Transcript Purification of Proteins Associated with Specific Genomic

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 telomeres
2. 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 WI38VA13 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)
are telomere-interacting

proteins
• Seven out of eight novel proteins identified by PICh proved to be
telomere-associated
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.