteins, two most abundant proteins were COUP-TF2 (#17) and TR4 (#18).

• These transcription factors were specifically enriched at telomeres compared to other nuclear regions as shown in the figure

Novel proteins at ALT Telomeres

• Examined localization of orphan receptors (novel proteins) and telomeres using cells in metaphase. • Immunostaining of metaphase

chromosomes revealed prominent signals for orphan receptors co localized with shelterin proteins at the tips of chromosomes

• they wondered if the binding of orphan receptors to at telomeres might impact telomere maintenance.• According to the PICh data there are at least five out of seven

different orphan receptors bound to telomeres. • Knocking down the expression of all five of these proteins at the same

time is technically challenging, they knocked down the expression of the highest ranked associated protein (COUP-TF2) • Found subtle shortening of telomeres in cell with knockdown of

COUP-TF2• Conclude that there is a subtle but measurable impact on telomeres

caused by knocking down COUP-TF2

Conclusion

• PICh has proven to be effective for finding proteins that associated with telomeres and can extends to other loci. • The ability to identify proteins bound to a given sequence based

solely upon the identity of that DNA sequence will allow unbiased discovery of regulatory interactions at genomic loci.